Iron-containing Nanoparticles for the Treatment of ...

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Spring 4-15-2019

Iron-containing Nanoparticles for the Treatment ofChrionic Biofilm Infections in Cystic FibrosisLeisha M A MartinUniversity of New Mexico - Main Campus

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Recommended CitationMartin Leisha M A Iron-containing Nanoparticles for the Treatment of Chrionic Biofilm Infections in Cystic Fibrosis (2019)httpsdigitalrepositoryunmedunsms_etds52

i

Leisha Marie Martin Candidate

Nanoscience amp Microsystems Engineering

Department

This dissertation is approved and it is acceptable in quality and form for publication

Approved by the Dissertation Committee

Marek Osiński PhD Chairperson

Terefe Habteyes PhD

Erin Milligan PhD

Pavan Muttil PhD

ii

IRON-CONTAINING

NANOPARTICLES FOR THE TREATMENT OF

CHRONIC BACTERIAL BIOFILM INFECTIONS

IN CYSTIC FIBROSIS

by

LEISHA MARIE MARTIN

BS Biology University of New Mexico 2010

MS Nanoscience amp Microsystems University of New Mexico 2012

DISSERTATION

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Doctor of Philosophy

The University of New Mexico

Albuquerque New Mexico

May 2019

iii

DEDICATION

To my children Jonathan and Isadora who have gone without many things so that this

project could be completed

사랑해

I love you very much

iv

ACKNOWLEDGEMENTS

Foremost I would like to thank my children Jonathan and Isadora for sacrificing after

school activities for hours in the lab thus solidifying their unwanted however thorough

advanced knowledge of materials science I want to also acknowledge the children I have

had or otherwise acquired between the time that this work was done and the time that this

paper was completed Tobias Hadassah Hannah and Joshua I would like to thank my

parents Robert and Anita Armijo for their continual support and of course their help with

the children I would also like to thank my advisor Dr Marek Osiński first of all for his

mentorship longsuffering and dedication to my work and also for teaching me what

veritable patience is Dr Maggie Werner-Washbourne for her above and beyond tireless

support lessons in truth and life Without her there is no way this work could have been

brought to completion She is truly the encompassment of a wise woman I kindly thank

Dr Sang Han for his mentorship and taking the time to personally review this dissertation

and keep me on track Another person who has contributed significantly to the completion

of this work Ms Linda Stewart who I sincerely thank for her hours of dedication and

overall generous contribution of personal time and effort committed to this project I thank

Dr Nathan Withers for sharing his wisdom and mentorship regarding everything science

Dr John Plumley for his assistance in mentoring students Dr Antonio Rivera and

Nathaniel Cook for their contribution in providing outstanding electron microscope

images Thank you to Dr Hugh Smyth for his mentorship and contributions to this work

Additionally I am grateful to Dr Yekaterina Brandt for her general mentorship in the field

of biology Many thanks to Stephen J Wawrzyniec not only for his perpetual support but

v

also for working alongside me over five exhausting 42-hour shifts in order to ensure that

our green chemistry solid-gas procedure was up and running I would also like to recognize

all of Dr Osińskirsquos lab members past and present whom have aided in one way or

another Dr Gennady Smolyakov Dr Erum Jamil Mallal Dr Farhana Anwar Brian

Akins Darcy Kruse and Shayla Nahar Bhuiya I must also acknowledge our collaborators

at the Sandia National Laboratories Center for Integrated Nanotechnology (CINT) Dr

Dale Huber Dr Todd Monsoon Dr Sergei Ivanov Dr Erika Cooley Vreeland and Dr

John Reno Also thank you to my students Jocelyn Baca Christian Carrillo Salomon

Maestas Cody Kamrowski Anna Sharma Shaheen Ahghar Alicia and Megan Williams

Madalyn Fetrow Michael Kopciuch Zuzia Olszoacutewka Qaiser Zaidi Allison Hayat F Zuly

Fornelli Annaka Westphal Abhyudai Nouni Surabhi Yadav Angelina Malagodi Gema

Alas Jane Nguyen Rana Chan Dennis Huang and all the other bright ambitious scientists

I have had the pleasure of working with over the years Thank you to the entire UNM

Neuroscience Department Dr Dan Savage Dr Martina Rosenberg and Dr Linda Saland

Many thanks to the Milligan lab and the previous Milligan lab members Wolfgang Scott-

Cohen Ellen Dengler Jenny Wilkerson and Audra Kerwin I thank Dr Natalie Adolphi

for her mentorship in physics electricity and magnetism and nanomagnetics I would like

to acknowledge my dissertation committee members and express my sincere thanks for

their review of this work Dr Terefe Habteyes chemist optics expert and recipient of the

National Science Foundationrsquos (NSF) Faculty Early Career Development (CAREER)

award for his project ldquoNear-Field Imaging for Nanoscale Visualization of Exciton-

Plasmon Energy Transferrdquo Dr Erin Milligan who mentored me during my time as an

vi

undergraduate student and taught me many things some of the most important being hard

work and attention to detail Dr Milligan was awarded the Regentrsquos Lectureship Award in

the Neurosciences Department in 2013 and has authored over 88 publications Dr Pavan

Muttil an expert in inhaled pharmaceuticals has authored over 50 publications disclosed

nine inventions and has two pending patent applications for his inhaled and oral vaccine

technologies I would like to thank all the scientists and staff at Lovelace Respiratory

Research Institute specifically Dr Phil Kuehl Dr Melanie Doyle Maurice Newton and

Aimee Kowell I also want to thank Dr Kevin Lind for his friendship and mentorship I

deeply thank my husband Joel Martin for his support and sacrifice Above all I thank God

for life for giving and taking away and for the fruits of the Spirit which are love joy

peace patience goodness gentleness faithfulness and self-control

This work was supported in part by the National Institutes of Health (NIH) under

the Grant No 1R21HL092812-01A1 ldquoMultifunctional Nanoparticles Nano-Knives and

Nano-Pullies for Enhanced Drug Delivery to the Lungrdquo Leisha Armijo was supported in

part by the NIH under the Grant No GM-060201 Initiatives to Maximize Student

Diversity (IMSD) the NSF IGERT program on ldquoIntegrating Nanotechnology with Cell

Biology and Neurosciencerdquo Grant No DGE-0549500 and by the More Graduate

Education Mountain States Alliance (MGEMSA) program through Arizona State

University This work was performed in part at CINTSNL under Project No U2010B1079

ldquoCharacterization of Multifunctional Nanoparticles for Enhanced Drug Delivery to the

Lungrdquo funded by DoE contract No DE-AC04-94AL85000

vii

IRON-CONTAINING NANOPARTICLES FOR THE

TREATMENT OF CHRONIC BIOFILM INFECTIONS

IN CYSTIC FIBROSIS

by

Leisha Marie Armijo

BS Biology

MS Nanoscience amp Microsystems

Doctor of Philosophy Nanoscience amp Microsystems Engineering

ABSTRACT

Cystic fibrosis (CF) is the most common genetic disease resulting in the morbidity and

mortality of Caucasian children and adults worldwide Due to a genetic mutation resulting

in malfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

protein CF patients produce highly viscous mucus in their respiratory tract This leads to

impairment of the mucociliary clearance of inhaled microbes In addition to reduced

microbial clearance anoxic environmental conditions in the lungs promote biofilm-mode

growth of the pathogenic bacterial species Pseudomonas aeruginosa Chronic infections of

P aeruginosa begin in early childhood and typically persist until respiratory failure and

viii

death result The average life-expectancy of CF patients is only about 40 years with

extensive treatment

Although the introduction of inhaled antibiotics has increased the life expectancy of

CF patients the thick mucus and biofilm formation contribute to the failure of inhaled

antibiotic drugs In order to address these issues we have synthesized and characterized

nanoparticles and nanoparticle-drug conjugates for magnetic gradient guided drug delivery

alone or in combination with medical magnetic hyperthermia to increase local temperature

and decrease the viscosity of these layers In the absence of the medical magnetic

hyperthermia application under static magnetic field the NP drug conjugates may be

gradient guided through the mucus and biofilm barriers to treat the P aeruginosa infection

directly We synthesized and characterized iron oxide (magnetite) and iron nitride

(martensite) nanoparticles as candidate nanomaterials for this application We synthesized

these materials using environmentally friendly green chemistry methods in multiple

nanoscale size ranges The NPs were synthesized using solvothermal methods and

characterized by transmission electron microscopy (TEM) energy dispersive x-ray

spectroscopy (EDS) x-ray diffraction (XRD) and direct current (DC) and alternating

current (AC) magnetometry These nanocomposites demonstrate observable bacterial

growth and biofilm inhibition even at surprisingly low (10 ngmL) concentrations making

them ideal candidates for incorporation into a low-cost treatment regime In vitro

cytotoxicity testing of the iron oxide nanoparticles shows low dosage dependent

cytotoxicity in human lung adenocarcinoma cells making the iron oxide nanoparticles an

ideal candidate material for this application

ix

Table of Contents

Dedicationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipiii

Acknowledgementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip iv

Abstracthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipvii

Table of Contentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipix

List of Figureshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxv

List of Tableshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxix

List of Abbreviationshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxx

Chapter 1 Introduction to Cystic Fibrosis Current Treatment Options and

Proposed Novel Treatment Method helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip1

11 The Epidemiology of Cystic Fibrosishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip2

12 Inheritance of Cystic Fibrosis Disease Heterozygote Advantage and

Persistence in the Populationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip6

13 The CFTR Gene and Different Mutation Typeshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip8

14 Current Therapeutic Regimeshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip13

141 CFTR Mutation Types and Personalized Medicinehelliphelliphelliphelliphelliphellip14

142 Complications in Gene Therapyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18

143 Summary of Treatment Failurehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21

15 The Rode of Pseudomonas aeruginosa in the Morbidity and Mortality of

Cystic Fibrosis Patientshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21

x

16 Antibiotic Drug Resistance and Biofilmshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip24

17 Proposed Universal Treatment Method Using Superparamagnetic

Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip27

171 Particle Transporthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip29

172 Biocompatibilityhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32

173 Biofilm Considerationshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34

174 Critical Parametershelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35

18 Overview of Dissertationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36

Chapter 2 Synthesis and Characterization of Iron Oxide Nanoparticles38

21 Synthesis of Colloidal Magnetite Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip40

211 Materialshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip40

212 Synthesis of iron oleate precursor complexhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip41

213 Synthesis of cube-shaped and polymorphous nanoparticleshelliphelliphellip42

214 Synthesis of nanowireshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip43

215 Synthesis of spherical nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip44

216 Summary of green chemistry modificationshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip46

217 Cost reductionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip46

22 Structural Characterizationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip47

23 Summary of Findingshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53

Chapter 3 Magnetic Characterization of Iron Oxide Nanoparticles and

Magnetic Hyperthermia Experimentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip55

31 Theoryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip57

xi

32 Experimentalhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip59

321 SQUID Magnetic Characterization of Iron Oxide Nanoparticleshellip59

322 Magnetic Hyperthermia Experimentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64

323 AC Susceptometryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip70

Chapter 4 Synthesis and Characterization of Iron Nitride (Fe16N2)

Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip73

41 Introduction to Fe16N2helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip74

43 Synthesis of Iron Nitride (Fe16N2) and Zero-Valent Iron (Fe0)

Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76

431 Materialshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77

432 Synthesis of Iron Oleate Precursor Complexhelliphelliphelliphelliphelliphelliphelliphellip77

433 Synthesis of Iron Oxide Precursorhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78

434 Removal of Oleic Acid Caphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip79

435 Production of Zero-valent Iron Nanoparticles helliphelliphelliphelliphelliphelliphellip79

436 Production of Iron Nitride Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip80

44 Structural Characterization of Iron Nitride Nanoparticleshelliphelliphelliphelliphellip80

45 Magnetic Characterization of Iron Nitride Nanoparticleshelliphelliphelliphelliphelliphellip82

46 Summary of Findingshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip85

Chapter 5 Hydrophilization and Bioconjugationhelliphelliphelliphelliphelliphelliphelliphellip87

51 Experimentalhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip88

xii

512 Removal of Oleic Acid Caphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip89

513 Citrate cappinghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip93

514 Alginate Cappinghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip93

515 Polyethylene Glycol (PEG) Succinylationhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip94

516 Polyethylene Glycol (PEG) Capping of Iron Oxide

517 Conjugation to Tobramycinhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip98

52 Characterization of Functionalized Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip100

521 Size Determination helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip100

522 Zeta Potential Measurementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip102

523 Fourier Transform Infrared (FTIR) Spectroscopyhelliphelliphelliphelliphelliphelliphellip103

53 Summary of Findingshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip103

Chapter 6 Determination of Minimum Inhibitory Treatment Concentrations

and Bacterial Sensitivitieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip105

61 Microbiological Methodshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip109

612 Minimum Inhibitory Concentration of Tobramycin

Determinationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip109

613 Establishment of Biofilm Communitieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip110

614 Motility Testinghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip112

615 Disk Diffusion Method helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip112

xiii

616 Biofilm and Mucus Model and Static Magnetic Field

Applicationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip115

617 Determination of Minimum Inhibitory Concentration (MIC) of Test

Articleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip116

618 Graphical and Statistical Analysishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip119

62 Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip119

621 Determination of Minimum Inhibitory Concentration (MIC) of

Tobramycinhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip119

622 Interpretation of Disk Diffusion Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip121

623 Disk Diffusion Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip124

624 Biofilm and Mucus Model and Static Magnetic Field Application

Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip129

625 Motility Testing Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip132

626 Comparison of Inhibition in Liquid Cultureshelliphelliphelliphelliphelliphelliphelliphelliphellip132

Chapter 7 Cytotoxicity of Iron Oxide Nanoparticleshelliphelliphelliphelliphelliphelliphellip138

71 Experimental Procedurehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip140

711 Materials and Reagentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip141

712 Dynamic Light Scattering (DLS)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip141

713 UV-vis-NIR Spectroscopyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip142

714 Human Lung Adenoarcinoma Cell Growthhelliphelliphelliphelliphelliphelliphelliphelliphellip142

715 Cytotoxicity Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip143

716 Viability Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip144

xiv

717 Apoptosis Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip145

718 Statistical Analysis Correction Factor and Mathematical

Methodshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip147

721 Dynamic Light Scattering (DLS) Size Distributionhelliphelliphelliphelliphellip149

722 UV-vis-NIR Spectroscopy Absorbance Measurementshelliphelliphelliphelliphellip150

723 Cytot oxicity Assay Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip152

724 Viability Assay Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip153

725 Apoptosis Assay Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip155

73 Discussionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip158

Chapter 8 Conclusions and Future Workhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip163

81 Importance of Green Methodologyhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip164

82 Bacterial Sensitivity Discussionhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip165

83 Conclusionshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip169

84 Future Workhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip170

Referenceshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip173

Publications (JournalsConferencesPatents) helliphelliphelliphelliphelliphelliphelliphelliphelliphellip214

APPENDIX I List of Chemicals Physical Properties and

Classificationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip219

APPENDIX II Protocol for Drug Conjugationhelliphelliphelliphelliphelliphelliphelliphelliphellip229

APPENDIX III MagneThermtrade Inductive Heater Tunable Frequencies

Magnetic Field Capabilities and Derivation of Working Equation helliphellip231

xv

List of Figures

11 Statistical transmission of the CFTR gene from parents to offspringhellip7

12 Chromosomal location of CFTR genehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9

13 Normal chloride ion channel function at the cell membranehelliphelliphelliphellip12

14 Barriers to drug diffusion surrounding biofilms of Pseudomonas

aeruginosa in the lungs of CF patientshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip24

15 Alginate moleculehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip26

21 Ion-exchange reaction between iron(iii) chloride and sodium oleatehellip41

22 Morphology alterations of iron oxide nanoparticles via additional

nucleation event(s)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip43

23 Active nanoparticle synthesis in the Schlenk linehelliphelliphelliphelliphelliphelliphelliphellip44

24 Summary of morphology controlhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip45

25 TEM images of 250 nm cubic and 15 nm spherical NPshelliphelliphelliphelliphelliphellip47

26 TEM images of magnetite nanoparticles capped with oleic acid showing

different morphologieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip47

27 TEM image of spherical magnetite nanoparticles capped with oleic

acidhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip49

28 HRTEM image and FFT of iron oxide monodisperse sphereshelliphelliphellip50

29 EDS spectrum of monodisperse spherical nanoparticleshelliphelliphelliphelliphellip51

210 X-ray diffraction (XRD) spectrum of 17 nm spherical

nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip52

xvi

211 XRD spectrum of iron oxide spherical nanoparticles using

monochromatorhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53

31 Mechanisms of energy loss leading to heat production in magnetic

hyperthermiahelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip58

32 Magnetization vs temperature for polymorphous Fe3O4

nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip60

33 Ferromagnetic hysteresis loophelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip61

34 Superparamagnetic hysteresis loophelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62

35 MagneThermtrade inductive heater setup in its entiretyhelliphelliphelliphelliphelliphelliphellip63

36 Inside of MagneThermtrade inductive heaterhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63

37 Magnetic Hyperthermia Results for NPrsquos in the ferroferrimagnetic size

range at two frequencies and field

strengthshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip65

38 Hyperthermia results for superparamagnetic NPs in water and glycerol

mixturehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip69

41 HRTEM image of Fe16N2 nanoparticles showing excellent

crystallinityhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip81

42 XRD spectrum for iron nitride nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip82

43 Magnetization vs temperature for Fe16N2 nanoparticleshelliphelliphelliphelliphellip83

44 Comparison of hysteresis loops of nanocrystalline samples of iron oxide

and iron nitride of similar grain sizehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip84

45 Close-up of hysteresis curvehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip85

51 Removal of oleate caphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip92

xvii

52 Citrate moleculehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip92

53 Monomer of alginic acidhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip94

54 Dialysis of succinylated PEG 5000helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip95

55 PEG succinylation overall reaction helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip95

56 Dried and purified succinylated PEG 5000helliphelliphelliphelliphelliphelliphelliphelliphelliphellip96

57 Absorbance spectra for succinylated PEGhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip97

58 Tobramycin Moleculehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip99

59 EDCSulfo-NHS crosslinking reaction schemehelliphelliphelliphelliphelliphelliphelliphelliphellip100

510 DLS size distribution histogramhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip101

61 Pyocyaninhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip108

62 Agar plates inoculated with P aeruginosa colonies taken from biofilm

cultures showing impregnated diskshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip114

63 Pole orientation for ring magnetshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip116

64 Illustration of serial dilution procedurehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip117

65 Schematic diagram of MIC determinationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip118

66 MIC of tobramycin over timehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip120

67 Agar cultures for susceptibility testinghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip122

68 Results of motility testhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip132

69 Optical density for liquid cultureshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip133

610 Percent bacterial inhibition vs treatment concentrationhelliphelliphelliphelliphellip134

xviii

71 Reduction of fluorescence signal in magnetite NPshelliphelliphelliphelliphelliphelliphellip148

73 Absorbance spectrum for magnetite NPshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip151

74 Absorbance spectrum for succinylated PEGhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip151

75 Cytotoxicityhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip152

76 Cell viability over timehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip154

77 Apoptosis luminescencehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip156

78 Apoptosis time curve helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip157

81 Mechanisms of cell damage and response after exposure to iron-

containing nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip168

xix

List of Tables

11 Birth prevalence of cystic fibrosis worldwidehelliphelliphelliphelliphelliphelliphelliphelliphelliphellip3

12 Classes of cystic fibrosis transmembrane receptor (CFTR) mutations17

61 Guidelines for understanding susceptibility results using disk diffusion

methodhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip123

62 Comparison of 3-day old biofilm sensitivities to magnetic nanoparticles

(MNPs) capped with polyethylene glycol (PEG) tobramycin

ciprofloxacin and nanoparticle-drug conjugateshelliphelliphelliphelliphelliphelliphelliphellip124

63 Susceptibility of Pseudomonas aeruginosa biofilms to various treatments

after 3 and 60-days of growthhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip126

64 Results of cystic fibrosis (CF) biofilm model with applied magnetic

fieldhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip130

65 Results of cystic fibrosis (CF) biofilm model no magnetic field

appliedhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip130

66 Summary of biofilm modelhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip131

III1 Tunability specifications for magnetherm inductive heaterhelliphelliphelliphellip234

xx

List of Abbreviations

ABC adenosine triphosphate binding cassette

AI auto-inducer

ATP adenosine triphosphate

cAMP cyclic adenosine monophosphate

CDC Centers for Disease Control and Prevention

CF cystic fibrosis

CFTR cystic fibrosis transmembrane receptor

CLSI clinical and laboratory standards institute

DI deionized

DNA deoxyribonucleic acid

EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

EDC EDAC 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide

hydrochloride

EDS energy-dispersive x-ray spectroscopy

ENaC epithelial sodium channel

EPA Environmental Protection Agency

EPS extracellular polysaccharides

I intermediate

ICU intensive care unit

LB Luria-Bertani

LPS lipopolysaccharide

MIC minimum inhibitory concentration

MNP magnetic nanoparticle

m-PEG methyl-terminated polyethylene glycol

xxi

mRNA messenger ribonucleic acid

NABF nucleotide-binding

NABF+R nucleotide-binding and regulatory domain

NBD nucleotide binding domain

NBF nucleotide binding factor

NOAEL no observed adverse effects level

NP nanoparticle

OD optical density

PEG polyethylene glycol

PEG-OH hydroxyl-terminated polyethylene glycol

QS quorum sensing

R resistant

r radius

RNA ribonucleic acid

ROS reactive oxygen species

rpm revolutions per minute

rRNA ribosomal ribonucleic acid

S sensitive

SNP single nucleotide polymorphism

SPION superparamagnetic iron oxide nanoparticle

Sulfo-NHS N-hydroxyl sulfosuccinimide

TEM transmission electron microscope

TSCA toxic substance control act

UV-VIS-NIR ultraviolet-visible-near infrared

Vp-p peak-to-peak voltage

xxii

XRD x-ray diffraction

1

Chapter 1

INTRODUCTION TO CYSTIC FIBROSIS

CURRENT TREATMENT OPTIONS AND PROPOSED

NOVEL TREATMENT METHOD

The most common genetic disease resulting in the morbidity and mortality of Caucasian

children and adults worldwide is cystic fibrosis (CF) [Wood 1976] [Hodson 2007]

[Feuchtbaum 2012] CF results from a mutation on the gene that codes for a specific ion

channel in the epithelial cells The faulty ion channel alters normal function in multiple

organ systems most significantly affecting the respiratory system Thick viscous mucus

secreted by the goblet epithelial cells coat the cilia in the upper respiratory tract reducing

mechanical (ciliary) clearance of inhaled microbes (viruses bacteria and allergens) thus

resulting in a chronic inflammation condition The chronic inflammation state in CF is

believed to be caused in part by autophagy frustration due to reactive oxygen species

(ROS)-mediated sequestration of the beclin 1ndashclass III PI(3)K complex in perinuclear

aggregates which redirect it from the autophagy active site at the endoplasmic reticulum

(ER) [Luciani 2010] [Leivine 2011] Death occurs when pathogenic bacteria and viruses

set up residence in the respiratory mucus eventually leading to respiratory failure and

death

2

The first recorded case of CF was in the 1930rsquos [Davis 2006] at which time

abnormal mucus plugging of the exocrine ducts was believed to be the major cause of the

pathology [Davis 2006] Autopsies performed on malnourished babies showed abnormal

mucus plugging of the glandular ducts which is why the disease was initially called

ldquocystic fibrosis of the pancreasrdquo [Davis 2006] During that time the life expectancy for a

CF patient was only 6 months [Davis 2006] Despite this being the first recorded case a

genetic disorder as prevalent and widespread as CF has most likely existed in the human

gene pool for a considerable amount of time before being identified and classified

Evidence supporting this theory comes from documented European folklore [Busch 1989]

In 1838 it was held that a parent should lick across a childrsquos forehead a child who tasted

salty was believed to be ldquobewitchedrdquo and was feared to soon die [Busch 1989] Another

paper published elsewhere theorizes that CF is much older than that [Mateu 2002] The

age of the most common mutation resulting in CF (ΔF508) is debatable with estimates

ranging from more than 40000 years ago (upper Paleolithic era and pre-Neolithic era)

[Morral 1994] to 3000 years ago (post-Neolithic era) [Serre 1990] Significant evidence

exists that the disease we would one day be officially named ldquocystic fibrosisrdquo may have

existed for thousands of years prior to recorded history

11 The Epidemiology of Cystic Fibrosis

The description of the distribution and determinants of any disease frequency in the human

population is referred to as the disease epidemiology The incidence of the disease is

defined as the number of new cases per 100000 people The birth prevalence is defined as

3

the number of people born with CF per 10000 live births Population prevalence is defined

as the number of people with CF per 100000 It is important to note that an accurate

calculation of incidence and prevalence of CF depends strongly on the existence of a

complete and accurate registry [Hodson 2007] Population prevalence depends on both

birth prevalence and survival therefore birth prevalence will give the best estimate of the

incidence of CF in a population since survival depends on access to adequate medical care

Table 11

Birth Incidence of Cystic Fibrosis Worldwide Incident case per number of live births adapted from [Hodson 2007] [Farrell 2008] According to this table the lowest incidence in the world by country is Japan with only one

incident case per 355000 live births [Fredericksen 1996] The highest incidence in the world by

country is The Republic of Ireland with once case per 1353 live births [Farrell 2007] The

highest birth prevalence is reported for relatively isolated populations such as the Zuni tribe of

New Mexico and the Amish in Ohio [Wood 1976] [Stutman 2002]

Region CF Incidence Europe

Austria

13500 [Southern 2007]

Belgium

12850 [Lucotte 1995] [Chung 2002]

Bulgaria

12500 [Chung 2002]

Czech Republic

12833 [Romeo 1989] [Lucoette 1995]

Denmark 14760 [Batten 1965]

14700 [Lucoette 1995] [Klaassen 1998] [Nielsen 2002]

Faroe Islands

11775 [Kaplan 1968]

Finland

125000 [Denning 1968]

125000 [Kere 1994] [Klaassen 1998]

France

12667 West Brittany [Lev 1965] 12838 Brittany

11972 (including terminated pregnancies) [Danes 1968]

14700 [Southern 2007]

Germany

13300 [Romeo 1989] [Lucoette 1995] [Schulz 2006]

4

Italy

14238 [Siegel 1960]

14238 [Bossi 2004]

Italy (Milan)

13170 [Chernick 1959]

Netherlands

14750 [Spock 1967]

14750 [Slieker 2005]

Northern Ireland (UK)

11857 [Noblett 1969]

Norway

16574 [Johnson 1984]

Poland

15000 [Southern 2007]

Republic of Ireland

11353 [Farrell 2007]

Romania

12056 [Popa 1997]

Scotland

11984 [Hide 1969]

Slovakia

11800 [Kadasi 1997]

Spain

13750 [Lucotte 1995] [Chung 2002]

Sweden

12200-4500 [Rosan 1962]

15600 [Lannefors 2002]

United Kingdom

12415 [Gracey 1969]

12381 [Dodge 2007]

North America

United States

13200 Caucasian 110500 Native American

111500 Hispanic 114000-17000 African American

[Bowman 1969]

12380-2630 Caucasian 16800-27000 Hispanic

113300-114800 African American 11790-2880

Ashkenazi Jewish 113700-128000 Asian American

[Palomaki 2004]

Canada

12500 [Mearns 1974] 13608 [Dupuis 2005]

Saguenay-Lac St Jean

(Quebec)

1895 [Weaver 1994]

Amish OH USA

1569 [Stutman 2002]

5

Zuni Tribe NM USA

1333 Native American [Wood 1976]

Middle East

Ashkenazi Jews and Arabs

14000-18000 [Crozier 1974]

Bahrain

15800 [Corey 1988]

Jordan

12560 [Nielsen 1982]

Oceana

New Zealand

13179 (non-Maori) [Szaff 1983]

Australia

12021 (British) 13625 (Italian)13726 (Greek)

[Jensen 1987]

Other

Japan

1355000 [Frederiksen 1996]

South Africa

12000 (Caucasian) [Allan 1973] 1784-13924

(African) [Chase 1979]

Although the birth prevalence is higher in Caucasians than other ethnic groups

estimated to be roughly 1 in every 2500 people [Hodson 2007] statistics studies done in

2011 identified a considerable number of CF patients with mixed African-Caucasian

Mexican-Caucasian and Indian-Caucasian ancestries [Li 2011] The CFTR mutation has

been found in South Africans of pure African decent and did not arise from mixing with

European populations [Maek 1997] Another recent study compared CF prevalence in

newborns of various races and revealed somewhat surprisingly the highest prevalence in

Native Americans [Wood 1976] [Feuchtbaum 2012] In some populations the birth

prevalence is much higher than expected such as Ohio (Amish) and Saguernay-Lac St

Jean Qubec [Hodson 2007] This is attributed to the founder effect these populations are

6

culturally or geographically isolated Whereas in other populations the birth prevalence is

much lower than expected such as Norway and Finland [Hodson] Developing countries

such as Africa or India do not maintain sufficient records on CF incidence as detection of

CF is a low priority compared to other substantial health problems leading to infant

mortality Per the cystic fibrosis foundation there are approximately 30000 people in the

United States living with CF and 1000 new cases are diagnosed annually [Cystic Fibrosis

Foundation 2015]

A more recent publication ldquoGeographical distribution of cystic fibrosis the past 70

years of data analysisrdquo [Mirtajani 2017] also cites the same references for incidence due

to a lack of updated data in peer-reviewed scientific literature Mirtajani also notes that

African Asian and the South American continents have limited or no CF registry and

estimates that more than 50 of countries provide no data on CF incidence at present

[Mirtajani 2017] We have provided some relatively older and newer reported values for

CF incidence and some researchers have noted a slight decrease in CF cases [Massie

2010] these decreases are attributed to screening followed by pregnancy termination and

do not represent an actual decrease in the incidence of the CF mutation or mutations in the

population The rate of CF in the population has relatively remained consistent over time

as far as we can tell by considering and comparing the available published data

12 Inheritance of Cystic Fibrosis Disease Heterozygote Advantage and Persistence

in the Population

7

The gene that codes for the faulty receptor responsible for the pathology associated with

CF disease is the cystic fibrosis transmembrane receptor (CFTR) gene The CFTR gene is

recessive therefore in order to have clinically diagnosable CF disease a person must

inherit two defective copies of the CFTR gene one from the mother and one from the

father A carrier is a person who has inherited one defective gene and one normal gene and

would typically present as healthy although there are some exceptions depending on the

specific mutation type Carrier individuals also called heterozygotes exist in a much

higher frequency in a population as they only possess a single defective gene

Heterozygotes differ in phenotype from homozygotes in that they may be short in stature

[Aitken 2003] and they may exhibit increased upper respiratory inflammation than non-

carriers [Kilbourn 1968] Despite these documented features heterozygotes exhibit normal

life expectancies In order to be clinically diagnosed with CF disease a person must

possess two defective genes one from each parent (see Figure 11)

Figure 11 Statistical transmission of the

CFTR gene from parents to offspring Green

represents normal gene red represents the

defective gene Top left shows a carrier

father top right shows a carrier mother

Below are the statistically predicted

offspring each having a 25 chance of

inheriting CF Genotype is the gene

combination present for example

heterozygous homozygous recessive or

homozygous dominant and corresponding

phenotype or expression of the defective

gene of heterozygote parents and four

statistically predicted offspring Image by L

Armijo 2016

8

The life expectancy of a CF patient with extensive treatment at the time of this

publication is less than 40 years [Anderson 2016] Prior to the discovery of antimicrobial

drugs the life expectancy was much lower Recall the life expectancy of children

diagnosed with CF in the 1930rsquos was only six-months [Davis 2006] Accordingly it would

stand to reason that if CF sufferers died in childhood before reaching reproductive age

that the disease should have been completely eradicated by natural selection This

however has not been the case Before the late 1960rsquos scientists investigated possible

benefits of the defective gene which may have allowed it to persist in the population

Finally 1967 studies confirmed that the mean number of live offspring of the grandparents

of CF patients was higher than for the grandparents of the healthy control group [Knudson

1967] These findings suggest a heterozygote advantage A so-called heterozygote

advantage occurs when a carrier individual demonstrates a selective advantage over the

rest of the population Findings confirmed that heterozygote carriers are resistant to

cholera toxin [Gabriel 1994] Just four years after that another paper reported that the

single defective CF gene imparts resistance to typhoid fever [Pier 1998] Considering the

long history and persistence of CF from the consequences of these long-term selective

advantages CF is significantly likely to continue to persist in the population long into the

future

13 The CFTR Gene and Different Mutation Types

The CFTR gene encodes the instructions for the cell to manufacture the CFTR protein The

CFTR protein is a cyclic adenosine monophosphate (cAMP) regulated chloride ion

9

channel found virtually exclusively in the secretory epithelial cells [Frizzel 2012] The

CFTR protein is encoded by the DNA and transcribed into messenger RNA (mRNA) The

mRNA is translated and the CFTR protein is manufactured in the rough endoplasmic

reticulum of the cell The assembled CFTR migrates to the cell membrane where it exerts

its function A mutation in the DNA coding for the CFTR protein could lead to a premature

stop codon in which case the message would never be translated and a CFTR protein

would never be produced

Figure 12 Chromosomal location of the CFTR gene locus on the q arm of

chromosome 7 in region 3 band 1 and sub band 2 hence the location

designation 7q31_2 Image after [NIH 2016]

10

Alternatively a different mutation could change the code such that a different

protein is produced resulting in either a faulty or a non-functioning CFTR Even though the

defect is found on a single gene there are many different mutations on that gene that can

cause CF disease All the mutations documented correspond to the same location on

chromosome 7 the difference in the type of mutation is characteristic of the code that was

inserted in this region Evidence of a tight linkage between the CF locus and a DNA

sequence polymorphism in the center third of the long arm of chromosome 7 between

bands q21 and q31 was found in 1985 drawing more attention to this region [White 1985]

Others identified the CF locus more specifically on human chromosome 7ce-q22 in that

same year [Wainwright 1985]

It was not until much later when it was shown that several different mutations

could result in a faulty or missing CFTR protein (see Table 12) [Peebles 2005] This is

significant because variations in the type of mutation the presence of some type of

defective CFTR or the absence of a CFTR protein complicate current standard treatment

regimes Treatment is most effective when catered to the patientrsquos specific mutation type

For practicality specific mutations are given a class number corresponding to a recognized

treatment regime (see Section 14) Since different mutations result in different pathologies

and severities thereof optimization of treatment requires a personalized approach A

universal treatment method is needed

In all cases the major underlying issue is either a malfunctioning or non-

functioning chloride ion channel at the epithelial cell membrane resulting in a decreased

volume of periciliary fluid in the lower respiratory tract This in turn leads to impaired

11

mucociliary clearance of inhaled microbes which colonize and ravage the lungs causing

child-onset chronic infections chronic inflammation tissue damage and eventual

respiratory failure and death Therefore in order to increase the life-expectancy of CF

patients we must uncover a reliable method to annihilate the bacterial species that

overwhelms the already compromised respiratory system in these patients

The CFTR protein is an ATP-binding cassette (ABC) transporter-class ion channel

(Figure 13) ABC transporters are classified as proteins based on the sequence and

organization of their domain or domains For example the CFTR has Nucleotide Binding

and Regulatory Domains 1 and 2 (NBD1 and NBD2 +R respectively) areas where

nucleotides bind to regulate function In general the CFTR is simply a protein that

conducts chloride (Cl-) [Riordan 2008] and thiocyanate (SCN-) [Childers 2007] anions

across epithelial cell membranes A normally functioning CFTR protein acts as an ion

pump channeling chloride ions (Cl-) from inside the cell across the cell membrane and

into the extracellular space in order to maintain healthy salinity levels within the cell In

addition the CFTR protein can inhibit the epithelial sodium channel (ENaC) when

activation is triggered by nucleotide binding to NBF1 The ENaC is a separate channel

through which sodium ions (Na+) are transported A healthy CFTR protein influences a

low-level sodium intake by regulation of NBF1 [Annereau 2003] When a defective CFTR

protein or no CFTR protein is produced the Cl- concentration within the cells is

compromised ENaC is activated and a subsequent increase in sodium transport into the

cell results

12

Figure 13 Normal chloride ion channel function at the cell membrane CFTR is the

cystic fibrosis transmembrane receptor shown in active transport of chloride ions

through its channel NBD1 is the first nucleotide-binding domain and NBD2 is the

second nucleotide-binding domain where nucleotides can bind to regulate function

Image by Armijo L 2014

Mutations of the CFTR gene altering chloride ion channel function cause

dysregulation of epithelial fluid transport in the lung pancreas and other organ systems

Clinical pathologies include thick condensed mucus in the lungs and recurrent respiratory

infections causing chronic disability and reduced life expectancy CF patients also suffer

from pancreatic insufficiency which results in malnutrition and diabetes Abnormal ion

13

regulation also causes the salty epithelial excretions which promote bacterial colonization

of the lungs

14 Current Therapeutic Regimens Personalized Medicine and Investigational

Treatments

The discovery of antibiotics in 1928 was undoubtedly one of the most important

developments in medicine to date responsible for saving millions of lives by making

formerly deadly infections curable [Tan 2015] Antibiotic reliability is the foundation for

modern medicine and has facilitated the development of numerous formerly impossible

medical procedures Virtually every aspect of what we call modern medicine treatment of

autoimmune diseases and allergies therapeutic use of corticosteroids or other

immunosuppressant drugs chemo- and radiation therapy any and all surgical procedures

burn and wound treatment to include any procedures or accommodations in which stents

catheters orthodontic wires ventilators staples sutures bandages clamps belts implants

or virtually any procedure in which an inert object-biological interface exists they all put

the patient at risk for infection The development of antibiotic drugs made all this possible

On the other hand researchers and medical professionals alike continue to struggle with

the intensifying issue of antibiotic resistance especially prominent in healthcare

environments which threatens to collapse the crucial foundation on which modern

medicine was built

Since death from respiratory failure is preceded by bacterial colonization of the lungs

of CF patients most treatment regimens include the use of aminoglycoside antibiotics

14

[Peebles 2005] [De Boeck and Amaral 2016] The most common pathogenic bacterial

species having the ability to form biofilm colonies and causing respiratory failure in CF

is Pseudomonas aeruginosa [Govan 1996] The next most important adversary in the war

on morbidity and mortality in CF is Burkholderia cepacia Burkholderia cepacia is

believed by some to be the most significant and provocative new opportunistic pathogen to

torment the CF community [Govan 1996] Other microbiological organisms contributing to

pulmonary disease in CF lungs by predisposing the patient to Pseudomonas aeruginosa

colonization are often referred to as nonpseudomonal CF pathogens The following

nonpseudomonal pathogens are known Staphylococcus aureus and Haemophilus influenza

(common) Streptococcus pneumoniae Legionella species viruses (in particular

respiratory syncytial virus (RSV) various glucose nonfermenters mycobacteria fungal

agents (less-common) [Gilligan 1991 Govan 1996] Because of the infectious disease

aspect antibiotic therapy is a common component of the current CF treatment regime

141 CFTR Mutation Types and Personalized Medicine

CF is a genetic disease that can manifest differently depending on the type of

mutation of CFTR gene Therefore treatments are optimized when they are catered to a

specific CFTR gene mutation In the interest of treatment optimization the CFTR

mutations resulting in CF disease have been traditionally been separated into V classes

[Peebles 2005] A new class class VI was later described and is distinguished by rapid

CFTR turnover at the channel surface [Zielenski 2000] Even more recently a new

classification based on therapeutic strategies and accounting for the potential of

15

personalized medicine and targeted drugs was proposed [De Boeck and Amaral 2016] In

this model De Boeck and Amaral separated the class I mutations into class I (stop-codon)

and a new class class IV (no mRNA transcription) due to the differing successful

treatment options for the two [De Boeck and Amaral 2016]

One example of mutation type is caused by a single nucleotide polymorphism

(SNP) A SNP occurs when a single base (nucleotide) along the DNA ladder is replaced by

a different one Another mutation type called a nonsense mutation converts a codon (a

triplet of bases that codes for an amino acid) into a stop codon (a triplet of bases encoding

the termination of translation) A nonsense mutation is responsible for the pathology

described in a CF class I mutation For example an adenosine molecule replaces a cysteine

molecule resulting in synthesis of a faulty protein or no protein synthesis at all A

missense mutation as in CF mutation classes II III IV or V occurs when a SNP results in

the substitution of a different amino acid in the amino acid chain It should be noted that

overlaps between different classes of mutations can also exist For example the delta-F508

(ΔF508) mutation can cause reduced chloride channel opening time in addition to

abnormal CFTR processing Occasionally the CFTR mutation can be modified by another

mutation or polymorphism on the same allele (a modifier gene)

The most common therapeutic regime for class I mutations includes

aminoglycoside antibiotics Aminoglycosides are antibiotics traditionally used for the

treatment of gram-negative bacterial infections (such as P aeruginosa infection) They are

named as such because they contain as a portion of the molecule an amino-

modified glycoside an aminoglycoside This family of antibiotics consists of tobramycin

16

streptomycin gentamycin and the neomycins The drug tobramycin which we have

chosen for our investigations annihilates bacterial cells in a synergistic manner Initially it

electrostatically binds the negatively charged lipopolysaccharide bacterial membrane

compromising membrane integrity and thus resulting in its degradation [Shakil 2008]

Once internalized acting from the inside of the bacterial cell tobramycin inhibits

ribosomal translocation thus interfering with protein synthesis [Saiman 2004] This

treatment is used for the chronic bacterial infections of respiratory tract characteristic of

CF

For a class II mutation a faulty CFTR is produced in the endoplasmic reticulum

where it remains Butyrates are a popular treatment for class II mutations Butyrate is the

generic name for the conjugate base of hydrocarbons containing butanoic acid (C4H7O2minus)

somewhere in their structure These agents cause a reduction in CFTR current amplitude

suggesting a kinetically fast blocking mechanism [Linsdel 2001] thus artificially

regulating that component of the CFTR

In a class III mutation a faulty CFTR causes inappropriate activation and

regulation of ion transport Despite expression of the full-length protein at the apical

plasma membrane class III mutations change CFTR gating which results in decreased Clminus

transport [Kreindler 2010] Genistein supplementation has been recommended for class III

mutations Genistein has been demonstrated to alter the maturation cell surface expression

and single-channel function of CFTR protein [Schmidt 2008] Genistein is a phytoestrogen

(plant-derived xenoestrogen) belonging to the category of isoflavones Although it has

17

many uses in hormone modulation in this case it is exploited for its ability to modulate the

CFTR channel potentiating its opening at low concentration and inhibiting at higher doses

Table 12

Classes of CFTR Mutations This table summarizes the recognized classes of mutations that cause cystic fibrosis disease There

are IV mutation classes each resulting in a specific alteration to or absence of the CFTR protein

Due to the unique resulting manifestations each mutation class has a specific treatment regime

Proposed class [De Boeck and Amaral 2016] Approved therapy Adapted from [Peebles 2005]

and [De Boeck and Amaral 2016]

In class IV mutations a faulty CFTR reduces chloride conductance and transport is

altered Milrinone is used for the treatment of class IV mutations Milrinone marketed

Class

Effect on CFTR

Types of Mutation

Therapy Potential

Therapy

I Defective synthesis of

message (messenger RNA)

causing absence of CFTR

Premature stop codon

(nonsense or frame

shift)

Aminoglycosides

Gene transfer read-

through compounds

II Abnormal CFTR produced

which fails to leave

endoplasmic reticulum

Amino acid deletion

(∆ F508 or missense

mutation)

Correctors

Butyrates

Gene transfer

III Abnormal CFTR causing

disruption of activation and

regulation at cell membrane

impaired gating

Missense mutation

(ie G551D)

Potentiators

Genistein

Gene transfer

IV Abnormal CFTR reducing

chloride conductance

Missense mutation

(ie R117H or

R347P)

Milrinone

Potentiators

Gene transfer

V Reduced or absent synthesis

of CFTR due to decreased

splicing of normal CFTR

Missense mutation or

splice site mutation

(ie A445E or 5T)

Aminoglycosides

Antisense

oligonucleotides

Correctors Gene

transfer

VI Absence of CFTR No mRNA

transcription

Aminoglycosides

Stabilizers

Gene transfer

VII Absence of CFTR No mRNA Aminoglycosides

Bypass therapies

18

under the brand name Primacorreg Milrinone is an inhibitor of phosphodiesterase 3 a

vasodilator Although class V mutations can lead to the production of normal CFTR the

same mutation can also result in a reduced or absent CFTR A limitation of transcriptional

regulation results in a reduced quantity of the protein being produced As with the other

mutations resulting in an absent CFTR (I VI and VII) the only approved treatment is

aminoglycoside antibiotic and supportive therapy

Many of the identified CFTR gene mutations can be placed into one of the six

classes thus accounting for approximately 80 of all CF patients [Rogan 2011] However

of the gt1900 CFTR mutations that have been identified there are only roughly 20

mutations have a frequency greater than 01 [Rogan 2011] CF disease can result from

any one of those numerous mutations on a single gene Despite the considerable number of

mutations the encoded gene product is one and the same the CFTR protein The most

common mutation accounting for 70 of the disease alleles leads to a single amino acid

deletion (∆F508) [Zielenski 1995] As presented in Table 12 the class II mutation which

includes the ∆F508 deletion is responsible for 85 of cases in Europe [Peebles 2005] It is

important to note that the percentage of CF patients expressing the most common mutation

type varies among ethnic groups For example only 30 of Israelis with CF have the most

common mutation (∆F508) [Shoshani 1992]

142 Complications in Gene Therapy

Because CF is a genetic disease it was initially believed that gene therapy would

be the most effective treatment for all classes of CFTR mutations In gene therapy correct

19

copies of the CFTR gene are transferred to the respiratory epithelial cells where the gene

can be translated and a functional CFTR can be synthesized [Burney 2012] Previous

studies have focused on increased chloride secretion out of the cell demonstrating positive

results of some normal CFTR function however clinical efficacy has not yet been

achieved [Burney 2012] Despite the vast knowledge obtained by research focused on

understanding the genetic defect underlying CF this understanding has been referred to as

only ldquohalf the battlerdquo in finding the cure for this disease [Hearst 1995] Anxiously awaited

cures focused on the gene therapy approach have failed to materialize in spite of the

significant amount of research performed in this field Complementation of CF using gene

transfer or gene therapy methods specifically focusing on the delivery of a CFTR cDNA

to the airway epithelium seemed appealing initially since the proposed target cells are

accessible by aerosol delivery approaches (or other direct instillation) however since the

first human gene therapy trial in 1993 realization of this goal has proved challenging [Sinn

2011] The use of the previous gold standard in CF gene therapy adenoviral vectors has

decreased recently due to low transduction efficiency weak promoter activity and

incapability for re-administration due to the development of an anti-viral vector immune

response [Griesenbach 2006] The adenovirus package is also very small and packaging

the large CFTR gene has proven difficult One group attempted to package the gene by

cutting it in half and using two separate viral vectors (each carrying half the gene) [Song

2009] Another study pointed out immune responses to the viral vector may be enhanced if

the patient already has an established P aeruginosa infection [Tosi 2004] A similar

problem has been reported when the alternative Sendai virus (SeV) vector is used for gene

20

transfer Although the SeV is an efficient gene transfer agent the gene expression is

transient and requires repeat administration as with the adenovirus vectors re-

administration of SeV vectors also results in an immune response [Griesenbach 2006] A

more serious problem with SeV vectors is that they have demonstrated the induction of

oncogenesis in certain trials [Hacein-Bey-Abina 2008] The developments of novel non-

viral methods for gene transfer have been slow One report on NP-mediated gene transfer

did show increased chloride transport however vector-specific mRNA was could not be

detected [Konstan 2004] Another problem with non-viral gene transfer is caused by the

heightened inflammatory state further frustrated by the introduction of plasmid DNA

[Burney 2012] The unmethylated nucleotide sequence in the plasmid DNA is identified as

an antigen by the immune system thus causing further inflammation in the lower

respiratory tract [Zabner 1996] [Schwartz 1997] CRISPRCas9 has demonstrated the

ability to repair a single-gene hereditary defect causing CF in murine and human stem cells

[Schwank 2013] and this treatment may become available soon However a recent paper

published in Nature Communications shows that CRISPRCas9 causes numerous

unwanted insertions and deletions (up to 600 bp) in the mouse genome [Shin 2017] and

may need significantly more investigation before it is used to treat human patients It is

also unlikely that gene transfer would be a viable option for patients with more than a

single mutation or patients with a class VII mutation alone or in combination with other

mutations A class VII mutation results in the total absence of a CFTR as well as an

absence of mRNA

21

143 Summary of Treatment Failure

Yet another hurdle was realized after treatment data for a larger population was

available patients with the same CFTR mutation genotype often respond differently to

drug treatments [Amaral 2015] [Marson 2015] This data suggests an even deeper level of

personalization may be necessary to achieve sufficient efficacy of current therapeutics

Personalized medicine despite presenting significant benefits is also costly and may not

become available in developing countries for quite some time

15 The Role of Pseudomonas aeruginosa in the Morbidity and Mortality of Cystic

Fibrosis Patients

The most frequently reported pathogenic microbial species colonizing the lungs of

CF patients is Pseudomonas aeruginosa P aeruginosa has been cultured from the

respiratory tract of 61 of all patients (ranging from 21 of patients under 1 year of age

to ˃80 of patients 26 years or older) [FitzSimmons 1993] P aeruginosa is also the top

etiology of all gram-negative nosocomial (acquired in hospitals) infectious bacteria with a

striking mortality rate of 50 or more [Baltch 1994] [Hauser 2003]

Pseudomonas aeruginosa is one of the notorious ESKAPE pathogens (a group

consisting of Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae

Acinetobacter baumannii Pseudomonas aeruginosa and Enterobacter species) which

have developed resistance to the bulk of our current antimicrobial regimes and instead

ldquoescaperdquo the lethal action of antibiotics [Rice 2008] More specifically many highly

resistant Gram-negative bacteria from the ESKAPE group including P aeruginosa are

22

emerging as exceptionally noteworthy pathogens in threatening public health in United

States as well as other parts of the world [Boucher 2009] The ESKAPE bacteria are of

tremendous concern because they are responsible for causing the overwhelming majority

of nosocomial infections Several reports identify significant limitations in current

treatment options for these pathogens that force medical professionals to settle on the use

of previously discontinued drugs having documented toxicity and unclear dosage and

administration guidelines [Bradford 2004 Cardo 2004 Falgas 2007 Urban 2008] They

also provide complex models of pathogenesis transmission and drug resistance [Rice

2008 Boucher 2009] Treatment regimens found to exhibit success against the ESKAPE

bacteria can be applied to virtually any other species Successful treatment of these species

alone will result in significantly safer healthcare environments more suitable for treating

disease and illness

This member of the Gammaproteobacteria class and the Pseudomonadaceae

family is a deadly pathogen responsible for the morbidity and mortality of CF and

oncology patients as well as burn unit patients and infects up to two-thirds of ICU

patients with nosocomial pneumonia [Torres 1990] It is responsible for more than 90 of

respiratory failure cases in CF patients [Gilligan 1981] P aeruginosa has inherent as well

as acquired resistance to many drug classes In addition it possesses the ability to quickly

alter its genetics to impart resistance to the presence of new unrecognized treatments [Lee

2007] Despite its classification as an ldquoopportunistic pathogenrdquo [Fick 1992] [Campon

1993] it remains a major worldwide public health problem due to its ubiquity in the

environment its ability to colonize virtually all regions of the body and its overall vitality

23

which has allowed it to adapt to a wide range of environmental conditions The pathogen

possesses the ability to grow with limited nutrients and can metabolize some unusual

organic molecules as carbon sources some examples are acetate and citrate It can grow

without oxygen if NO3 is available as an electron acceptor for cellular respiration This

species is so robust that it is one of the few extremophiles that can colonize deionized

water

Many issues arise when attempting to treat P aeruginosa infections in the

respiratory tract of CF patients using conventional methods First CF sputum is highly

viscous interfering with normal oxygen diffusion thus hypoxic conditions exist The

hypoxic environment promotes biofilm formation by P aeruginosa [Worlitzsch 2002] as

low-oxygen conditions trigger the phenotypic switch to biofilm mode Once in the biofilm

mode of growth the bacterial colony produces a protective alginate layer around itself At

this point two barriers to drug diffusion exist physically blocking the aerosol antibiotics

from reaching the target the viscous mucus layer and the biofilm layer (Figure 14) No

antimicrobial agent can penetrate the biofilm unless the microorganisms form aggregates

that affect its diffusion [Stewart 2001]

While numerous microbial species can successfully colonize the respiratory tract of

CF patients P aeruginosa ultimately dominates the microbial flora becoming the chief

contributor to disease severity and respiratory failure The phenotypic switch of P

aeruginosa microcolonies from a planktonic (non-mucoid) to a biofilm (mucoid) state is

characterized by both antibiotic resistance and accelerated pulmonary decline [Govan

24

1996] Therefore an artificial active transport method is needed to deliver antibacterial

drugs to the bacterial cells

Figure 14 Biofilm and mucus barriers in a CF lung infection Illustration of

biofilm layer fixed to infected tissue protecting bacterial cells and CF mucus layer

inhibiting penetration of antibiotics and antibodies

16 Antibiotic Drug Resistance and Biofilms

Many bacterial species in response to the presence of antibiotics or bacteriophages or in

low oxygen or low nutrient conditions switch to the biofilm mode of growth These initial

bacteria release chemical signals inducing the switch in neighboring populations as well

Biofilm mode consists of a phenotypic switch from planktonic (free) cells by means of

gene regulation [An 2007] To form a biofilm planktonic cells first adhere to a surface via

van der Waals forces then by using flagella or cilia as an anchor Quorum sensing (QS) is

used to recruit other bacterial cells and promote expression of the genes necessary for cell

25

aggregation and subsequently biofilm production An inducer binds the bacterial QS

receptor triggering transcription and translation of necessary genes

Once a colony is established the anchor cells produce exopolysaccharides which

form the protective biofilm layer around the bacterial colonies N-acyl homoserine lactones

are signaling molecules called auto-inducers (AIs) used in QS [Smith 2002] It is

interesting to note that compounds with similar structures may be of interest for blocking

QS (receptor antagonists) [Sio 2006] Antibiotic resistance typically results from a transfer

of antibiotic resistance genes through bacterial conjugation gene regulation or other

modes of gene transfer However a major factor contributing to antibiotic drug resistance

in P aeruginosa is the production of biofilms The production of a biofilm results in a

slower growth combined with bacterial production of extracellular polysaccharides (EPS)

which form a physical barrier that limits the ability of antibiotic drugs to interact with the

bacteria The EPS biofilm is mainly composed of alginate a slimy anionic co-block

polymer which forms a viscous gum when in the presence of water hence the designation

ldquomucoidalrdquo Alginate or alginic acid is a linear copolymer consisting of homopolymeric

blocks of (1-4)-linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G)

residues [Gacesa 1990] (see Figure 15) The M and G residues are covalently linked

together in different sequences or blocks The monomers may be homopolymeric in

blocks of consecutive G-residues or consecutive M-residues co-block alternating M and

G-residues The known and proposed roles of alginate in biofilm infections include

generation of an alginate covering forming a direct barrier to phagocytosis and

26

opsonization immunomodulatory effects and other biofilm-related phenomena such as

bacterial adhesion and antibiotic resistance [Govan 1996]

Figure 15 Alginate molecule Carbon skeleton showing the homopolymeric

blocks of (1-4)-linked β-D-mannuronate (M) (upper ring) and its C-5 epimer α-

L-guluronate (G) (lower ring) Image created with MarvinSketchtrade

Bacterial biofilm infections in general are a significant public health problem

Specifically P aeruginosa biofilms cause infections in indwelling catheters burns open

wounds orthodontic wires CF lungs and stents and can infect virtually any part of the

body As discussed earlier bacterial biofilms reduce the efficacy of therapeutics due to

their physical interference with drug diffusion by blocking diffusion of the drug to the

target bacterial colonies [Govan 1996] In addition with regards to the more than 20 genes

that are differentially expressed in tobramycin-treated biofilms sheer existence in a biofilm

27

indicates moderate resistance to all antibiotic drugs [Whiteley 2001] Regarding the P

aeruginosa species interference of the alginate barrier with antibiotic penetration to the

strain and thus antibacterial action has also been thoroughly investigated [Kumon 1994]

Interestingly when bacterial cells are released from a biofilm they typically experience an

abrupt increased susceptibility to antibiotics This suggests that the antibiotic resistance of

biofilm bacteria was not acquired through mutations or incorporation of mobile genetic

elements into the bacterial genome [Anwar 1989] Since the most common cause death for

CF patients is respiratory failure from chronic bacterial infections and P aeruginosa is the

top etiology responsible for such infections annihilation of P aeruginosa is a fundamental

step in increasing the life expectancy of CF patients

17 Proposed Universal Treatment Method Using Superparamagnetic Nanoparticles

Significant improvements have been made in the treatment of CF over the past 30 years

Direct drug delivery via inhalation aerosols have increased the average life expectancy of

CF positive children born in developed countries to approximately 40 years [Elborn 1991]

[Staab 1998] Despite this the life expectancy of CF patients could still stand to improve

The efficacy of inhaled therapies still remains marginal due to the presence of the viscous

mucus barrier within the airways extensive degradation and metabolism of inhaled drug

prior to exerting its pharmacological action and the development of mucoid P aeruginosa

biofilm colonies Therefore an adequate active transport method is necessary to deliver

28

antibiotic drug to the bacterial colonies below the mucus layer within the protective

biofilm

The possibility of using magnetic gradient guided active transport of antibiotic

drug using superparamagnetic nanoparticles was investigated further since the barriers to

diffusion of therapeutic drug or gene through mucus and biofilm are the principal bases for

treatment failure Nanoparticle carrier mediated of drug or gene delivery based on passive

transport have demonstrated inadequate penetration efficiencies [Sanders 2000] Similar

passive transport-based nanocarrier methods perform insufficiently and are unlikely to

enhance the penetration efficiencies to clinically relevant levels Frequently drugs or gene

vectors are unable to reach the intended target prior to their activity being diminished or

eliminated Poor transport efficiencies in drug delivery have resulted in the inadequacy of

therapies since the mucus and biofilm barriers to drug diffusion result in sub-therapeutic

levels of drug at the infected area These low-levels of antibiotic drug near the bacterial

colonies further leads to drug resistant bacterial strains as the colonies become sensitized to

the drug Because the use of nanomagnetic materials bound to antibiotic drug would allow

us to guide the magnetic nanoparticles (MNPs) to the area of interest by using an external

magnetic field the particles could be guided deeper into the respiratory tract than

inhalation alone would allow Particularly of interest is the capability of MNP systems to

put forth robust influences on their local environment by means of heat under an

oscillating magnetic field In other words MNPs once guided via directed motion under

an inhomogeneous static magnetic field to an area of interest can be placed in an

oscillating magnetic field and raise the local temperature by means of inductive heating A

29

local temperature increase is anticipated to reduce the viscosity of the mucus and biofilm

layers facilitating delivery of the antibiotic drug We have demonstrated the ability of the

iron oxide NPs to increase local water temperature in vitro under AC magnetic field These

are attractive functional attributes for fostering transport and drug distribution in CF-

related lung infections Therefore utilizing the unique transport and inherent

superparamagnetic properties of selected nanoscale systems provides a promising strategy

for overcoming the biological mucus and biofilm barriers in CF lung disease

171 Particle Transport and Drug Delivery

Our group has previously demonstrated marked increases in particle transport of

nanoparticles can be attained using a static non-uniform magnetic field [Smyth 2008]

[McGill 2009a] in Chapter 3 we show that both ferromagnetic iron oxide NPs as well as

superparamagnetic iron oxide nanoparticles (SPIONs) can be heated using an external AC

magnetic field under which the SPION could cut through biopolymers such as alginate

and DNA which are responsible for the diffusion-limiting properties of the biofilm In

addition in Chapters 2 and 4 we demonstrate our ability to synthesize several different

types of magnetic nanoparticles (MNPs) to optimize the physical properties and chemical

stability We synthesized and characterized iron oxide NPs having various morphologies

iron nitride NPs and zero-valent iron NPs These particles are surface-biofunctionalized

for drug conjugation We then attach a model drug to the surface using a biocleavable

conjugation scheme (see Chapter 5) Drug release could potentially be triggered by

30

external magnetic fields in a non-invasive manner if necessary Many researchers have

reported the use of external magnetic fields to achieve controlled drug delivery using

hyperthermia via two general methods Hyperthermia-based controlled Drug delivery

through Bond Breaking (DBB) and Hyperthermia-based controlled Drug delivery through

Enhanced Permeability (DEP) [Kumar 2011] The first successful demonstration of DBB

was reported using radiofrequency EMF activation of release of fluorescein-labeled 18 bp

in a model tumor near the posterior mammary fat pad of mice [Derfus 2007] Our

laboratory later confirmed this concept by triggering the release of fluorophore bimane

amine from the surface of SPIONs under external oscillating magnetic fields [McGill

2009b] The first report was by Kost and others who demonstrated insulin release from a

magnetic composite of ethylene vinyl acetate under a low frequency magnetic field [Kost

1987] A commonly proposed approach is to use a composite carrier consisting of a

magnetic iron oxide core inside any thermally sensitive polymer having a temperature-

dependent drug release profile then when the core is self-heated drug release is triggered

[Liu 2008] [Liu 2008] reported the successful triggered delivery of Vitamin B12 within

minutes between 40-45 degC using poly(ethylene-oxide)-poly(propylene-oxide)-

poly(ethylene-oxide) block copolymers 4-nitrophenyl chloroformate gelatin and 1-ethyl-

3-(3- dimethylaminopropyl) carbodiimide self-assembled nanocapsules and magnetic iron

oxide NP cores which were responsible for the heating [Liu 2008] Triggered drug delivery

would be necessary if the required therapeutic dose is found to be higher than the dose

found to be cytotoxic to healthy cells In this case the overall environment could be kept

at a safe drug concentration while the highest concentration would be released specifically

31

at the infection site reducing collateral damage Finally when loaded with drug the MNPs

will be incorporated into inhalable microparticles suitable for lung targeting This will

initiate simultaneous highly efficient transport and highly specific lung deposition

Additionally these systems will transport inhibitory drug concentrations directly to the site

of action and will therefore facilitate improvements in drug and gene therapies in CF

prolonging survival and enhancing quality of life

The physics of particle delivery to the lower respiratory tract has been well

characterized Further engineering of the particle or particles into a stable micron-range

polymer matrix in a stable dry-powder form is necessary for successful pulmonary

delivery Many factors impact the performance of a particle system such as mass median

aerodynamic diameter (MMAD) particle size distribution dispersibility particle

morphology and thermodynamic stability [Chow 2007] [Hickey 2007b] The combination

of two specific parameters size and surface roughness greatly influence performance It is

known that the particles must be further engineered to increase the diameter from the

nanoscale to the microscale range to avoid deposition in the throat [Hickey 2003] Previous

research has also demonstrated that particles with MMADs 1-2 μm deposit in the smaller

(lower) airways and 5-10 μm deposit in the larger (upper) airways [Vehring 2007]

Particles having a high degree of surface roughness exhibit increased dispersibility due to

decreased interparticulate interactions consequently resulting in significantly decreased

particle aggregation resulting in a larger aerodynamic size (for the agglomerate) [Gilani

2005] Typically lactose [Kaialy 2012] or mannitol [Hamishehkar 2012] is used as a

carrier because it has a sweet taste Some other polymers which have been previously

32

investigated for this application are the FDA approved polymer poly(lactic-coglycolic)

(PLGA) [Tomoda 2009] poly(ethylene glycol)-co-poly(sebacic acid) (PEG-PSA) [Tang

2010] and dipalmitoylphosphatidylcholine (DPPC) with dipalmitoyl phosphatidylethanol

aminemethoxy-polyethylene glycol (DPPE-PEG) [Meenach 2013]

172 Biocompatibility

Previous work on biocompatible magnetic materials has focused on the iron oxides [Gupta

2005] [Xie 2009] [Xie 2010] iron core-iron oxide shell particles [Qiang 2006] cobalt

[Bao 2005] [Xu 2007] [Lukanov 2011] iron core gold shell particles [Chen 2003] or the

rare-earth elements [Meiser 2004] [Setua 2010] [Dobson 2006] However the iron oxides

have shown the greatest potential as biofilm inhibitors having low cytotoxicity [Johannsen

2007] Significant research on silver NPs as antimicrobial agents has been reported in the

literature [Sondi 2004 Morones 2005 Cho 2005 Kim 2007 Pal 2007 Shrivastava 2007

Duraacuten 2007 Martiacutenez-Castantildeoacuten 2008 Rai 2009 Chudasama 2010 Lara 2011 El-Kheshen

2012 Dong 2012 Prabhu 2012 Le 2012 Sadeghi 2012 Rai 2012 Emeka 2014 Losasso

2014 Agnihotri 2014 Franci 2015 Cavaliere 2015 Lara 2015 Giessen 2016 Russol

2017 Patra 2017 Shaker 2017] and much research has also been done on the efficacy of

silver NPs against P aeruginosa [Afreen 2011 Eid 2013 Palanisamy 2014 Singh 2014a

Anasari 2014 Mushin 2014 Singh 2014b Mapara 2015 Raza 2016 Haghighi 2016

Nasiri 2016 Kasitherar 2017] Due to their undisputable antibacterial properties silver

NPs are among the most commonly exploited nanomaterials in commercialized products

[Beer 2012] Although silver NPs have demonstrated antimicrobial properties against

many bacterial species silver is costly and is also known to exhibit toxicity in multiple

33

species [Asharani 2008] including in vitro cytotoxicity in various human cell lines

[Kawata 2009 Beer 2012 Foldbjerg 2011] Most researchers attribute the observed

toxicity either to silver ions [Asharani 2008] or the combination of silver NPs and silver

ions [Bilberg 2011 Foldbjerg 2011] An ideal bactericidal agent should be lethal to

bacteria but safe to human cells One such candidate is iron and its compounds Iron-oxide

NPs have been shown to be non-toxic [Sumanta 2008 Sun 2010 Prodan 2013 Grottone

2014] For example ferahemeferumoxytol containing superparamagnetic iron-oxide NPs

was approved by the US Food and Drug Administration as an iron supplement for

treatment of iron deficiency in patients with renal failure [Provenzano 2009 Coyne 2009

Lu 2010] According to a previous report iron-oxide in NP form is not only non-toxic but

its byproduct degraded iron from the cores apparently accumulates in natural iron stores

in the body [Weissleder 1989] Properly biofunctionalized iron-oxide NPs have been

shown to inhibit growth of Staphylococcus aureus [Tran 2010 Darwish 2015 Shi 2016]

and Escherichia coli [Darwish 2015 Chatterjee 2011] prevent biofilm formation by P

aeruginosa [Niemirowicz 2015] and Streptococcus mutans [Javanbakht 2016] and exhibit

bactericidal activity against a range of Gram-negative and Gram-positive bacterial species

[Behera 2012 Prodan 2013 Thukkaram 2014 Prabhu 2015 Arakha 2015 Nehra 2018]

While these are very encouraging results more work is necessary in the investigation of

iron-oxide NPs as a feasible alternative to silver NPs in the treatment of bacterial infections

and for biofilm disruption

34

173 Biofilm Considerations

According to a previous report there are no clinically effective inhibitors of biofilm

formation presently available [Musk 2005] However iron salts appeared to inhibit biofilm

formation in a concentration-dependent manner Investigations into the P aeruginosa

genetics show that elevated iron concentrations repress the expression of certain genes

essential for biofilm production in P aeruginosa [Musk 2005] To address the biofilm

problem we have synthesized and characterized iron oxide (magnetite) NPs capped with

biodegradable short-chain carboxylic acid derivatives conjugated to the most common

antibiotic arsenal for the treatment of gram-negative bacteria The functionalized

nanoparticles may carry the drug past the mucus and biofilm layers to target the bacterial

colonies via magnetic gradient-guided transport Additionally the magnetic ferrofluid may

be used under application of an oscillating magnetic field to raise the local temperature

causing biofilm disruption slowed growth and mechanical disruption P aeruginosa can

sustain normal growth at temperatures up to 42 ˚C therefore an increase in the local

temperature may increase the bacterial susceptibility to the antibiotic drugs if not

destroying them This temperature increase would not harm local healthy cells as a

temperature reached by natural fever does not harm healthy tissue It is well-known that

hyperthermia increases the penetration of cytostatic drugs into tissuecells [Witkamp 2001]

and may also increase penetration of drug into biofilms and bacterial colonies In this case

because the drug we are using is beta lactam antibiotic drug which works by interfering

with production of peptidoglycan cell walls increased influx of beta lactam antibiotics into

of healthy mammalian cells would have no effect as they do not have peptidoglycan cell

35

walls Caution must be used however if this technology was used in the delivery of a

chemotherapy agent The healthy tissue (along with the cancerous tissue) would become

more susceptible to the toxic effects of the chemotherapeutic agent [Witkamp 2001]

[Koning 2010] These abilities of the ferrofluid would also treat multi-drug resistant

strains which appear to be increasing in many nosocomial as well as acquired

opportunistic infections

174 Critical Parameters

Particle size prior to polymer engineering is a crucial parameter as polymer

coating and drug conjugation will increase particle diameter Previous studies have shown

that although conventional particles are often entrapped in mucus small sized particles

(120 nm) exceeded the rate of diffusion through mucus when compared to larger particles

(560 nm) [Sanders 2000] These findings are significant since it is now known that the

maximum pore size in CF sputum is 400 nm Therefore an ideal drug carrier would have

to be significantly smaller than 400 nm to enhance the rate of free diffusion of the particles

through mucus pores Our group has previously shown that superparamagnetic iron oxide

nanoparticles (SPIONS) exhibit enhanced diffusion through alginate biofilms using

magnetic field gradient guiding in vitro [McGill 2009a] In addition to magnetic field

guided transport capability MNPs are capable of releasing heat upon placement in an

external oscillating magnetic field [McGill 2009b] Three potential mechanisms are

implicated in heating in the frequency range suitable for human patient treatment Neacuteel

relaxation Brownian motion relaxation and hysteresis losses in the ferro (ferri) magnetic

36

size range This phenomenon is exploited in the application of hyperthermic tumor

destruction or thermotherapy an experimental cancer treatment in which heat released

from MNP placed in an AC magnetic field may be used to kill tumor cells We expect heat

released from MNP hyperthermia would further enhance the magnetic-field-guided particle

movement through the mucus and EPS matrix in the lower respiratory tract by reducing

their viscosity These methods would provide a viable universal treatment method which

would likely increase life expectancy for all CF sufferers without regard to the mutation

type or severity of the disease

18 Overview of Dissertation

In Chapter 2 we describe the synthesis and characterization of iron oxide nanoparticles

(NPs) of which we investigated several sizes and morphologies iron martensite NPs

(Fe16N2) and zero-valent iron NPs (Fe0) These samples were either uncapped or capped

with polyethylene glycol (PEG) for structural and magnetic characterization and either

uncapped or capped with alginate or PEG for in vitro bacterial sensitivity studies Capping

is done by attaching a water-soluble molecule or polymer to the positively-charged NP via

a negatively-charged terminal carboxyl group This is done to enhance solubility of NPs in

water When the iron oxide NPs come out of synthesis they are coated in the metal carrier

molecule oleic acid Oleic acid is a long chain hydrocarbon with a terminal carboxyl group

that attaches to the positively charged metal (Fe+) The long chain hydrocarbon which

remains surrounding the metal NP after its formation contains no other carboxyl carbonyl

or hydroxyl groups and is therefore hydrophobic In order to prevent NP oxidation in air

37

or aqueous solution prevent particle aggregation and allow for drug conjugation the NPs

should be coated with a passivation layer If the NPs are to be used in vivo it is necessary

to coat them with a water-soluble substance otherwise entropic forces would cause them

to aggregate in the aqueous environment of the body For these studies we chose alginate

citrate or polyethylene glycol coatings because they are FDA approved for human

consumption in food and pharmaceuticals The samples were characterized by transmission

electron microscopy (TEM) X-ray diffraction (XRD) and energy dispersive X-ray

spectroscopy (EDS) (Chapters 3 and 4) and tested for magnetic hyperthermia using the

NanoTherics Ltd MagneThermtrade as described in Chapter 4 Spherical magnetite (Fe3O4)

NPs having high iron content and a mean radius between 15 and 25 nm were found to

exhibit the best magnetic properties (Chapter 4) The NPs having a radius lt19 were

superparamagnetic The NPs were further functionalized and conjugated to tobramycin

using EDCsulfo-NHS cross-linking discussed in detail in Chapter 5 The drug-loaded NPs

as well as NP samples with different capping agents were investigated alone Antibiotic

drug was used to test the sensitivities of mucoidal colonies of P aeruginosa at time

intervals from 3-60 days to determine if growth time alters the dosage response the results

of these experiments are described in Chapter 6 Cytotoxicity viability and apoptosis

assays in a human adenocarcinoma cell line were performed on two concentrations of iron

oxide NPs and the results are described in chapter 7 Overall the iron oxide NPs did not

exhibit statistically significant cytotoxicity in this cell line

38

Chapter 2

SYNTHESIS AND CHARACTERIZATION OF IRON

OXIDE NANOPARTICLES

The iron oxides exist naturally the most common phases being hematite (α-Fe2O3)

maghemite (γ-Fe2O3) and magnetite (Fe3O4) [Cornell 2006] Magnetite exhibits the

strongest magnetic properties of all phases of iron oxide [Cornell 2006] [Majewski 2007]

[Teja 2009] which is why it was selected for this application The concept of magnetic-

field-guided drug delivery has existed for over 30 years [Indara 2010] Aside from our

antibacterial application colloidal suspensions of (SPIONs) called ferrofluids have been

proposed for a range of biomedical applications such as magnetic gradient-guided drug

carriers for targeted drug delivery [Sahoo 2003] [Veiseh 2010] cancer thermotherapy

[Hirsch 2003] [Thiesen 2008] and magnetic resonance imaging (MRI) contrast agents

[Kim 2005] [Alexiou 2006]

We have synthesized and characterized magnetic nanoparticles (MNPs) to

overcome the existing barriers and achieve critical improvements in CF therapy which will

increase the life expectancy of CF patients Antibiotic conjugated nanomaterial systems

will facilitate significant enhancement of the efficacy of model therapeutic agents due to

increased diffusion and penetration through mucus and biofilm barriers in cystic fibrosis

when administered directly to the lung as an inhalation aerosol Along with the numerous

39

applications numerous methods for synthesis of SPIONs have been previously published

[Laurent 2008] Various methods include thermal or sonochemical decomposition of iron

pentacarbonyl (Fe(CO)5) [Shafi 2001] [Hyeon 2003] [Wu 2008] microemulsions [Loacutepez

Peacuterez1997] [Santra 2001] [Chin 2007] sol-gel synthesis [Gash 2001] [Lu 2002]

hydrothermal reactions [Hu 2007] [Takami 2007] [Ge 2009] hydrolysis and thermolysis

of precursors [Iida 2007] flow injection syntheses [Salazar-Alvarez 2006] and

electrospray syntheses [Kruis 1998] [Basak 2007]

We have selected a green chemistry solvothermal method for our syntheses due to

the flawless crystallinity morphology control and monodispersity Green chemistry

applied to the practice of synthetic materials engineering focuses not only on minimizing

waste reducing energy use and recycling but also using natural water-soluble non-toxic

or reduced toxicity precursors and reagents When possible petroleum products are

replaced with natural lipids and toxic nitrates are replaced with chloride salts

Iron oxide NPs were synthesized in a high boiling point solvent consisting of inert

hydrocarbons The NP growth was facilitated and somewhat controlled by the organic

carrier molecule oleate At the end of synthesis the NPs remained capped with oleate

Later cap exchange may be performed using either alginate citrate PEG-OH (hydroxyl-

terminated polyethylene glycol) or PEG-COOH (carboxyl-terminated polyethylene

glycol) for water solubility as discussed further in Chapter 5 The synthesis is a

modification of a procedure published elsewhere [Park 2004] Our modifications to this

popular method yielded NPs of various sizes and morphologies achieved by changing the

boiling point of the solvent or reflux time These methods were not previously reported in

40

the literature Additional modifications were made to reduce cost while developing green

chemistry methods Spherical cube-shaped and polymorphous NPs as well as nanowires

were obtained by varying the reaction time and reflux temperature This was achieved by

using higher boiling point organic solvents for higher reaction temperatures In addition

green chemistry and lower-cost alternative chemicals were also investigated

21 Synthesis of Colloidal Magnetite Nanoparticles

The procedure consisted of two steps as described by [Park 2004] synthesis of the iron

oleate precursor complex and synthesis of the iron oxide NPs The precursor was iron

oleate (iron(II III) [(9Z)-9-octadecenoate] n) where n is the coordination number of iron

and could form a monomer dimer or trimer [Bronstein 2007] [Palchoudhury 2011]

produced in our laboratory using a modified procedure of Bronstein et al [Bronstein

2007] The iron oleate complex was formed from the combination of sodium oleate salt

(sodium (9Z)-9 octadecenoate) and iron(III) chloride hexahydrate (FeCl3middot6H2O) The

precursor preparation was modified by washing with water ethanol and acetone to

remove additional contaminants before aging in the oven overnight

211 Materials

FeCl3middot6H2O (97) was purchased from Sigma-Aldrich n-docosane (99) and n-eicosane

(99) were purchased from Alfa Aesar n-dodecane (gt99) was purchased from Fischer

Scientific sodium oleate (gt97) was purchased from Tokyo Chemical Industry Co

41

hexanes (95) ethanol (99) and acetone (99) were purchased from EMD Chemicals

Inc All chemicals and their physical properties may be referenced in Appendix I

212 Synthesis of Iron Oleate Precursor Complex

In a standard reaction 675 g of FeCl3middot6H2O was combined with 25 mL of deionized

water and vacuum-filtered through 022 μm filter paper The mixture was then combined

with 2435 g of sodium oleate in a three-neck round-bottom flask 150 mL of a stock

solution consisting of a 246 mixture of deionized water ethanol and hexane was added

to the flask Under argon flow the mixture was vented and filled

Figure 21 Ion exchange reaction between iron(III) chloride and sodium oleate producing

iron oleate and the byproduct sodium chloride Image by L Armijo 2012

42

for three one-minute intervals to remove all oxygen from the reaction flask The solution

was then slowly (5 degCmin) heated to 50 degC under vigorous stirring

Once the solid sodium oleate had completely melted and the reflux had begun

(around 50ndash60 degC) the temperature was further increased (3 degCmin) to 70 degC and the flask

was kept at this temperature for four hours ensuring that the total reflux time was 4 hours

The mixture was then cooled to 60 degC and washed three times with deionized water in a

separatory flask if necessary additional hexane was added to dissolve the organic layer

The product was then washed twice with 12 mL aliquots of acetone and ethanol The

organic layer was placed in a rotary evaporator (Rotovap) with the water bath set at 30 degC

until the hexane and ethanol were evaporated away The resulting waxy complex was then

dried in a vacuum oven for 24 hours at 70 degC The final product was a waxy dark-brown

solid The overall reaction is illustrated in Figure 21

213 Synthesis of Cubic Polymorphous and Spherical Nanoparticles

Using a 500 mL three-neck-flask attached to the Schlenk line (Figure 23) the reaction was

carried out In a standard reaction 5 g of iron oleate (washed with water for cubes and

water acetone and ethanol for spheres) was combined with 56 mL of oleic acid and

1315 g of n-eicosane (boiling point 3427 degC) The mixture was slowly heated (3 degCmin)

to 50 degC under argon flow and vigorous stirring Once the reactants had dissolved the

temperature was further increased to 342 degC at a heating rate of 30 degCmin For 19 nm

cubes the mixture was refluxed for 30 minutes For larger particles the reflux time was

extended with an average growth rate of 22 nm per minute The maximum size

43

achievable without adding additional reagents was 250 nm after 99 min If the solution was

allowed to cool below the nucleation temperature (~ 200 degC) [Bronstein 2007] for any

amount of time before being refluxed at the same maximum temperature again the NP

growth favored spherical morphology in which polymorphous NPs represented an

intermediate morphology (see Figure 22) It appears from these results that the spherical

morphology is thermodynamically favored exhibiting stability at high temperatures After

30 minutes the spheres were highly monodisperse

Figure 22 Morphology alterations of iron oxide nanoparticles via

additional nucleation event(s)

214 Synthesis of Iron Oxide Nanowires

In a standard reaction 5 g of iron oleate was combined with 16 mL of oleic acid and

1315 g of n-dodecane (boiling point 2162 degC) The mixture was slowly (3 degCmin)

heated to 50 degC under argon flow and vigorous stirring For ~55times2 nm wires once the

reactants had dissolved the temperature was further increased to 216 degC at a heating rate

44

of 3 degC per minute and the mixture was refluxed for 60minutes For smaller wires ~25times2

nm the reflux was carried out at 150 degC for the same time These findings confirm those

reported by [Palchoudhury 2011]

Figure 23 Active iron oxide nanoparticle synthesis in the Schlenk line

215 Synthesis of Spherical Nanoparticles

45

1315 g of n-docosane (boiling point 370 degC) The mixture was slowly heated to 50 degC at a

heating rate of 3degC per minute under argon flow and vigorous stirring Once the reactants

had dissolved the temperature was further increased to 370 degC at a heating rate of

3degCmin For ~20 nm particles the mixture was allowed to reflux for 32 minutes For

larger particles the reflux time was extended with an average growth rate of 16 nm per

minute The maximum size without adding additional reagents was 158 nm after 99 min

Figure 24 Summary of morphology control time and temperature

parameters established by this study for the synthesis of Fe3O4 NPs

46

216 Summary of Green Chemistry Modifications

The sustainability of novel materials is crucial to human progress Ensuring environmental

friendliness the engineering process and integrating natural compounds into the materials

was a priority in these studies Naturally existing molecules may have lower cytotoxicity

compared with synthetic products and are less likely to detrimentally affect the delicate

ecosystem upon disposal In addition the procedure for manufacturing many synthetic or

purified compounds is typically not environmentally friendly The Environmental

Protection Agency (EPA) standards for green chemistry [EPA 2015] are very clear reduce

waste maximize yield use less hazardous materials minimize accident risk By simply

replacing the popular metal nitrate precursors with water soluble chloride salts many

environmental benefits are suggested Our environmentally-friendly carrier molecule and

stabilizing agent oleic acid is a derivative of vegetable oil

217 Cost Reduction

A lower cost and environmentally sound modification may be made to the aforementioned

procedures by simply replacing the high molecular weight hydrocarbon solvent with

paraffin wax (bp gt370 ˚C) or the natural solvent beeswax Paraffin wax or beeswax may

be thermally separated for reuse as well rather than disposing of solvents after each

synthesis This simple green chemistry modification resulted in the same monodisperse

NPs above Docosane costs $7860 for 100 g or ~$079 per gram (Sigma-Aldrich)

paraffin wax costs $5 for 453 g (1 lb) This roughly corresponds to ~$001 per gram

compared to the cost of docosane thereby reducing the cost by 987 Beeswax costs ~$9

47

for 453 g (1 lb) or approximately $002 per gram resulting in a cost reduction of 974

In addition we have used re-distilled solvents and saved them for use in future NP

syntheses These solvents were processed and purified in-house to further reduce cost and

eliminate toxic waste

22 Structural Characterization

The transmission electron microscope (TEM) images in Figures 25 through 27 show the

various morphologies and sizes of Fe3O4 NPs we were able to obtain using this method

Cubic NPs having a maximum size of 250 nm were obtained after a 99-minute reflux

Figure 25 Transmission electron microscope image of 250 nm magnetite nanocube

(left image) formed after 99-minute reflux scale bar is 100 nm and ~15 nm spherical

NPs (right image) scale bar is 10 nm

Cubic and spherical NPs were easily produced with high monodispersity in sizes ranging

from 16 to 250 nm Polymorphous NPs shown in Figure 26a were obtained by allowing the

cubic NPs to cool below their nucleation temperature of 200 ˚C prior to refluxing above the

48

nucleation temperature Interestingly while attempting to measure particle size during

synthesis by taking aliquots of the NPs as time went on the temperature controller failed

triggering the power supply to shut-off and the particles cooled to room temperature In hopes

of salvaging the experiment we returned the temperature to 340 ordmC We found that the aliquot

taken after a 3-minute reflux performed after allowing the sample to cool below the nucleation

temperature was polymorphous

Figure 26 Transmission electron microscopy (TEM) images of magnetite

nanoparticles capped with oleic acid a) Polymorphous NPs scale bar is 100 nm

b) monodisperse spheres formed from refluxing of polymorphous NPs scale bar

is 100 nm c) monodisperse spherical NPs ~22 nm in diameter scale bar is 100

nm d) nanowires scale bar is 50 nm [Armijo 2012a]

49

A second aliquot taken after 30 minutes of refluxing consisted of monodisperse

spherical NPs These findings suggest that the spherical morphology may be favored at

higher temperatures due to growth on all faces Monodisperse spheres with a diameter

of ~30 nm in Figure26b formed from polymorphous NPs shown in Figure 26a

when the reaction mixture was allowed below the nucleation temperature of 200 ˚C

for approximately 30 minutes before being refluxed again Spheres of ~22 nm in

diameter (Figure 26c) and 55times2 nm nanowires (Figure 26d) were made in n-

docosane (boiling point 370 degC) and n-dodecane (boiling point 2162 degC)

respectively [Armijo 2012a] We performed high-resolution (HR) TEM to

characterize morphology and to confirm high crystallinity of the NPs

Figure 27 Transmission electron microscope (TEM) image of Fe3O4 spherical

superparamagnetic nanoparticles capped with oleic acid This sample was chosen for

bacterial sensitivity studies discussed in Chapter 6 due to its excellent monodispersity

and superparamagnetic properties scale bar is 50 nm

50

Figure 28 High-resolution transmission electron microscope

(TEM) image and its fast Fourier transform (FFT) of the iron

oxide monodisperse spheres (shown in Figure 26b above) scale

bar is 5 nm

The image in Figure 28 represents fringes observed for the monodisperse spheres from

Figure 26b The TEM images demonstrate the wide range of NP sizes and morphologies

attainable with minor time and temperature modifications to the procedure

Elemental composition of the Fe3O4 NPs was verified with energy dispersive x-ray

spectroscopy (EDS) and example is shown in Figure 29 Magnetite samples of all

morphologies gave the same spectrum in EDS therefore presented the same elemental

composition Iron and oxygen are present in the monodisperse spheres from Figure 26b

The carbon and copper peaks are due to the carbon-coated copper grid

The x-ray diffraction (XRD) data for iron oxide polymorphous nanoparticles

(Figure 210) and the XRD data for the ~17 nm spherical particles are similar and suggests

51

that the composition of the nanoparticles synthesized by this method to be ~70 (plusmn5)

magnetite Fe3O4 with space group Fd3mF41d32m due to a perfect card match to the

major peaks in the crystallography database

Figure 29 Energy dispersive x-ray spectroscopy (EDS) spectrum of magnetite

nanoparticles This particular spectrum was taken from the monodisperse spherical

NP sample imaged in Figure 26b

However it is important to note that the several of the peaks assigned [220] [311] [400]

[440] [422] and [511] which match magnetite in the database correspond to the spinel

phase Spinel phase peaks are present in XRD spectra of both γ-Fe2O3 and Fe3O4 as well

as multiphase crystals containing these phases [Casula 2006] [Bronstien 2007] who also

characterized SPIONS synthesized by this method attributed these peaks to (likely) being

Fe3O4 as do we The remaining 30 of the crystal appears to be composed of ferrous oxide

wuumlstite (Fe1-xO) where x can be between 005 and 017 and α-Fe2O3 The Wuumlstite is a

52

phase of iron(II) composing meteorites The presence of this highly dense highly

magnetic phase is typical of iron oxides produced under low oxygen conditions [Casula

2006] There are small peaks at ~56deg and 84deg which match to the [116] and [128] of α-

Fe2O3 possibly the result of surface oxidation Since the wuumlstite phase is metastable it is

known to convert to α-iron and magnetite or a mixture of wustite α-iron and magnetite

[Redl 2004] The α-iron is reported to accumulate on the shell where on exposure to

atmosphere it oxidizes [Bronstein 2007] which would explain why it is not detected on

the XRD however α-Fe2O3 is Wuumlstite and magnetite are structurally similar and likely

compatible in a multiphase crystal therefore it is not uncommon to observe both phases

together [Bronstein 2007] Magnetite and magemite are indistinguishable from one another

by XRD analysis [Bronstein 2007] It is important to note that due to the similarity in space

groups and lattice constant the oxidation state of iron oxide phases is difficult to determine

with absolute certainty using XRD

Figure 210 XRD spectrum of polymorphous nanoparticles (NPs) (pictured in

Fig 26a) The majority of the prominent peaks in this spectrum correspond to

magnetite or spinel phase iron oxide

53

12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

0

1000

2000

3000

4000

5000

Inte

nsity (

cps)

2-Theta (degree)

[111]

[110]

[311]

[220][422]

[011]

Figure 211 X-ray diffraction (XRD) spectrum of 17 nm spherical NPs taken

with monochromator attached for noise reduction This spectrum also shows

spinel phase hematite and magnetite peaks

23 Summary of Findings

We synthesized and characterized magnetite NPs having various sizes and morphologies

using green chemistry methods Our synthesis method significantly reduces cost while

producing superior nanomaterials while exercising environmental consciousness We were

able to synthesize iron oxide nanowires at a temperature that was not previously believed

to facilitate NP formation [Palchoudhury 2011] We have also shown that spherical

particles are the most thermodynamically stable

54

Although we thoroughly investigated the physical properties of all three NP

morphologies it was decided that the magnetic properties of spherical NPs in the

superparamagnetic size range (lt20 nm) were best for our specific application For

magnetic characterization refer to Chapter 3 In addition because this material will be

administered to the lung lower aspect ratios NPs (spheres rather than nanowires) are

anticipated to have lower cytotoxicity than high aspect ratio NPs Furthermore the

procedure for synthesizing spherical NPs is much simpler than the procedures for the

synthesis of other morphologies because the temperature range required for successful

synthesis is not as narrow Spherical NPs having an easily reproducible synthesis

procedure can be used to provide for further studies thus ensuring minimal variation

between batches For these reasons the 16-18 nm spherical NPs (Figure 27) were used in

the bacterial sensitivity studies discussed in Chapter 6

55

Chapter 3

MAGNETIC CHARACTERIZATION OF IRON OXIDE

NANOPARTICLES AND

MAGNETIC HYPERTHERMIA INVESTIGATIONS

The history of magnetism in medicine is extensive The first report of the use of magnetite

powder for in vivo medical treatment of iron poisoning was in the 11th century AD by the

Persian polymath Avicenna [Haumlfeli 1998] Since the dawn of the era of nanotechnology

thousands of papers have been published proposing uses of nanoscale grain sized magnetic

powders for many biomedical applications One important application which has arisen

only after the modern medicine acknowledged the fact that magnetic fields are not

especially contraindicated for humans is medical hyperthermia [Mornet 2004] also called

thermotherapyThe therapeutic potential of heat has been known for a very long time

beginning with the recognition that fever enhancement promotes faster recovery from

illness by homeopaths it is now known that heat can be used to cure a variety of different

diseases [Andrauml 2007] Heat use for cancer therapy and tumor cauterization has been

proposed since as early as 3000 BC [Strohbehn 1984] A more contemporary historical

account actually suggested the use of lower temperatures which would not cause damage

to healthy tissues (hyperthermia vs cauterization) [Busch 1866] In the past 150 years

much work has been done attempting to maximize heat effects in a local area of interest

56

with varying degrees of success [Streffer 1987] [Baronzio 2010] [Minev 2011] [Moros

2013]

In magnetic thermotherapy the response of MNPs to oscillating magnetic field

causes thermal energy to be dissipated into the surroundings killing the adjacent cells

Additionally hyperthermia can be used to enhance radiation and chemotherapy treatment

of cancer [Praetorius 2007] [Krishnan 2010] [Maier-Hauff 2011] As mentioned in

Chapter 1 one of our original aims was to use the hyperthermic heat evolved to thin the

alginate biofilm characteristic of chronic Pseudomonas aeruginosa lung infections and the

thick CF mucus barrier in conjunction with magnetic gradient guided drug delivery to

deliver antibiotic drug to the infected area Magnetic hyperthermia results from domain

switching upon AC electromagnetic (EM) radiation application Our group previously

investigated iron oxide nanoparticles for heating applications [Armijo 2012a] [Armijo

2012b] however the major mechanism involved in the temperature increases in these

specific nanomaterials have only now been uncovered Biomedical applications require a

material with a large magnetic moment as well as the control of the magnetic properties

imparted by superparamagnetism The attractive property of superparamagnetic materials

relates to the ability of the physician to induce their magnetic properties only after the

magnetic nanoparticles have arrived at the area of interest by application of an external

magnetic field This allows for venous delivery without agglomeration within the blood

vessels Iron-containing nanomaterials having high saturation magnetic moments in the

SPM size-range are attractive for in vivo use The iron oxides specifically have

demonstrated high biocompatibility and low systemic toxicity [Maier-Hauff 2011]

57

[Soenen 2010] [Soenen 2011] as well as having received FDA approval for use as contrast

agents in magnetic resonance imaging (MRI) [Shieh 2005] [Veiseh 2005] We have

investigated magnetic properties of iron oxide nanomaterials in the 15-30 nm size range for

this potential application This size range was chosen because it is close to the single-

domain multi-domain size limit for iron oxides 20-30 nm This size range has shown the

greatest temperature increase under oscillating magnetic field application at many of the

frequencies being investigated for medical hyperthermia in our case 1111 kHz with a

magnetic field of 25 mT [Hergt 2006]

31 Theory

Considering nanoscale colloidal suspensions of superparamagnetic NPs also called

ferrofluids the dominant relaxation mechanism resulting in heat generation could be due to

Brownian motion [Maier-Hauff 2011] or Neacuteel relaxation [Shieh 2005] Figure 31A

illustrates Neacuteel losses magnetic losses owing to domain wall displacements Figure 31B

(lower image) shows Brownian losses energy loss from mechanical rotation of the

particles in a colloidal suspension acting against viscous forces of medium Heat energy

generated results from the contribution of both energy loss effects Figure 31 is the

analysis of the AC data allows us to determine which of these mechanisms dominates

Brownian or Neacuteel relaxation of the particles

Single domain particles have a magnetic moment mp given by

mp = Msυ (31)

58

Where Ms denotes the saturation magnetization and υ is the magnetic volume of the

particle The Brownian relaxation time τB is given by

τB = 4πr3η=kBT (32)

Where r is the hydrodynamic radius η is the dynamic viscosity of the solvent kB is

Boltzmanrsquos constant and T is absolute temperature (K)

For uniaxial anisotropy the Neel relaxation energy barrier is given by Kυ where K

is the anisotropy value of the particle The associated magnetic moment direction reversal

or domain switching time is given by [Fannin 1989] [Fannin 1994] [Neel 1949] [Preacutevot

2001]

τN = τ0exp(σ) (33)

Where τ0 is a damping time having an average value of 10-9 s and σ=KυkT

Figure 31 Mechanisms of energy loss leading to heat production in magnetic

hyperthermia A) Models Neacuteel relaxation the magnetic field flipping within the

NP B) Models Brownian motion the entire particle moves within the solvent

59

For polydisperse samples combinations of the mechanisms respond to the heating in AC

fields This is why it is crucial to use a monodisperse sample for magnetic characterization

These samples were characterized using an effective relaxation time as follows

τeff=τNτB(τNτB) (34)

In which the dominant mechanism is the one with the shortest relaxation time [Fannin

1989] [Shliomis 1974] [Shliomis 1993] This is analogous to the dominant rate of a

chemical reaction being the slowest step

32 Experimental

A typical feature of magnetic nanocrystals is their irreversible ferromagnetic behavior

below the blocking temperature TB and reversible magnetization above it caused by

superparamagnetic behavior of the nanocrystals We investigated the magnetic

properties blocking temperature magnetic saturation and coercivity (hysteresis) using

a superconducting quantum interference device (SQUID) magnetometer We investigated

the heat evolved at two different frequencies and magnetic field strengths using the

nanoTherics Ltd MagneTherm trade Later we looked at the dominant loss mechanism

under AC field at room temperature using the DynoMagreg AC Susceptometer (IMEGO

AB Sweden)

321 SQUID Magnetic Characterization of Iron Oxide Nanoparticles

The blocking temperature can be found experimentally by measuring

magnetization under field-cooling (FC) and zero-field cooling (ZFC) conditions Below

60

TB the Neacuteel relaxation time τN is larger than the measurement time τm (typically 100 s)

and magnetization depends strongly on the field history Above TB magnetization is

strongly affected by thermal fluctuations (τmgtτN) making FC and ZFC curves coincide In

other words for a given measurement time τm hysteretic behavior observed below TB

would not be observed above TB

0 50 100 150 200 250 300 350

00004

00006

00008

00010

00012

00014

00016

Field-Cooled

Zero Field-Cooled

Mag

ne

tizatio

n (

em

u)

Temperature (K)

Figure 32 Magnetization vs temperature for polymorphous Fe3O4 nanoparticles

(NPs) zero-field cooled (circle symbols) and field cooled (square symbols) We

measured temperature dependence of magnetization for the Fe3O4 NP samples under

ZFC and FC conditions The DC (τm = 100 s) magnetization of the ferrofluid

samples was measured with a dc field of 100 Oe in the temperature range between 9

K and 350 K Data shown in Figure 32 for 22 nm spherical particles [Armijo

2012a]

In the entire temperature range up to 350 K the Fe3O4 NP samples demonstrated strong

ferriferromagnetic behavior as evidenced by the gap between the ZFC and FC curves

61

persisting even at 350 K (Figure 32) From the ZFC curve we can loosely estimate TB to

be ~175 K but even above that temperature equilibrium magnetization of the nanocrystal

sample was not reached and superparamagnetic behavior of the nanocrystals was not

observed

Figure 33 Ferromagnetic hysteresis loops for (a) Fe3O4 polymorphous nanocrystals

and (b) ~22 nm spherical Fe3O4 nanospheres τm= 100 s Left full sweep of magnetic

field measured at 293 K showing saturation Right enlarged loop measured at 293K

at low field [Armijo 2012a]

Strong ferromagnetic behavior of the Fe3O4 nanocrystal samples was confirmed in

magnetic hysteresis measurements Consistent with the results of dc magnetization

measurements magnetic hysteresis measurements at 293 K performed on Fe3O4

62

polymorphous NPs (Figure 33a) find large coercivity ~37 mT (~29 kAm) at 100 s

measurement time Even larger coercivity of ~119 mT (~947 kAm) was measured for ~22

nm Fe3O4 nanospheres

An additional sample which consisted of 17 nm spherical particles displayed no

hysteresis under full magnetization vs field strength (MH) sweep (Figure 34) This

demonstrates the significance of a 5 nm size difference on the magnetic properties Zero

coercivity is a typical feature of superparamagnetic materials [Cai 2007] Magnetite NPs in

this size range (10-20 nm) were the only samples shown to be truly superparamagnetic

and therefore are of the greatest interest for in vivo use

Figure 34 Superparamagnetic hysteresis loop for 17 nm spherical particles

showing no coercivity (hysteresis) thus superparamagnetic properties

63

Figure 35 MagneThermtrade inductive heater setup in its entirety Shows

MagneThermtrade function generator DC power supply oscilloscope and infrared

thermometer

Figure 36 Inside of MagneThermtrade inductive heater with front cover removed

shows inductor (coil) and capacitor (black box on right) clear hoses above and right carry

cooling water

64

322 Magnetic Hyperthermia Experiments

Magnetic hyperthermia for the Fe3O4 NP samples was tested using the nanoTherics Ltd

MagneThermtrade which operates at frequencies between 100 and 1000 kHz The

MagneTherm is frequency tunable changing capacitor and or inductors out The ranges of

frequencies and magnetic field strengths that may be achieved using this equipment were

calculated and may be referenced in Appendix III

In Figure 36 you can see the number of coils on the inductor by changing out coils

and capacitors you can tune to a range of frequencies (and magnetic field strengths) The

temperature of the sample was measured using the Omega HHTFO-A portable fiber optic

data logger thermometer version 1025 with 01 degC resolution Samples were prepared as

described in Chapters 2 and 4 coated with water soluble polymer as described in Chapter

5 and dispersed in deionized water All concentrations were 30 mgmL and sample

volumes were 5 mL The NPs compared in the following graphs were 22 nm spheres

polymorphous nanocrystals and 55 times 2 nm wires Later we investigated the heating of ~17

nm superparamagnetic spherical particles in water and in viscous (glycerol) media The

heating efficiency of the NP samples was tested at frequencies of 1111 kHz and 6292

kHz Data acquisition for hyperthermia was started at ambient temperature Figure 37a

shows the heating of NPs of various morphologies 22 nm spheres 25 nm polymorphous

NPs and 55x2 nm wires at a frequency of 1111 kHz (magnetic field of 25 mT) Figure

37b shows the heating of the same NPs at a frequency of 6292 kHz (magnetic field of 9

mT) The spherical and polymorphous particles follow a similar trend consistent with their

similar morphology and particle volume Although they do heat at the frequency of 1111

65

kHz the observed heating was relatively low Interestingly the total increase in

temperature after 40 minutes was 6 degC for spheres whereas it was only 1 degC for

Wire (black)

0 5 10 15 20 25 30 35 40 4515

18

21

24

27

30

33

36

39

42

45

48

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

Frequency f =1111 kHza)

0 5 10 15 20 25 30 35

24

26

28

30

32

34

36

38

40

42

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

Frequency f=6292 kHzb)

1111 kHz

6292 kHz

Spherical (blue)

Poloymorphus (red)

Figure 37

Magnetic hyperthermia

results for particles in the

ferriferromagnetic size

range in AC field comparing

the frequency dependence for

different MNP morphologies

22 nm spheres

polymorphous MNPs and

55x22 nm wires taken at at

the following frequencies

magnetic field strengths

(a) 1111 kHz 25 mT and

(b) 6292 kHz 9 mT

Adapted from [Armijo 2012a]

66

polymorphous NPs However as shown in Figure 38 the 17 nm spherical particles

performed significantly better at low frequency with a total temperature increase of 25 ˚C

When the frequency of the oscillating magnetic field was increased to 6292 kHz the

spheres and polymorphous NPs showed increased heating

This temperature increase can likely be attributed to a size effect However unlike

the data obtained at 1111 kHz the total temperature increase was similar for the

polymorphous NPs and NPs of spherical shape 16deg C for spheres and 15 degC for

polymorphous NPs As for the nanowires the observed trend was just the opposite The

total temperature increase at 1111 kHz after 40 minutes was a remarkable 30deg C and

notably saturation of the temperature was not reached in this timeframe At 6292 kHz

however the increase in temperature generated by the wires was much less than the one

obtained by spheres and polymorphous NPs representing the total temperature increase of

4 degC

It has been shown that the transition from ferromagnetic to superparamagnetic

behavior is associated with the change of the loss mechanism and accordingly of the

heating effect of magnetic NPs in hyperthermia experiments Hysteresis losses dominate in

ferromagnetic nanocrystals whereas heat production in superparamagnetic ones is due to

relaxation losses Since the blocking temperature TB explicitly depends on the

measurement time τm (inversely proportional to the frequency of the oscillating magnetic

field) superparamagnetic NPs as measured in DC magnetization experiments become

ferromagnetic at sufficiently high frequencies gt 1τN (or gt 1τ where τ = τΝτΒ (τΝ + τΒ) if

both Neacuteel and Brownian relaxation mechanisms are present) and generate heat due to

67

hysteresis losses With the Fe3O4 NP samples demonstrating strong ferromagnetic behavior

in DC magnetization and hysteresis measurements hysteresis losses are expected to be the

main mechanism of heating in the operating frequency range of our hyperthermia

experiments

We estimated the Neacuteel relaxation time τN at room temperature for the Fe3O4

polymorphous NPs (Figure 31) as follows

τN = τ0exp(EbkT) (34)

Where Eb is the magnetic anisotropy energy barrier k is the Boltzmann constant and

τ0=10minus10 s is the attempt time Eb is related to the blocking temperature TB as Eb =

kTBln(τmτ0) = 276 kTB and we arrive at τN = τ0exp(276 TBT) for the Neacuteel relaxation

time At T = 300 K τN asymp 0001 s At the frequencies of interest ω gtgt 1τN which is far

from the relaxation resonance the Neacuteel relaxation losses saturate at a level that is

negligible for large enough τN [Hergt 1998] Therefore we will interpret our results based

on the mechanism of hysteresis losses prevailing for NPs in this size range When

hysteresis losses are the main heating factor heating power is proportional to the area of

the hysteresis loop and to the frequency of the applied magnetic field An approximately 6-

fold increase in the heating power is expected when the frequency is changed from 1111

kHz to 6292 kHz The observed increase in heating power from the Fe3O4 polymorphous

NPs and nanospheres is not that large We note however that both the frequency and

amplitude of the magnetic field were changed in our experiments and the magnetic field

strength was reduced from 25 mT at 1111 kHz to 9 mT at 6292 kHz which can explain

68

the heating power increase not being proportional to the frequency for the Fe3O4

polymorphous NPs and nanospheres The higher temperature increase of 6 degC for the

spheres compared to 1 degC for the polymorphous NPs at 1111 kHz can be explained by a

significantly larger area of their hysteresis However the difference in the specific heat

production between the spherical and polymorphous NPs at 6292 kHz is not significantly

pronounced

We consider hyperthermia experiments with nanowires separately as their

morphology differs dramatically from that of polymorphous NPs and nanospheres and may

be the decisive factor Fine magnetite particles of needle shape with high aspect ratio have

been investigated previously by [Hergt 1998] High potential for hyperthermia was noted

there for the particles that possess very high shape anisotropy and hence high-energy

barrier for remagnetization resulting in a wide hysteresis and high hysteresis losses It was

concluded however that strong magnetic fields very often unacceptable for human

patients are required to fully utilize their potential Very strong nonlinear dependence of

the hysteresis loss on the strength of the applied magnetic field was reported Comparison

was made among particles of different shapes and it was found that needles were by far

superior when applied magnetic field exceeded ~35 kAm (4389 mT) while below that

value the magnetic field was not strong enough to open the hysteresis loop in needles and

their hysteresis losses were by several orders of magnitude lower compared to particles of

other shapes with low aspect ratio We expect similar effects to be observed in nanowires

that are characterized with even higher aspect ratios of their shape We believe that our

results for hyperthermia in nanowires can be explained by similar superlinear dependence

69

of their hysteresis loss on the magnetic field strength with that superlinear dependence

being much stronger than mere proportionality of the heating power to the frequency of the

applied magnetic field

Figure 38 Hyperthermia results for superparamagnetic NPs having

an average diameter of 17 nm in water and waterglycerol mixture

having high viscosity Data was taken at a frequency of 1111 kHz

with a magnetic field of 25 mT in the inductor

We note that the remarkable 30 degC temperature increase was observed in nanowire

sample at 111 kHz and magnetic field of 25 mT (196 kAm) which is very close to the

typical values used in medical treatments 100 kHz and 20 mT [Wust 2006] [Mehdaoui

2011] Smaller spherical particles having an average diameter of 17 nm were shown to

perform comparably (see Figure 38) at low frequency In order to verify our findings in

viscous media an additional sample consisting of 16-20 nm spherical magnetite particles

0 10 20 30 40 5020

30

40

50

Tem

per

ature

[d

egre

es C

elsi

us]

Time [min]

Fe3O

4 NPs in water

Fe3O

4 NPs in glycerol mixture

70

was characterized Figure 38 shows the summary of hyperthermia experiments with the

SPIONs dispersed in water and in aqueous glycerol (50 ww) mixture six times more

viscous than water alone

Additionally no temperature increase was observed for the control experiments

using DI water under the same AC field and under identical experimental conditions with

no NPs (not shown) With this sample we were able to achieve a total temperature increase

in excess of 25 degC and the initial fast heating rate of ~4 degCmin decreased to ~02 degCmin

after 12 minutes This confirms the heat-generation is a result of the magnetic AC energy

absorption by the magnetic component of the ferrofluid samples Due to the similar heating

trends in water and high viscosity aqueous glycerol we attributed most of the heating

losses to a Neacuteel process This further verifies our susceptometry findings (recall that NP

samples below 20 nm in size displayed no coercivity (hysteresis losses) in MH curves)

323 AC Susceptometry

Measurements of the frequency-dependent volume susceptibility in the frequency range

1 Hz to 100 kHz were performed using the DynoMagreg (IMEGOAB Sweden) with a

frequency range from 1 Hz to 200 kHz a resolution magnetic moment of 3times10-11 Am2 and

excitation amplitude of 05 mT The ferrofluid magnetite (Fe3O4) sample 1 and 2 in water

solvent at a concentration of 130 M was measured using a 200-microL sample Measurements

were performed on a sample which consisted of the base ferrofluid colloidal suspension of

magnetite (Fe3O4) particles having spherical morphology of mean particle diameter 15 nm

in deionized water solvent with succinylated PEG as a capping agent

71

Susceptometry data verify the magnetic hysteresis measurements in which we

found that the sample was superparamagnetic at room temperature The susceptometry

measurements demonstrate a single peak which we attribute to a Neacuteel process in which

τN=129x10-6 ms Assuming the superparamagnetism the Neacuteel relaxation time of moment

rotations activated by thermal fluctuation is given by Eq (43) and (44) with where V =

1767x10-24 m3 for the 15 nm diameter spherical SPIONs When kBT gt KuV the magnetic

moment flips during the measurement time demonstrating zero coercivity Presently the

effective anisotropy energy (Ku) of the iron oxide sample may be estimated to be 42 times 105

ergscc by the relation KuV = 25kBTB (assuming TB = 215 K) [Zhang 2010] higher than the

Ku of bulk Fe3O4 (Ku = 64 times 104) due to additional anisotropies which agrees with the

findings of [Zhang 2010] for particles of similar size The effective anisotropy energy of

the iron nitride sample was calculated to be 56 x105 ergscc A reference value for bulk

Fe16N2 is not presently available in the literature The real part of the susceptibility (χrsquo)

values for both samples was greater than zero a typical feature of ferriferromagnetic

materials Despite this the χrsquo value for iron nitride is two times higher than the value for

iron oxide As expected the real part of the susceptibility (χrsquo) curve remains above zero for

both materials a typical feature of ferriferromagnetic materials

We characterized the magnetic properties of iron oxide NPs of various morphologies in the

paramagnetic to ferromagnetic size range thus allowing for further functionalization and

drug conjugation DC magnetization and AC heating power (hyperthermia characteristics)

72

of the Fe3O4 NPs in water have been studied The Fe3O4 NPs samples having a mean

diameter gt20 nm demonstrated strong ferromagnetic behavior and hysteresis losses were

identified as the main mechanism of heating in hyperthermia experiments Whereas the

NP samples having a mean diameter of 15-17 nm demonstrated superparamagnetism and

Neacuteel relaxation appears to be the dominant heating mechanism Our hyperthermia data

shows that all three NP morphologies spheres polymorphous NPs and wires are good

candidates for thermotherapy Significant heating was observed well within the limits for

oscillating magnetic field parameters established for biological applications The observed

temperature increase for 22 nm Fe3O4 nanospheres at 1111 kHz and 25 mT after 40 min

was 6 degC If the corresponding temperature increase took place from normal human body

temperature (366 degC) as a starting point it would bring the local temperature up to 426

degC which is right within the desirable temperature limits for the applications of medical

hyperthermia (41ndash45 degC) [Hergt 2006] In the waterglycerol study we showed that the

initial fast heating rate of ~4 degCmin decreased to ~02 degCmin after 12 minutes (when the

temperature reached 45 degC) Although the heating rate is not linear and appears to slow

down as a function of temperature (when the slope between one point and the next is

considered) the data points considered to be within the initial fast heating rate are the data

points of interest for medical hyperthermia (36-42 degC) Of special interest for hyperthermia

applications the nanowires demonstrated a remarkable 30 degC temperature increase and the

superparamagnetic (~17 nm) spherical particles demonstrated a 25 degC temperature increase

under magnetic field conditions that were very close to (or lower than) the typical values

used in medical treatments

73

Chapter 4

SYNTHESIS AND CHARACTERIZATION OF HIGHLY

SUPERPARAMAGNETIC IRON NITRIDE

NANOPARTICLES (Fe16N2)

The Fe16N2 (martensite) phase is of interest for our application and many others not just

because it is in-fact the most magnetic material in the world [Kim 1972] [Ji 2010] but

also because it is free from toxic cobalt and the costly rare-earth elements Using a stronger

magnet has many benefits for gradient-guided drug delivery one obvious one being an

anticipated increased in the active transport rate due to a stronger interaction with the

external applied field Zero-valent iron is another highly magnetic phase which serves as

an intermediate in the synthesis of iron nitride described in Section 43 Samples of zero-

valent iron were taken from this procedure for use in bacterial sensitivity studies described

in Chapter 6

Over 20 years ago the iron nitride phase having the empirical formula Fe16N2 and

the specific phase of αrdquo was claimed to possess a giant saturation magnetization (Msat)

[Metzger 1994] [Ji 2010] At that time it was well established that iron cobalt having the

formula Fe65Co35 composed the strongest magnet in the world [OrsquoHandley 2000]

However it was suggested that Fe16N2 might possess a saturation magnetization far

exceeding the iron-cobalt alloy [Metzger 1994] The αrdquo-Fe16N2 phase was first discovered

74

prior to 1950 the procedure having been published in 1951 when researchers initially

characterize the temperature dependence on the formation of different phases of iron

nitrides [Jack 1951] In this paper phase diagrams for iron-nitrogen systems were

proposed and the αrdquo-Fe16N2 phase was described as a metastable crystal formed from rapid

quenching of γ-FeN (austenite) [Jack 1951] Unfortunately the magnetic properties were

not measured and it was not until 1972 after the giant saturation magnetization was

finally measured that interest in this material finally peaked [Kim 1972] Since then there

have been many attempts to synthesize this material as the sole phase in a crystal system

however typically these findings report the presence of a phase of αrdquo-Fe16N2 mixed in with

other phases of iron nitride iron oxide or alpha iron [Comstock 2002] Even now more

than 40 years later a need still existed to engineer single-phase crystals on the large-scale

This method was developed in the interest of solving that problem while promoting the use

of green chemistry methods

41 Introduction to Fe16N2

Iron nitride magnets offer a low cost however superior alternative to rare earth

magnets In addition the questionable stability of rare earth magnets on the nanoscale is

avoided in the binary iron phases It has been shown that the low nitrogen content phases

such as γ-Fe4N ε-Fe2ndash3N αrsquo-Fe8N and αrsquorsquo-Fe16N2 are ferromagnetic compounds having

exceptionally well characterized stoichiometry [Wang 2003] and electronic properties [Eck

1999] are attractive compounds for magnetic functional nanomaterials [Grachev 2001]

The synthetic routes for commercial production are also well-documented In order to

75

create a timeless protocol for large-scale manufacturing of these nanomaterials we must

anticipate the future regulations requiring green-chemistry procedures for the production of

all synthetic materials By making minor though profound modifications to known

methods using known physical and chemical properties we can be environmentally

conscious while continuing to engineer superior materials Fe16N2 being a phase of iron

nitride being a superior material reported to possess a very high magnetic moment even

greater than pure iron [Cadogan 1997] and iron cobalt [Hattori 2001] The saturation

magnetization of Fe16N2 powder with the largest specific surface area at room temperature

was previously reported to be a striking 200 emug with a maximum coercive force 2250

Oe [Hattori 2001]

42 Theory

The Fe16N2 phase is considered ferromagnetic [Wang 2003] meaning it consists of an

array of atomic moments exhibiting very strong interactions These interactions stem from

electronic exchange forces quantum mechanical phenomenon resulting from the relative

orientation of the electron spins These spin orientations result in either parallel or

antiparallel atomic moment alignment Exchange forces are exceptionally large in

magnitude on the order of 100 T or 1x108 times the strength of the Earthrsquos magnetic field

[Wang 2003] It is important to note that this field is detectable with simple low-cost

equipment Ferromagnetic materials exhibit parallel alignment of moments resulting in

large net magnetization even in the absence of a magnetic field The αrdquo-Fe16N2 phase in

particular being the most important new material of interest for high-density magnetic

76

recording due to its exceptionally high magnetic moment which as previously mentioned

is larger than α-iron [Sugita 1991] [Bao 1994] The coercivity and saturation

magnetization (Msat) of these phases incorporated into thin films have been investigated by

many researchers

The saturation magnetization Msat of the other ferromagnetic phases is generally

lower than that of the α-Fe except for the phases of αrdquo-Fe8N and αrdquo-Fe16N2 which have

been demonstrated by the above-mentioned researchers Although others appear to have

achieved a phase of αrdquo-Fe16N2 mixed with other phases we believe that their ultra-high

temperature synthesis to be unfavorable to the stability of the αrdquo-Fe16N2phase and the sole

parameter (aside from accidental oxidation) being responsible for the existence of mixed

phase crystals Producing these crystals at a temperature higher than 400deg C facilitates

formation of the more thermodynamically stable phases of iron nitride γ-Fe4N and ε-Fe3N

These other phases have signature saturation magnetizations lower than that of α-Fe which

makes the Msat measurement an essential tool for differentiating between phases [Wang

2003]

43 Synthesis of Iron Nitride (Fe16N2) and Zero-valent (Fe0) Iron Nanoparticles

This green-chemistry procedure consisted of five-steps 1) synthesis of the iron oleate

precursor complex 2) synthesis of the iron oxide NPs 3) oleic acid cap removal and

purification of iron oxide NPs 4) reduction to α-iron and 5) nitrogenation under ammonia

gas

77

431 Materials

(99) were purchased from Alfa Aesar n-dodecane (gt99) and hydrochloric acid (1N

certified) were purchased from Fischer Scientific sodium oleate (gt97) was purchased

from Tokyo Chemical Industry Co UHP hydrogen gas (999999 ) and UHP ammonia

gas (999999 ) were purchased from Matheson Tri-Gas All chemicals were used as

received without purification Chemicals and their physical properties may be referenced

in Appendix I

The precursor was iron oleate (iron(II III) [(9Z)-9-octadecenoate]n) where n is the

coordination number of iron and could form a monomer dimer or trimer [Bronstein

2007] [Palchoudhury 2011] as described in detail in Chapter 2 Iron oleate is produced in

our laboratory using a modified procedure published elsewhere [Bronstein 2005] The

formation of the complex was verified with UV-Vis-NIR spectroscopy The iron oleate

complex was formed from the combination of sodium oleate salt (sodium (9Z)-9-

octadecenoate) and iron(III) chloride hexahydrate (FeCl3middot6H2O) In a standard reaction

675 g of FeCl3middot6H2O was combined with 25 mL of deionized water and vacuum-filtered

through 022 μm filter paper The mixture was then combined with 2435 g of sodium

oleate in a three-neck round-bottom flask 150 mL of a stock solution consisting of a 246

mixture of deionized water ethanol and hexane was added to the flask Under argon flow

the mixture was vented and filled for three one-minute intervals in order to remove all

78

oxygen from the reaction flask The solution was the slowly (5deg Cmin) heated to 50deg C

under vigorous stirring Once the solid sodium oleate salt had completely melted and the

reflux had begun (around 50ndash60deg C) the temperature was further increased (3 degCmin) to

70deg C and the flask was kept at this temperature for four hours ensuring that the total

reflux time was 4 hours The mixture was then cooled to 60deg C and washed three times

with a 11 mixture of hexane and deionized water in a separatory flask The organic layer

was placed in a rotary evaporator (Rotovap) with the water bath set at 30deg C until the

hexane and ethanol were evaporated away Wet iron oleate complex (the hydrate form) as

obtained from the procedure described above was a reddish-brown highly viscous liquid

The precursor was further purified with ethanol acetone hexane and water washes and

dried in the oven at 70deg C for 24 hours After drying the product was a dark-brown waxy

solid

433 Synthesis of Iron Oxide Precursor

Subsequently iron oxide nanoparticles were prepared using a modification of a procedure

published previously [Park 2004] 148 mmol (5 g) of iron oleate were combined with 16

mL (50 mmol) of oleic acid and 1315 g (465 mmol) of n-docosane (for spherical

particles) or a molar equivalent of eicosane (for cubic morphology) The mixture was

slowly (3 degCmin) heated to 50 degC under argon flow and vigorous stirring Once the

reactants had dissolved the temperature was further increased to 370deg C with a heating

rate of 30deg Cmin For 20 nm particles (plusmn14 nm) the mixture was allowed to reflux for 30

79

minutes For larger particles the reflux time may be extended with an average growth rate

of 16 nm per minute The particles were washed three times with hexane and acetone

434 Removal of Oleic Acid Cap

As discussed in Chapter 2 the iron oxide NPs come out of synthesis capped with oleic

acid The presence of the cap may introduce unwanted contaminants into the new iron

nitride crystal and must be removed Either of two methods may be used to remove this

cap Chemically the coating is removed by adding 1M solution of hydrochloric acid drop-

wise until the carboxyl group of the oleic acid is protonated (pHlt5) and detaches from the

NPs The uncapped particles are then isolated using the standard methanol and hexanes

extraction An alternative method which does not require hazardous reagents is to simply

anneal the oleate coated NPs above the melting point of oleic acid allowing the capping

agent to evaporate off (Tgt 250ordm C) Annealing is typically done for 20-30 minutes

435 Production of Zero-valent Iron Nanoparticles

The iron oxide NP powder sample is reduced under UHP hydrogen gas overnight at 300-

350 degC Then the sample is exposed to ammonia gas for 2-24 hours at a temperature

between 250-400 degC This temperature is below the ammonia decomposition temperature

however recall that iron catalyses the decomposition of ammonia making the lower

temperature sufficient A sample of zero-valent iron NPs was preserved for bacterial

sensitivity studies reported in Chapter 6 For this study we produced zero-valent iron using

a hydrogen gas reduction (above) however other options exist for the synthesis of zero-

valent iron NPs Zero-valent iron nanoparticles may be produced from iron pentacarbonyl

80

in sonicated in a medium molecular weight alcohol under air-free conditions In addition

zero-valent iron NPs may be produced by mixing iron oxide NPs with a molar equivalent

of sodium borohydride then annealed in a high boiling point inert hydrocarbon under inert

gas for 30 minutes

436 Production of Iron Nitride Nanoparticles

Iron nitride NPs were produced using zero-valent iron nanoparticles as a precursor Any

capping agents are removed as described in Section 434 Then the sample is exposed to

ammonia gas overnight at a temperature between 250-400 degC for 2 to 24 hours

44 Structural Characterization of Iron Nitride Nanoparticles

Iron nitride NPs were characterized by XRD and TEM magnetic characterization was done

by SQUID magnetometry For structural characterization TEMEDS samples were

prepared by placing a drop of the colloidal solution onto a 200-mesh carbon-coated copper

grid The solvent was allowed to evaporate away thus fixing the sample on the grid The

JEOL-2010F transmission electron microscope was equipped with an OXFORD Link ISIS

energy dispersive spectroscopy (EDS) apparatus which determined elemental

composition The electron beam was focused on a single nanocrystal and the characteristic

X-ray peaks specific to each element were identified using the OXFORD Link ISIS

software EDS showed the presence of iron and a small peak corresponding to nitrogen

The iron binary phase and crystal structure were determined using a Rigaku Smartlabreg X-

Ray Diffractometer (XRD) with a Cu Kα source (0154 nm) and attached monochromator

81

It is important to note also that the TEM analysis was difficult due to the strong magnetic

interaction between the material and the electron beam The strong magnetic properties of

the sample caused the beam to oscillate interfering with the analysis Both the XRD and

TEM show a body centered tetragonal (BCT) crystal system This system would be

expected for Fe16N2 thus differentiating it from iron or iron oxide Excellent crystallinity

is demonstrated in the TEM image shown in Figure 41

Figure 41 High-resolution TEM image of Fe16N2 NP showing crystallinity

Figure 42 shows the XRD spectrum for the uncapped iron nitride NP sample The Jade

softward automatched the spectrum to the iron nitride (martinsite) phases Fe8N

ICDDICSD card number 01-070-6150 and Fe16N2 ICDDICSD card number 01-078-

1865 both tetragonal crystals with lattice constants a=571 Å b=571 Å c=6016 Å and

82

a=572 Å b=572 Å c=629 Å respectively The scan also reveals some magnetite

(Fe+2Fe2+3O4) ICDDICSD card number 00-019-0629 which is a cubic crystal with lattice

constants a=838 Å b=838 Å c=838Å This iron oxide likely resulted from surface

oxidation of the uncapped NP sample which was set onto the slide using ethyl alcohol

chloroform and heat

Figure 42 XRD spectrum for iron nitride NPs taken with CuKα having a 0154 nm

wavelength and using attached monochromator

45 Magnetic Characterization of Iron Nitride NPs

We measured temperature dependence of magnetization for the Fe16N2 NP samples under

zero-field cooled (ZFC) and field cooled (FC) conditions The DC (τm = 100 s)

magnetization of the samples was measured with a DC field of 100 Oe in the temperature

range between 10 K and 350 K In the entire temperature range up to 350 K the Fe16N2 NP

samples demonstrated strong ferromagnetic behavior as evidenced by the gap between the

83

ZFC and FC curves persisting even at 350 K From the ZFC curve we can loosely estimate

TB to be ~350 K but even above that temperature equilibrium magnetization of the NP

sample was not reached Superparamagnetic behavior of the nanocrystals was observed in

this sample but not observed in larger samples (gt20 nm)

Figure 43 Magnetization vs temperature for Fe16N2 NPs (blue) compared to magnetite

(red) Zero-field cooled (lower curves) and field cooled (upper curves) Magnetization measured

with a DC field of 100 s We measured temperature dependence of magnetization for the Fe16N2

NP samples under ZFC and FC conditions The DC (τm= 100 s) magnetization of the ferrofluid

samples was measured with a dc field of 100 Oe in the temperature range between 9 K and 350 K

Iron nitride appears to block around 350 K whereas iron oxide blocks around 210 K The

elevated blocking temperature of iron nitride makes it attractive for many applications that

presently require supercooling

Superparamagnetic behavior of the Fe16N2 NP samples was observed in magnetic

hysteresis measurements Consistent with the results of DC magnetization measurements

84

magnetic hysteresis measurements at 293 K performed on Fe16N2 NPs find no coercivity

verifying that the magnetic hyperthermia results from a Neacuteel process We were unable to

find saturation Msat with the field strengths presently attainable by the equipment (Figure

45) Extrapolating the line gives a loose estimate of Msat ~ 100 emug The DC (τm = 100

s) magnetization of the ferrofluid samples was measured with a dc field of 100 Oe in the

temperature range between 9 K and 350 K using a Quantum Designtrade magnetic property

measurement system (MPMS) superconducting quantum interference device (SQUID)

magnetometer

Figure 44 Comparison of

hysteresis loops of

nanocrystalline samples of

iron oxide (red) and iron

nitride (blue) of similar

grain size showing the

significantly stronger

magnetic properties of iron

nitride Upper image shows

entire hysteresis loop of iron

nitride Lower image is a

close-up of the same

showing hysteresis loop of

iron oxide

-50E4 00 50E4-1

0

1

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

-50E4 00 50E4-80

-60

-40

-20

0

20

40

60

80

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

85

00 20E4 40E40

20

40

60

80

100

(

Am

2k

g)

H (mT)

Figure 45 Close up of hysteresis curve (positive axis) showing

that Msat was not reached in the 50 T applied field at room

temperature

Iron nitride NPs were synthesized via solvothermal and solid-gas phase reaction in which

iron oxide powder as an intermediate The composition structure was characterized using

x-ray diffraction (XRD) Saturation magnetization (Msat) and coercivity of NPs was

determined using superconducting quantum interference device (SQUID) We found that

the successful formation of the Fe16N2 phase is strongly dependent on temperature and

reducing agent selection Fe16N2 exhibits saturation magnetizations larger than that of α-Fe

86

The highly magnetic Fe16N2 phase of iron nitride may be produced in high yields having

good resistance to oxidation exceptionally high blocking temperatures and depending on

the precursor some control of particle morphology [Armijo 2012a] This material has a

high magnetic moment though it contains no costly rare earth elements or toxic cobalt

Additionally the green chemistry procedure produces minimal toxic waste It still remains

unclear whether this material is safe for use in vivo

87

Chapter 5

HYDROPHILIZATION AND BIOCONJUGATION

All charged (metal) nanoparticles (NPs) require an organic or non-organic polymer shell

to prevent aggregation potential oxidation and allow for further conjugation In the case

of ferriferromagnetic NPs the coating of magnetic nanoparticles (MNPs) must also be

sufficient to prevent magnetic interactions between particles In general to keep the

particles from interacting magnetically in such a manner that they agglomerate the polymer

shell should have a thickness equal to at least half the radius of the magnetic NP In the

case of superparamagnetic NPs no magnetic interaction in the absence of an external

magnetic field occurs When the application is biomedical the organic coating or polymer

shell must be water-soluble in order to be used in the aqueous biological environment

Many FDA-approved polymers are available for use such as poly(lactic-co-glycolic acid)

(PLGA) and polyethylene glycol (PEG) are often chosen simply because of their

confirmed safety rather than their physical or chemical properties Some other attractive

options are natural polymers which are anticipated to be biocompatible simply due to their

existence in other biological systems Many are produced by plants algae or fungal

species and must simply be purified for use [Lehr 1992] [Dang 2006] These are typically

water-soluble and happen to possess many useful functional groups which allow for further

88

conjugation to a gene or drug Any organic molecule or polymer having a negatively

charged terminal functional group (OH- or COOH- are ideal) may be used to

electrostatically bind a positively charged NP The stronger charge on the carboxyl group

will hold stronger especially in high salinity The colloidal stability of the NPs depends on

the ability of the polymer to maintain a strong ionic interaction with the NP as charged salt

ions can easily electrostatically bind to the charged functional groups on an organic

molecule or polymer Shelf life as well as the systemic half-life of nanomaterials are

strongly dependent on and are highly controllable by this one parameter [Braatz 1993]

[Prencipe 2009]

NOTE The terms ldquoorganic moleculesrdquo or ldquoorganic polymersrdquo were not used to describe potential

NP passivation coatings because silicon-based polymers may also be used We use standard

chemistry terminology in which ldquoorganicrdquo refers to ldquocarbon-basedrdquo

51 Experimental

Prior to engineering polymer or other organic coatings the stabilizing agent oleic acid

must be removed from the surface of the NPs Afterwards water soluble polymers

presenting additional functional groups for bioconjugation may be attached We

investigated the FDA approved polymer PEG as well as the naturally occurring

biodegradable capping agents citrate and alginate for this application These capping

agents present carboxyl terminal groups for conjugation to the amine group of the

tobramycin molecule

89

511 Materials

m-PEG 5000 (methyl-terminated PEG) powder and sodium alginate from green algae

(medium molecular weight) succinic anhydride (gt99) phosphate buffered saline (PBS)

powder and TRIS hydrochloride (PharmaGrade) were purchased from Sigma Aldrich

anhydrous citric acid (995 ) chloroform (999 ) hexane (99) acetone (99) and

hexanes (99) pyridine (99) methanol (99)were purchased from EMD Chemicals

Inc 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (cat 22981)

and N-hydroxysulfosuccinimide Sulfo-NHS (cat 24510) were purchased from

ThermoFisher Scientific Chemicals and their physical and chemical properties can be

referenced in Appendix I

The NPs come out of synthesis described in Chapter 2 capped with oleate (oleic acid) As

discussed earlier in Chapter 2 the major reagent is iron oleate an oleate molecule acting as

the organic carrier facilitating high temperature (solvothermal) crystal growth Since iron

oleate served as the organometallic (metal carbonyl) complex by which the iron was

delivered to the iron-oxide crystal [Bronstein 2007 Palchoudhry 2011 Armijo 2012a] The

oleic acid on the NP surface consists of a hydrocarbon chain and a single carboxyl group

that is electrostatically bound to the metal oxide NP (Figure 51) it has no functional

groups for drug conjugation and is not water soluble Due to these significant limitations

many researchers simply coat the NP with an additional water-soluble polymer (over the

existing oleate coating) [Liu 2012] in the case of Yb NPs Other groups have reported the

90

encapsulation of oleate-capped magnetite with a polystyrene layer [Ramirez 2003] or

chitosan [Shete 2014]

In order to ensure direct bacterial contact with the NP as well as sufficient charge

shielding for our application the oleic acid cap was removed using a hydrochloride

solution wash at a pH of 1 The carboxyl group of the oleic acid becomes protonated at a

pH around 5 because pKa is ~54 Fatty acids like oleic acid contain long carbon chains

and typically have Ka values near 1 times 10-5 (pKa ~ 5) The oleate cap was removed with a

hydrochloric acid wash The process of removing the cap is governed by the Henderson-

Hasselbalch equation [Henderson 1908a Henderson 1908b] which derives the pH as a

measure of acidity from pKa (the negative log of the dissociation constant) and the ratio of

the concentrations of an undissociated acid and its conjugate base [Brown 2012]

119901119867 = 119901119870119886 + 11989711990011989210([119860 minus]

[119867119860] (1)

where [A-] is the conjugate base (oleate anion) concentration and [HA] is the organic acid

(oleic acid in our case) concentration

The pKa is given by [Kanicky 2002]

119901119870119886 = minus11989711990011989210([1198673119874+][119860minus]

[119867119860] (2)

where [H3O+] is the hydronium ion concentration

When the pH is equal to the pKa there will exist in solution an equal amount of

protonated (acid) and deprotonated (conjugate base) molecules ([A-][HA] = 1 A typical

carboxylic acid has a pKa between 4 and 5 [154] however titration experiments have

shown that oleic acid has a much higher pKa of 985 [Kanicky 2002] An organic acid will

91

be significantly deprotonated in a solution if its pKa is two or more units lower than the pH

of the solution Although the reaction would have proceeded at a higher pH we used an

HCl solution having a pH of 1 to ensure a more rapid protonation and thus detachment of

oleate from the iron-oxide NP at 25 degC Inserting our pH value of 1 and the oleate pKa of

985 into equation 1 returns a value of 69743 for the ratio [HA][A-]

NOTE THIS PROCEDURE WAS DESIGNED USING BASIC

ORGANIC CHEMISTRY METHODS SPECIFICALLY THE

PKA OF THE OLEIC ACID MOLECULE WHICH IS KNOWN

AND CAN BE DETERMINED EXPERIMENTALLY WE CAN

DEDUCE FROM THE PKA OF THE TERMINAL CARBOXYL

GROUP THE PH AT WHICH IT IS PROTONATED AND WILL

DETACH FROM THE NANOPARTICLE THE METAL (M+)

BEING REPLACED BY THE HYDROGEN ION (H+) AND

BALANCING OUT THE NEGATIVE CHARGE ON THE

TERMINAL CARBOXYL GROUP

The oleate ion is the ionized form of oleic acid [Raymond 2010]

CH3 (CH2)7CH=CH(CH2)7CO2H + H2O CH3(CH2)7CH=CH(CH2)7CO2- + H3O+

oleic acid + water oleate ion + hydronium ion

119870119886 = [119900119897119890119886119905119890 119894119900119899][1198673119874+]

[119900119897119890119894119888 119886119888119894119889] = 1 times 10-5 pKa = 5

Upon reaching the pH which favors formation of the carboxylic acid group the

yellow-tinged transparent oleic acid could be visually observed to fall out of solution

Oleic acid is soluble in methanol so a standard methanolhexanes extraction removes the

oleic acid from the bare iron oxide NPs The NPs were separated in a 95

92

hexanesmethanol mixture in which the methanol solvated the oleic acid Acidic solution

slows oxidation dramatically so there is no need to use inert gas flow for this procedure

Once the two phases are separated the oleic acid is removed using a separatory funnel and

the NPs are isolated via centrifugation The NPs are then redispersed in chloroform

Capping should be done immediately to maintain passivation

Figure 51 Removal of oleate cap acid-wash facilitated removal of oleate cap

leading to uncapped NP and free oleic acid (the protonated form of the oleate

ion) Image by L Armijo 2014

Figure 52 Citric acid molecule the

protonated form of the citrate ion

3D image (upper image) and carbon

skeleton (lower image) drawn with

MarvinSketch

93

513 Citrate Capping

Citrate was the first biodegradable capping agent investigated The citrate molecule has 3

carboxyl groups and one hydroxyl group (Figure 52) available for attachment to the NP

and further conjugation to drug Attachment to a specific group cannot be controlled at

present Citrate was added to the NP solution in chloroform ensuring a (pH gt 6) by adding

drop-wise amounts of 1M sodium hydroxide solution to ensure that a free carboxyl group

is available for attachment to the colloidal NPs The sample was then sonicated and

washed The sample remained stable in water for more than one year when an excess of

citrate was used to ensure complete coverage The citrate cap may also be removed using

an acid wash which converts citrate to citric acid as in Figure 51

514 Alginate Capping

Because a major component of the P aeruginosa biofilm is alginate we anticipated that

alginate capping should facilitate transport through biofilms based on the principle that

ldquolike dissolves likerdquo The alginate monomer shown in Figure 53 has 2 carboxyl groups

and 4 hydroxyl groups contributing to electronegativity and facilitating attachment to the

positively-charged NP As with application of the other polymers alginate was added to

the NP solution in a basic aqueous chloroform solution (pH gt 6) to ensure a free carboxyl

group for attachment to the colloidal NPs The sample was then sonicated for 30 minutes

to an hour in order to keep the particles from agglomerating and then the NPs were

washed in water and removed via centrifugation This sample also remained stable in water

for more than one year when an excess of alginate was used to ensure complete coverage

94

Figure 53 Monomer of alginic acid the protonated form of the alginate ion

showing 3D image (left) and carbon skeleton (right) drawn with MarvinSketch

515 Polyethylene Glycol Succinylation

In order to enhance the binding affinity of PEG-OH to the NPs we further engineered

mPEG using a simple succinylation procedure mPEG-5000 was chosen as its use is

thoroughly documented for biomedical applications Succinylated PEG was produced in-

house from the PEG-OH terminal of mPEG (methyl terminated polyethylene glycol) in a

process during which the terminal hydroxyl group was converted by a small chain

extension to a more electronegative carboxyl group which enhances binding affinity and

thus promotes long-term colloidal stability even under increasing salinities In order to

keep a sealed pyridine bottle under close to atmospheric pressure 25 mL of nitrogen gas

were drawn up into a syringe through the septum of a nitrogen-filled three-neck flask

connected to the Schlenk line and injected into the pyridine bottle After injection 25 mL

of anhydrous pyridine (the solvent) were drawn up from the bottle and injected into the

nitrogen-filled flask The temperature controller was set to 50 degC the temperature at which

the solid mPEG dissolves Subsequently 25 g of succinic anhydride were added to the

three-neck flask This reaction process lasted for one hour at 50 degC The addition of

95

pyridine was repeated four more times using the same methodology as described above

and the reaction was allowed to continue for another 2 hours at 50 degC Pyridine was then

removed using three DI water washes using the rotary evaporator The material was then

re-dissolved in water and placed in 1 kDa cutoff dialysis tubing in a 1 L beaker of DI

water The DI water in the 1 L beaker was replaced after 2 4 and 8 hours The final

material is a light brown substance that originally was thought to be a contaminant of the

original synthesis

Figure 54 Dialysis of succinylated polyethylene 5000 glycol using

dialysis tubing (under stirring in deionized water at room temperature)

for removal of precursors and unreacted reagents

Figure 55 PEG Succinylation overall reaction Shows the initial PEG molecule

having the characteristic terminal hydroxyl group and the product of PEG

succinylation procedure now having a more electronegative terminal carboxyl

group

96

Figure 56 Dried and purified succinylated polyethylene glycol

(PEG) 5000 powder

In the first synthesis most of the succinylated PEG was lost through repeated

efforts to purify the material through crystallization and the use of activated charcoal

Once this was realized a second synthesis was performed by the original protocol that had

better results Shown in Figure 54 is the purification through dialysis with 1000 Dalton

(Da) dialysis bags The succinylated 5000 Da PEG is trapped inside of the dialysis tubing

while the lighter precursor materials are able to diffuse out of the bag into the surrounding

fluid called the dialyte After dialysis purification the mixture was dried with the rotary

evaporator with the water bath set to 50 degC the same temperature of synthesis The dried

succinylated polyethylene glycol was still liquid at this temperature and becomes a brown

waxy solid when cooled to room temperature as shown in Figure 57 Absorbance

measurements were performed on the succinylated polyethylene glycol using a Cary 5000

UV-VIS spectrophotometer It is known that iron oxide is a semiconductor having a band

gap of 22 eV The measurement of 1 by weight solution was performed from 200 to 700

97

nanometers which showed increasing absorption below 600 nm with a shoulder peak at

250 nm This data is especially important if this material is used in the future as a coating

for optically active materials such as quantum dots The graph of the absorbance curve is

shown below Figure 57

200 300 400 500 600 700

00

02

04

06

08

10

12

14

Abs

orba

nce

[OD

cm

]

Wavelength [nm]

1 Syccinylated mPEG 5000 in DI H20

Figure 57 Absorbance spectra for succinylated polyethylene

glycol (PEG) 5000

516 Polyethylene Glycol (PEG) Capping of Iron Oxide Nanoparticles

After succinylation PEG capping was performed using a modified procedure from

[Shtykova 2007] for our work we only used PEG as opposed to a combination of costly

polymers The iron oxide NPs were solvated in chloroform and combined with PEG using

a NP to PEG mass ratio of 12 The NP polymer solution was sonicated at 40 Hz for an

hour at room temperature The NPs were then washed three times with DI water via

centrifugation before being resuspended in DI water

98

517 Conjugation to Tobramycin

Drug conjugation to tobramycin was done using EDC with Sulfo-NHS Sulfo-NHS is a

chemical modification reagent used in the conversion of carboxyl groups to amine-reactive

esters in bioconjugation or crosslinking Sulfo-NHS is a charged analog of NHS (N-

hydroxysuccinimide) and like NHS facilitates control and alteration of carbodiimide

crosslinking reactions in which carboxylates (ndashCOOH) such as those present in the

polymer molecule are activated for conjugation with primary amines (ndashNH2) found on the

tobramycin molecule Such derivatives are synthesized by mixing the sulfo-NHS with a

carboxyl-containing molecule such as alginate citrate or carboxy-PEG with a dehydrating

agent such as the carbodiimide EDC (also abbreviated EDAC) EDC is a ldquozero-length

cross-linkerrdquo meaning that it acts by bringing the two molecules of interest together but

does not change the hydrodynamic size by increasing the polymer chain length In the first

step of the reaction the carboxylated particles are activated by addition of the EDC

followed by the formation of a reactive ester intermediate O-acylisourea After that the

ester will react with an amine group forming an amide however this amide is highly

unstable and will hydrolyze regenerating the carboxyl group if it does not encounter

another amine functional group Our procedure was adapted from a previous publication

[Hermanson 2013] In order to conjugate the SPIONs capped with any of the above-

mentioned organic molecules or polymers 100 mg of Fe3O4 NPs have been washed three

times with 10 mL of coupling buffer (50 mM phosphate buffered saline pH 72) and

removed by magnetic separation The purified NPs were then suspended in 5 mL of

coupling buffer To ensure an excess of the ligand 50 mg of tobramycin (50 mg

99

tobramycin per 100 mg NPs) was dissolved in coupling buffer thus making a 10 mgmL

tobramycin solution The detailed protocol for drug conjugation can be referenced in

Appendix II

Figure 58 Tobramycin molecule an aminoglycoside antibiotic having the formula

C18H37N5O9 shows 3D image (left) and carbon skeleton (right) drawn with

MarvinSketch

Under gentle stirring the NP solution was added drop-wise into a beaker containing

the tobramycin solution and allowed to sit for 2 min at 450 rpm 100 mg of EDC for each

100 mg of NPs were added to the reaction mixture under stirring until solvated The

conjugation reaction was allowed to proceed for 4 hours at room temperature under gentle

stirring Afterwards the NPs were washed twice with 5 mL of coupling buffer before

being resuspended in coupling buffer containing 35 mM Tris to block excess reactive sites

100

Afterwards the particles were washed twice again suspended in deionized water (DI

H2O) and stored in the refrigerator

Figure 59 EDCSulfo-NHS crosslinking reaction scheme in which the alginate coating

on the NP is conjugated to tobramycin thus binding drug to the NP via a new amide

linkage Image after [Conde 2014]

NOTE The sulfite in tobramycin sulfate completely ionizes in an aqueous environment it

is not covalently bound to the molecule and does not participate in the reaction

52 Characterization of Functionalized Nanoparticles

521 Size Determination

Hydrodynamic size distributions of the nanocrystals have been measured using a DynaPro

Titan Dynamic Light Scattering (DLS) module from Wyatt Technology Corporation In

101

order to reduce aggregation and maximize the accuracy of the measurement samples were

prepared for analysis by diluting the NPoleate stock solution to 50 microgmL in pure

chloroform The NPalginate stock solution was diluted in DI H2O The 1-mL samples

were vortexed then sonicated at 40 kHz for 5 minutes prior to analysis to separate

agglomerates and ensure that a more homogeneous solution was analyzed DLS results on

OA capped NPs right after synthesis returned an average diameter of ~16 nm (not shown)

in agreement with the TEM observations values PEG-5000 has a theoretical average

length of ~30 nm however it is important to note that the polymer length is just an average

value in addition the polymer chain can bend and twist resulting in a range of measured

Fig 510 DLS size distribution showing average hydrodynamic size of iron-oxide NPs

after alginate capping

102

In our experiments the succinylated PEG-5000 capping increased the hydrodynamic size

of the NPs from 16 to 4065 nm (not shown) Alginate capping using the natural alginate

also having a range of polymer lengths increased the hydrodynamic size of the NPs to

22971 nm (Fig 510) Tobramycin conjugation did not alter hydrodynamic size as

expected due to the small sizes of both the tobramycin molecule and the crosslinker

Tobramycin conjugation was confirmed by FTIR spectroscopy

522 Zeta potential measurements

Zeta potential measurements have been used to characterize the electrostatic potential at

the electrical double layer that forms at the interface of a colloidal NP and the dispersing

solvent Although the zeta potential measurement is often regarded as NP surface charge it

is not actually a measure of surface charge Zeta potential measures the potential difference

between the dispersion medium and the adsorbed layer of solvent ions surrounding the

particle This is not equal to the surface charge or the Stern potential [Kirby 2010] which

are defined at a different location Colloids with a zeta potential between -10 to +10 mV

are considered neutral while colloids with a zeta potential greater than 30 mV or smaller

than -30 mV are considered strongly cationic or anionic respectively [McNeil 2011]

Particles with a large measured value of zeta potential whether negative or positive are

electrostatically stabilized whereas particles with low absolute values of zeta potential

aggregate or flocculate [Greenwood 1999 McNeil 2011 Hanoar 2012] According to Liao

et al [Liao 2015] iron oxide NPs in water had a zeta potential of +161 mV (incipient

stability) which shifted to -601 mV (good-excellent stability) after capping with alginate

103

Because most cell membranes are negatively charged zeta potential is a key parameter in

membrane permeability and cationic particles tend to exhibit toxicity associated with

membrane disruption (lysis) [McNeil 2011] In our case the alginate coating will impart

the nanocomposites similar negatively charged electrostatic properties to the target

membrane and biofilm environment which should promote diffusion through the alginate

biofilms while also imparting the colloid significant stability at physiological pH

Additionally the average diameter of the functionalized NPs is still small enough to

diffuse through the pores in the mucus as long as they do not agglomerate

523 Forrier transform infrared (FTIR) spectroscopy

Was performed on tobramycin-conjugated NPs to confirm the successful conjugation of

the drug Since neither the tobramycin molecule nor the capping polymer have an amide

linkage preexisting in their structure the presence of an amide bond (1630-1681 cm-1) can

be used to verify a successful EDC conjugation The samples were dispersed in KBr pellets

for FTIR analysis The presence of an amide stretch visible on FTIR at 1630ndash1680thinspcmminus1

was used to verify the success of the crosslinking procedure Loading efficiency of

tobramycin calculated as mass of NP conjugates mass of alginate capped NPs was found

to be ~2

We report on a method for removal of the organic coating resulting from the carrier

molecule used in the solvothermal synthesis method We report a successful synthesis of

the more colloidally stable succinylated PEG from mPEG as well as a method for coating

104

uncapped NPs We have synthesized and water-solubilized magnetite NPs using various

organic shells We have conjugated drug (tobramycin) and verified conjugation to NP

samples capped with two biodegradable polymers alginate and citrate Using EDC

crosslinking in synergy with sulfo-NHS the NP samples were easily conjugated to the

amine groups on the tobramycin molecule The amide bond between the two groups is not

present on either organic molecule prior to conjugation and is visible on FTIR

spectroscopy We report an increase in colloidal stability and hydrodynamic size of

uncapped NPs (~60 nm) to ~230 nm for NPs capped in alginate

105

Chapter 6

DETERMINATION OF MINIMUM INHIBITORY

TREATMENT CONCENTRATIONS AND BACTERIAL

SENSITIVITY TESTING

Several different batches of NPs were used to thoroughly investigate against P aeruginosa

biofilms and liquid cultures uncapped magnetite NPs magnetite NPs capped with

alginate magnetite NPs capped with polyethylene glycol (PEG) zero-valent iron NPs

capped with alginate and magnetite NPs capped with alginate and conjugated to

tobramycin Citrate capped magnetite NPs were also produced however their use was

limited as they became oxidized and fell out of solution (lost colloidal stability) much

faster than the other compounds In addition initial investigations into the antimicrobial

properties of citrate-capped iron oxide NPs showed that they enhanced bacterial growth

[Brandt 2013] Iron oxide NPs were investigated in the uncapped form as well as with a

biodegradable (alginate) and a non-biodegradable (PEG) polymer coating because our

previous research showed that the PEG capped iron oxide did not inhibit bacterial growth

[Armijo 2014] and it was necessary to determine any inhibition without the contribution

of the cap The lack of inhibition observed in the PEG-capped sample is possibly due to the

non-biodegradable plastic PEG cap which kept the iron from ever interacting directly with

bacterial cells Despite the numerous investigations into the antibacterial effects of noble

106

metal and other nanomaterials described in the literature [Pal 2007] [Panaacutecek 2006]

[Shrestha 2009] and [Moritz 2013] not much focus has been placed on the role the

capping agent plays in the antibacterial properties Our findings demonstrate a major

dependence on the type of capping agent (if any) that is used We have investigated this

role by examining the same nanomaterial uncapped and capped with several different

polymers An ideal control would include a non-drug conjugated sample having the same

polymer cap used for drug conjugation Because it is well known that zero-valent iron

inactivates viruses [You 2005] gram negative E coli [Auffan 2008] [Lee 2008] and was

investigated in inactivating gram positive and gram-negative microbes as well as fungal

cells [Diao 2009] we investigated the antimicrobial properties of zero-valent iron NPs as a

positive control

Prior to the characterization of drug conjugates we investigated the bacterial

sensitivities to two FDA approved antibiotic drugs tobramycin (an aminoglycoside

antibiotic) and ciprofloxacin (a fluoroquinolone antibiotic) Proof of bacterial sensitivity to

tobramycin and determination of the minimum inhibitory concentration of tobramycin was

a necessary baseline establishment for the nanocomposite characterization studies since

tobramycin is the aminoglycoside antibiotic proposed for delivery by the nanosystem We

also investigated bacterial sensitivity to ciprofloxacin as an additional control in the event

that the bacterial colonies demonstrated resistance to tobramycin Although

fluoroquinolone antibiotics are not typically used in treatment due to their considerable

side-effects ciprofloxacin specifically is known to have a low MIC for the P aeruginosa

species Both the antibiotic drugs are known to be effective against P aeruginosa that has

107

not acquired resistance causing bacterial cell death via two different mechanisms of

action Therefore acquired genetic resistance to one mechanism should not impart

resistance to the other Aminoglycoside antibiotics possess several amino functional

groups becoming protonated in biological media thus resulting in a polycationic species

[Kotra 2000] The polycationic nature of the molecule imparts a high binding affinity to

negatively charged nucleic acids especially prokaryotic rRNA [Fourmy 1996] [Fourmy

1998] The simplest manifestation of resistance is posttranslational modification of the

rRNA target or to production of resistance enzymes [Kotra 2000]

It is important to note that P aeruginosa is a human pathogen therefore

appropriate biosafety practices need to be followed personal protective equipment used

and engineering controls must be in place and functional when working with this species

All experiments involving the use of live P aeruginosa must be performed in a biosafety

level 2 (BSL-2) laboratory Laboratory biosafety criteria detailing essential elements for

the BSL-2 and describing in detail required standard microbiological practices special

practices safety equipment and laboratory facilities can be obtained from the Centers for

Disease Control (CDC)

Although biomedical researchers typically limit their biofilm growth to 6 days

[Sauer 2012] [Benamara 2014] a previous report published [Moritz 2010] showed that

30-60-day-old biofilms have greater resistance to some stresses However another group

reports that after 7 days of biofilm formation the accumulation of biomass had not yet

reached a plateau [Klausen 2003] while a classic publication reports that 5 weeks of

growth was the optimal amount of time to achieve the maximum amount of biomass [Hays

108

1945] and yet another report in which the mucoid (biofilm) phenotype observed in P

aeruginosa typical of CF infections was investigated biofilm cultures were maintained for

90 days [Speert 1990] Because a typical P aeruginosa infection in CF patients is typically

thoroughly established we have chosen the extended growth period of 60-days We

anticipate that this model will more closely represent a classic CF infection Therefore

although much of the research on P aeruginosa is reported on biofilms which have

undergone shorter term growth it appears that short term cultures are only merited in in

vitro diagnostics as they were originally intended In diagnostic studies colonies are

allowed to differentiate just long enough to obtain diagnostic and sensitivity information

Figure 61 Pyocyanin

Image showing the

presence of pyocyanin

(blue-green) pigment

produced by P

aeruginosa cultures

grown on agar for

disk diffusion testing

Image by L Armijo

2014

Longer term studies although costlier are no doubt merited in research settings

due to the documented difference and robustness of established colonies Since

109

we are interested in modeling a typical P aeruginosa infection in CF patients

which is an established infection known to have more inherent resistance to

antibacterial agents we maintained our biofilms for a period of 60-days prior to

testing susceptibility to NPs and NP-tobramycin conjugates Established colonies

produced a thick alginate polymer matrix and secrete several pigments

characteristic of P aeruginosa pyocyanin (blue-green) pyoverdine (fluorescent

yellow-green) and pyorubin (red-brown) The optical color changes were noted in

the biofilm communities The blue-green pyocyanin can clearly be seen in figure

61

61 Microbiological Methods

611 Materials

Luria Bertani (LB) broth (cat 11006-004) and LB agar (cat 11006-001) were purchased

from IPM Scientific Inc Eldersburg Maryland USA Pseudomonas aeruginosa cultures

were purchased from ATCC (ATCC 27853)

612 Minimum Inhibitory Concentration of Tobramycin Determination

Because our proposed treatment method is based on the delivery of Tobramycin to P

aeruginosa colonies it was critical to first establish susceptibility to as well as the

inhibitory concentration of Tobramycin in this strain According to a previous report 1000

microg of tobramycin per mL was not sufficient to kill biofilm cells [Nickel 1985] however

others have reported minimum inhibitory concentrations (MICs) ranging from 05 microgmL

110

to 2 microgmL [Nichols 1981] Loosely considering these findings an initial range of 25-250

microgmL was selected for determination of the MIC of this strain To measure the MIC

tobramycin sulfate was first diluted with sterile DI H2O to 1 mgmL (stock solution)

Afterward tobramycin was serially diluted and added to the 1 mL aliquots of culture to

final concentrations between 25 and 250 microgmL with 25 microgmL increments 1 mL of sterile

DI H2O was added to the aliquot of the culture as a negative control The cultures were

then grown overnight on a rotary shaker at 37 ordmC and 150 rpm The next day 50 microL

aliquots of the cultures were diluted 12 with nutrient broth plated on the nutrient agar

plates and grown for 24 hours at 37 ordmC The next-day plates were examined for the

presence of bacterial colonies The MIC was accurately determined by using the dilution

series with 5 microgmL increments of tobramycin concentration ranging between its highest

concentration that still allowed the growth of P aeruginosa colonies on the plate and the

next lowest concentration that completely inhibited their growth [Brandt 2013] MIC of

tobramycin was measured over time beginning on day 1 after one overnight incubation (in

liquid culture without boiling stones) days 3 10 60 of biofilm growth

613 Establishment of Biofilm Communities

Cultures of Pseudomonas aeruginosa were maintained as a frozen stock (in 75 glycerol)

in a liquid nitrogen tank Two days before the experiment the broth medium was

inoculated and grown overnight on a rotary shaker at 37 ordmC and 150 rpm until the optical

density at 600 nm (OD600) reached 05-06 OD600 is a well-established method for

determination of bacterial cell concentration (mgmL) from the linear determination of

111

colony forming units (CFU) in the media The number of CFUs corresponding to the

optical density for P aeruginosa at an OD600=10 is 204x108 CFUmL which is equal to a

bacterial concentration of 2085 mgmL [Kim 2012] OD600 was determined using Cary

5000 UV-VIS-IR spectrophotometer against a blank cuvette containing the same volume

of the liquid medium This concentration was used to inoculate cultures in liquid media

P aeruginosa PAO1 biofilm communities were grown on sterile boiling stones in

liquid media for 60 days until firmly established P aeruginosa PAO1 biofilm

communities were grown on sterile boiling stones in liquid growth media for 60 days until

firmly established Other researchers have published protocols in which P aeruginosa was

grown for 3 days [Mandelbaum 1995] [Grassmeacute 2000] 6 days [Davey 2003] 8 days

[Suzuki 1973] or 7-10 days [Moreau-Marqiuis 2010] As mentioned above we not only

investigated these typical growth periods but investigated an extended growth period in

hopes of modeling an established (chronic) infection This significant amount of time for

biofilm establishment has not been previously reported Liquid cultures were grown in LB

broth at 37 ˚C for increments of 3 10 or 60 days Other researchers have reported

protocols for growth of P aeruginosa on sterile granite pebbles [Whiteley 2001] sterile

glass beads [Leboffe 2012] or glass wool [Benamara 2014] For our protocol biofilm

cultures were grown in LB broth on sterile boiling stones which have significant surface

area for nucleation events and conveniently biofilm attachment The liquid media was

decanted thus leaving only attached cells in the culture followed by replenishing cells

with fresh broth This method is a sufficient low-cost alternative to a flow chamber At the

end of the 60-day period the cultures were sonicated at 40 Hz for 15 minutes to remove

112

attached cells without damaging them similar protocols have been previously published

[Schmitt 1986] [Vandevivere 1993] The cells were then diluted to an optical density at a

600 nm wavelength (OD600) between 05 and 06 corresponding to about 102x107

CFUmL and a bacterial cell concentration of 10425 mgmL OD600 was determined using

the Cary 5000 UV-VIS-IR spectrophotometer against a blank cuvette which contained

only un-inoculated broth Once diluted the cultures were tested in liquid media or applied

to agar plates for susceptibility testing

614 Motility Testing

Motility testing was done on cultures after 1 3 and 60-days of growth Motility testing was

done by preparing agar in test tubes and inoculating the agar using the stab technique with

a sterile inoculation loop having a pointed end In this method the sharp end of the

inoculation loop is dipped into the cultures and stabbed into the agar inside of the test tube

one time The tubes are then incubated overnight and observed the next day Motile strains

can be seen to have disrupted the agar surrounding the place where the stab inoculation

was inserted into the agar This disruption of the agar is not detectable in non-motile

strains

615 Disk Diffusion Method

The disk diffusion method is one of the most popular approaches to bacterial sensitivity

testing due to its low cost and efficiency [Tendencia 2004] The disk impregnated with a

candidate antibiotic drug or compound of interest is placed on the inoculated agar which

contains a uniform layer of bacteria taken from liquid culture The disks are commercially

113

available containing the proper concentrations of antibiotic drugs based in moles per gram

As low-cost alternative disks may be prepared using filter paper soaked in the appropriate

aqueous concentrations of the antibiotic drugs of interest [Loo 1945] [Van Bijsterveld

1969] The underside of the plate is numbered for each sample to be tested The cultures

are distributed evenly onto a sterile agar plate using a sterile cotton swab to form a uniform

layer on the agar The disks containint the prescribed amount of antibiotic drug

recommended for susceptibility testing by the Clinical and Laboratory Standards Institute

(CLSI) the institution responsible for maintaining uniform standards for such research

Disks can also be made out of filter paper saturated in the standard dose of drug using a

known concentration and pipetting the corresponding volume onto the disk The underside

of the plate is numbered for each sample to be tested The method used was the agar disk

diffusion as described in CLSI where impregnated disks were applied to the cultured agar

plates overnight for 16-18 hours [CLSI 2014]

Approximately 108 CFUmL of bacterial cultures corresponding to an approximate 1

mgmL concentration determined by OD measurements was distributed evenly onto a

sterile agar plate using a sterile cotton swab to form a uniform layer on the agar The disks

impregnated with NPs drug or NP-drug conjugates were then placed on top of the agar

shown in Figure 62 A previous method of impregnation the dip method in which dry

disks were dipped into known concentrations by forceps and then placed onto the agar

cultures was found to produce inconsistent results because it was shown that the disks can

absorb different amounts of liquid introducing variability in the absorbed concentrations

[Simon 1970] Instead the more accurate drop method described by Sabath [Sabath 1976]

114

was used In this method the dry disks are placed on the agar plates then a known volume

The dry disks were placed atop the cultures and a 01 μL drop of the solution of interest at

the desired concentration was applied to the disk using a micropipette calibrated

micropipette

Figure 62 Agar plates inoculated with Pseudomonas aeruginosa colonies taken from

biofilm cultures showing disks impregnated with DI water NPs or NPs bound to

drug Image was taken prior to incubation [Armijo 2014]

This method eliminates variability in the total absorbed amount since a known volume is

applied Disk concentrations of tobramycin were initiated at the CLSI recommended disk

content for tobramycin corresponding to 10 μg absorbed into the disk when this mass

returned a negative susceptibility the concentrations were increased incrementally until a

susceptible mass was determined For the initial disk diffusion study investigating

115

tobramycin NPs and NP-conjugates the mass on the disk was determined from

concentration and applied volume For example a 01 μL aliquot of a solution having a

concentration of 100 mgmL corresponds to 10 μg in the disk (01 μL100 mg1 mL = 10

μg) a 50 mgmL concentration corresponds to 5 μg in the disk (01 μL100 mg1 mL = 5

μg) and a 25 mgmL concentration corresponds to 25 μg in the disk (01 μL25 mg1 mL

= 25 μg) and so on The cultures were grown under the previous conditions overnight (16-

18 hours) at 37 degC The diameter of zone of inhibition around the disc was observed and

recorded

A CF biofilm mucus model was also investigated on the 60-day-old biofilms in order to

determine whether magnetic field application susceptibility For this model the cultures

were prepared on solid agar in a petri dish as described above however 1 mL of either

prepared pig mucin aqueous alginate or both were applied on top of the plated colonies

The drug or NP-drug impregnated disks were applied over the barriers Half of the agar

plates were placed on top of a ring magnet composed of sintered neodymium iron and

boron magnetic alloy blendgrade N45 having a Gauss rating of 13500 Gauss a pulling

force of 282 lbs an axial pole orientation a NiCuNi coating and a tolerance of 0002

inches The magnets were left below the agar plate in the incubator for the entire overnight

growth period

116

Figure 63 Pole orientation options for ring magnets Left Axial Right Radial

Ring magnets having axial pole orientation were used for this study

617 Determination of Minimum Inhibitory Concentration of Test Articles

The diluted bacterial cultures were treated with various treatment concentrations The

concentrations were attained by performing a standard serial dilution A 1735 mgmL

stock solution was serially diluted by removing 05 mL from the stock tube and moving it

to the next tube containing the same total volume and so on as shown below Twelve

117

dilutions were done in total Serial dilutions of tobramycin tobramycin bound NPs or NP

suspensions were prepared

Figure 64 Illustration of serial dilution procedure Starting

concentration in the first vial (red) was 1735 mgmL and 05 mL was

transferred from the previous vial to the subsequent vial in line all of

which contained the same final volume

For the MIC measurements the compound of interest (NPs tobramycin or NP-

conjugates) were serially diluted in liquid growth media as shown in figure 62 inoculated

from cultures grown for a specific period and incubated in sterile 2 mL vials overnight

The cultures were then grown overnight on a rotary shaker at 37 degC and 150 rpm Optical

density (OD) of liquid cultures was compared to a control cuvette containing only growth

118

media and ODs comparable to the growth media alone were considered inhibited growth

OD typically increased with decreasing treatment concentrations as the bacterial cells

were increasingly able to differentiate at the decreasing treatment concentrations The MIC

was narrowed down by using the dilution series with even smaller increments of

tobramycin concentration ranging between its highest concentration that still allowed the

growth of P aeruginosa colonies and the next lowest concentration that completely

inhibited their growth The MIC experiments are schematically illustrated in Figure 65

Figure 65 Schematic diagram of minimum inhibitory concentration (MIC) determination

of tobramycin iron-oxide NPs tobramycin-NP conjugates and zero-valent iron NPs in P

aeruginosa liquid cultures

To verify inhibition an inoculation loop was used to plate samples from liquid

cultures having been incubated overnight with a known treatment concentration and

119

having an OD comparable to growth media alone The bacteria were allowed to grow on

the agar plates overnight at 37 degC MIC was determined by complete inhibition defined by

negative growth on agar as well as no apparent growth in liquid cultures determined by

OD For the control sterile DI water was added to the aliquot of the culture as opposed to

an investigational compound Due to the potential for interference of NPs with OD

measurements NPs were removed from solution by magnetic separation after inoculates

were plated on agar but prior to OD measurement

618 Graphical and Statistical Analyses

Graphical and statistical analyses analysis of variance (ANOVA) were performed on

Microsoft Excel and GraphPad Prizmtrade Average values and standard deviations being

calculated on Microsoft Excelreg and ANOVA performed on GraphPad Prizmtrade

62 Results

621 Minimum Inhibitory Concentration of Tobramycin Results

Using the procedure described in Section 61 and a tobramycin concentration curve we

determined the MIC of this particular strain of P aeruginosa in the initial pilot study to be

between 10-15 microgmL for planktonic cultures and averaged 50 microgmL for established one-

week-old old biofilms in liquid media (plusmn5 microgmL) These findings are similar to the

previously reported MIC of 35-50 microgmL (plusmn5 microgmL) found in planktonic cultures [Brandt

2013] Despite the documented need for a significantly increased drug concentration for

the treatment of bacterial biofilm infections (if drug susceptible at all) A previous report

120

found that 1000 microg of tobramycin per mL was applied to an established biofilm and a

significant proportion of the bacterial cells within the biofilm were found to remain viable

after 12 h of exposure to this very high concentration [Nickel 1985] The same group

reported the MIC in another study was found to be several orders of magnitude lower only

04 microgmL so MIC may differ tremendously from strain to strain and among different

growth modes Others have reported MICs ranging from 05-2 microgmL [Nichols 1981]

Another previous investigation found that oxygen limitation and low metabolic activity in

the interior of the biofilm not poor antibiotic penetration to be factors contributing to the

antibiotic tolerance of the P aeruginosa biofilm system [Walters 2003]

Figure 66 Minimum inhibitory concentration (MIC) of tobramycin to P aeruginosa

colonies as a function of growth time Please note that the cutoff concentration for

susceptibility of P aeruginosa to tobramycin in liquid cultures is le4 μgmL therefore

none of the cultures are tobramycin susceptible by CLSI standards

121

The MIC of tobramycin in this strain of P aeruginosa determined at several time

points during biofilm growth increased over time and was found to be 32 μgmL for 3-day

old biofilm cells 50 microgmL for 10-day old biofilms and 937 mgmL for 60-day old

biofilms The MIC of tobramycin differs significantly from strain to strain when

comparing planktonic vs biofilm cells and biofilm growth time These trends were not

observed for shorter periods of growth These findings add merit to our longer-term growth

period for the establishment of biofilm colonies According to the breakpoints

recommended by the CLSI for determination of MIC inhibition at a concentration le 4

microgmL of tobramycin means the strain is susceptible inhibition at a concentration of 8

microgmL is intermediate and inhibition at concentrations ge 16 microgmL means the strain is

tobramycin resistant [CLSI 2019] Therefore according to the CLSI breakpoints for

interpretation of MIC the cultures taken from biofilm communities were never found to be

susceptible to concentration of tobramycin defining susceptibility This strain exhibited

intermediate susceptibility in some cases in planktonic colonies (liquid cultures grown

overnight) with a MIC of 10-15 microgmL The biofilm cultures were found to be

tobramycin-resistant in all cases beginning on day 3 and becoming more resistant over

time

622 Interpretation of Disk Diffusion Results

The impregnated disks diffuse antibiotic drug with the highest drug concentration

assumed to be at the center of the disk and decreasing with the distance from that center

point According to the Clinical and Laboratory Standards Institute (CLSI) the investigator

122

must use the standards provided for the organism and the corresponding infected tissue or

organ CLSI tables provide the drugs and corresponding concentration dose for

susceptibility testing using the disk diffusion method for many bacterial species including

Pseudomonas The doses recommended by the CLSI were used for the initial studies

(Table 61) the recommended dose used for the susceptibility determination of P

aeruginosa is 2 microgmL for tobramycin and the 02 microgmL for ciprofloxacin Since the

plates were streaked using a sterile inoculation loop dipped in the liquid culture (having an

OD between 05 and 06)

Figure 67 Agar cultures used for susceptibility testing A) Agar plate with impregnated

disks prior to overnight incubation B) Image shows zone of inhibition (ZOI) halo around

disk impregnated with antimicrobial agent of interest a positive susceptibility result C)

Motility testing results in agar stab cultures after incubation upper tube is a negative

motility result and lower tube is a positive motility result

123

The effective doses for the susceptibility testing are much lower than the MIC reported for

cultures in liquid media because the number of bacterial cells is much lower when a plate

is inoculated For example in a 1 mL liquid culture tube having a concentration of 104

mgmL corresponds to a total biomass of about 1 mg whereas an inoculation loop is

dipped into the tube and used for streaking the plate only contains about 50 microL

corresponding to a total biomass of 005 mg If we examine as mg per mass of bacterial

cells it is apparent that the CLSI dose of 2 microgmL on the disk for a biomass of about 005

mg is close to our initial experimentally determined average MIC of 35 microgmL applied to

a 1 mg biomass

After overnight incubation the agar plates were examined The presence of a ldquohalordquo

around the disk suggests some degree of bacterial susceptibility to the compound applied

The halo surrounding the disk is a positive result for sensitivity called the zone of

inhibition (ZOI) The diameter of the ZOI is used for interpretation of these results based

on CLSI breakpoints This represents a concentration gradient with the maximum drug

concentration at the center of the disk The diameter of the halo was measured and

susceptibility was based on this measurement as follows

Table 61

Guidelines for interpretation of disk diffusion results

Method Susceptible Intermediate Resistant

Disk diffusion

[mm]

ge 15 13-14 le 12

Where R is resistant S is susceptible and I is intermediate

124

623 Disk Diffusion Results

The results of disk diffusion susceptibility studies are reported The first table 62 shows

the results of Fe3O4 NPs capped with PEG-OH Fe2O3 capped with PEG-OH Fe3O4 NPs

capped with alginate and bound to tobramycin Fe3O4 NPs capped with citrate and bound

to tobramycin tobramycin ciprofloxacin citrate and a DI water negative control

Table 62

Comparison of 3-day old biofilm sensitivities to MNPs capped with

PEG tobramycin ciprofloxacin and NP-drug conjugates

The bacterial colonies were susceptible to the CLSI concentrations for the treatment of P

aeruginosa 02 microgmL for ciprofloxacin The colonies did not demonstrate antibiotic

Disk

number chemical or drug Radius of

inhibition Sensitivity

1 NPs alone (Fe3O4) capped

with PEG-OH 17 mm S

with PEG-OH 0 R

3 NP (Fe3O4)-alginate-

tobramycin 17 mm S

4 NP (Fe3O4)-citrate-tobramycin 295 S

5 Tobramycin alone 10 mm R

6 Ciprofloxacin alone 40 mm S

7 Deionized water 0 R

8 Aqueous citrate 1 mm R

125

resistance to ciprofloxacin It is important to note that despite the effectiveness of the drug

ciprofloxacin this drug is not typically included in the normal treatment regime for P

aeruginosa infections Ciprofloxacin has been given a black box warning by the FDA [US

Food and Drug Administration 2008] due to its potential to cause permanent damage to

muscles tendons joints nerves and the central nervous system Its use is recommended

only when there are no other treatment options

The results of overnight sensitivity studies comparing different capping agents are

summarized in Table 62 Due to our previous findings in which citrate capped NPs

slightly promoted bacterial growth [Brandt 2013] we also tested citrate alone Although no

explanation for this was described by [Brandt 2013] it is possible that the citrate on the NP

surfaces was used as a source for pyruvate synthesis by the bacterial cells We were not

able to characterize increased bacterial growth on this solid agar as was observed

previously in liquid cultures however we did not observe inhibition by citrate or citrate-

capped NPs

These results were obtained on 3-day-old biofilms plated on LB agar using the

standard dose described in the introduction In this result we can see that the P

aeruginosa biofilm colonies did have an intermediate sensitivity to PEG-OH capped

magnetite in this initial study suggesting incomplete coverage of the NP by the non-

biodegradable polymer There was no inhibition by maghemite NPs capped with PEG-OH

either due to the lower iron content of the material or due to complete coverage by the

capping agent It is important to note that we have observed PEG-OH capped NPs to be

less colloidally stable than a capping agent that is attached to the NP via a COO- group

126

Table 63 Susceptibility of P aeruginosa biofilms to various treatments after 3 and 60 days of

growth by disk diffusion

Sensitivity is described with S for sensitive I for intermediate and R for resistant DI

water was used as a negative control and no ZOI was observed for DI water

Material Dose on disk ZOI (mm)

day 3

ZOI (mm)

day 60

Fe3O4 NPsdagger 10 μg 22S 21S

5 μg 175S 16S

25 μg 11R 10R

Fe3O4PEG NPs 10 μg 0R 0R

5 μg 0R 0R

25 μg 0R 0R

Fe3O4ALG NPs 10 μg 22S 22S

5 μg 16S 15S

25 μg 10R 8R

Fe3O4ALGTOBRA NPs 10 μg 23S 22S

5 μg 11R 15I

25 μg 7R 5R

ZVFeALG NPs 10 μg 25S 24S

5 μg 21S 22S

25 μg 20S 20S

Tobramycin 10 μg 10R 0R

100 μg 25R 15R

1000 μg 35R 32R

Interpretation R ndash resistant I ndash intermediate S ndashsusceptible daggerUncapped NPs CLSI breakpoint for susceptibility of tobramycin by disk diffusion is 10 μg therefore all colonies are found to be tobramycin resistant by CLSI standards Higher tobramycin doses in the disk were investigated to determine whether any susceptibility existed at higher doses At present there are no CLSI valuesbreakpoints for NPs as antimicrobial agents ZOI zone of inhibition PEG polyethylene glycol ALG alginate TOBRA tobramycin ZVFe zero-valent iron

127

It is probable that a percentage of the polymer is protonated and detached from the NP in a

colloidal suspension at an undetermined equilibrium concentration As is well known

water at equilibrium contains H+ and OH- at pH dependent concentrations so it is possible

that some of the PEG-O- is protonated in water even at a physiological (neutral) pH

Table 63 shows results of sensitivity testing and determination of MIC for experiments

using NP-drug conjugates on the 60-day old established biofilm colonies These colonies

were also grown as described in Section 61 For this study succinylated PEG (PEG-

COOH) was used to ensure complete continuous coverage of the NP samples For this

study we also investigated the inhibitory properties of zero-valent iron which is known to

inactivate microbes

Disk diffusion results for tobramycin were interpreted based on the 2019 CLSI

breakpoints for tobramycin in P aeruginosa [CLSI 2019] in which the mass of

tobramycin on the disk is 10 microg and a disk diameter ge15 mm is susceptible (S) 13-14 mm

is intermediate (I) and le12 mm is resistant (R) Since there are no established standards for

the investigation of iron oxide nanoparticle susceptibility in any microbes we used the

same cutoff values as we used with tobramycin in order to maintain consistency We also

investigated a range of concentrations of both tobramycin NPs and NP-conjugates in order

to determine susceptibility range The disk diffusion results (Table 63) taken together

with the MIC results over time demonstrate that the tobramycin susceptibility decreases

and resistance increases as the colonies are allowed to grow in biofilm mode for longer

periods of time despite being tobramycin naiumlve Therefore this is not due to exposure-

related resistance development It is important to note that the observed increase in

128

resistance is not due to a larger initial amount of CFUrsquos in the 60-day old biofilms because

cultures were diluted and identical concentrations of CFUs were used for inoculation and

plating for all time periods These findings suggest that the age of the infection alone (ie

establishment of a chronic infection) contributes to resistance This is possibly due to

broader genetic diversity in the population No comparable increase in resistance over time

was observed for the NP samples investigated suggesting that a genetic resistance

mechanism to counter the action of the compound may not exist We can speculate that the

mechanism of action of the iron-oxide NPs is not based on inhibition of genes or bacterial

protein synthesis which implies the toxicity may not be prokaryote-specific

For the iron-oxide NPs alone we found that inhibition of established biofilms on agar

plates was observed for low concentrations When capped with alginate the inhibition

remained low even though part of the mass of this core-shell type NP consists of non-

bioactive alginate In the case of iron-oxide NPs capped with succinylated PEG no

inhibition was observed possibly because the non-biodegradable nature of the capping

agent may keep the iron from interacting directly with the bacteria (see Table 63) If the

iron ions contribute to the toxicity it may be possible that in this case they were not

distributed to the colonies and therefore could not inhibit bacterial growth

These findings demonstrate that the crucial role of the capping agent to the impartation

of antimicrobial properties Therefore the capping agent also contributes to or negates the

toxicity of this material We can speculate that a complete succinylated PEG cap may also

reduce the toxicity of NPs known to exhibit cytotoxic effects in vivo since it appears to

limit interaction with the cells at least in this short exposure time frame

129

Even at high concentrations we might expect to observe some inhibition due to

incomplete coverage however that is not the case In the case of iron-oxide NPs

conjugated to tobramycin we find that the bacterial inhibition at these concentrations

mirrors the inhibition trend of iron-oxide NPs alone It is important to note that these

findings are characteristic of this particular strain after this period of growth and its

susceptibility to tobramycin Recall the previous study which found that after a 1000

μgmL concentration of tobramycin was applied to established biofilms a significant

proportion of the bacterial cells were still viable after 12 hours [Nickel 1985] This group

also reported that planktonic cells taken from the same strain was completely killed by

only 50 μgmL Another relevant study reports the MIC from their clinical isolates to be 8

μgmL [Shawar 1999] These published findings suggest a huge theoretical therapeutic

dose ranging from 8 microgmL to more than 1000 μgmL MIC and susceptibilities appear to

differ dramatically from strain to strain and in planktonic vs biofilm communities

Therefore it is probable that these susceptibilities may also differ from strain to strain and

under different growth conditions

624 Biofilm and Mucus Model and Static Magnetic Field Application Results

The CF disk diffusion model grown on solid agar in petri dishes in which artificial mucin

and alginate barriers were applied over the bacterial colonies cultured from established 60-

day old biofilms reveal that the application of an external magnetic field enhances

susceptibility to the iron-oxide NPs and NP-drug conjugates possibly by promoting

transport across the two barriers For this study 50 mgmL concentrations of NP conjugate

130

and NP solution was applied to the disk such that each disk contained 50 microg of test article

The results with (Table 65) and without (Table 66) magnetic field application

demonstrate zero susceptibility to tobramycin alone

Table 64

Results of CF biofilm model (magnet applied)

A mucin barrier an alginate barrier or both- were applied to 60-day-old biofilm

colonies For this study a magnet was placed below the petri dish

Disk Number Compound Mucin

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

1 Iron Oxide NPs dagger 30S 0R 20S

2 Zero-valent Iron NPs 5R 20S 20S

3 Iron Nitride NPs 30S 32S 15I

4 Iron Oxide NP-

Tobramycin

25S 19S 14I

5 Tobramycin (200 mg) 32R 30R 20R

Disk diffusion method was used Minimum concentrations demonstrating susceptibility in

previous disk diffusion studies were used for NPs and NP-tobramycin conjugates daggerUncapped

NPs Maximum CLSI cutoff concentration for susceptibility of tobramycin 10 μg absorbed onto

disk These doses of tobramycin shown are up to seven orders of magnitude higher than the CLSI

standard dose for disk diffusion therefore although inhibition was observed these colonies are

tobramycin resistant by CLSI standard

Table 65

Results of CF biofilm model

A mucin barrier an alginate barrier or both were applied to 60-day-old biofilm

colonies For this study a magnet was not applied

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

6 Iron Oxide NPs dagger 14I 0R 22S

7 Zero-valent Iron NPs 0R 0R 14I

8 Iron Nitride NPs 0R 0R 0R

9 Iron Oxide NP-

Tobramycin

0R 0R 0R

10 Tobramycin (200 mg) 30R 40R 40R Disk diffusion method was used Minimum concentrations demonstrating susceptibility in previous

disk diffusion studies were used for NPs and NP-tobramycin conjugates daggerUncapped NPs

Maximum CLSI cutoff concentration for susceptibility of tobramycin 10 μg absorbed onto disk

These doses of tobramycin shown are up to seven orders of magnitude higher than the CLSI

131

No CLSI breakpoints exist for NPs or NP conjugates at present however the CLSI

dose for susceptibility determination of P aeruginosa to tobramycin is 10 μg absorbed

onto disk with cutoff values are ge15 susceptible 13-14 intermediate and le12 resistant

[CLSI 2019] The same parameters were used for interpretation of the NP and NP

conjugate results Tables 64 and 65 demonstrate the highly statistically significant

contribution of the external magnetic field in enhancing susceptibility to the test articles

More work is needed to determine the exact role of the magnetic field in addition to

determining the minimum or maximum field strength necessary to achieve maximum

susceptibility It is possible that the pulling force of the magnet may relate in some way to

the thickness of the biofilm and mucus barriers

Table 66

Summary of biofilm model using alginate barrier mucin barrier or both

on 10-day-old biofilms Comparison between petri dishes in which a magnet was or was not applied

Alginate and

Mucin

(No magnet)

Alginate

(magnet)

Mucin

(magnet)

Alginate and

Mucin

(magnet)

DI Water R R R R

Tobramycin R S R S

NP-alginate-

drug

R I S S

NP-citrate-

drug

R I I I

A summary of the results of a pilot study presented in Table 66 summarizes the

results of a pilot study in which magnetite NPs conjugated to tobramycin was investigated

The results summarized in Table 66 shows that magnetic field application alone enhanced

132

susceptibility of biofilms to all the test articles including tobramycin Therefore magnetic

field may be acting as an antimicrobial facilitator by mechanism other than magnetic

gradient-guided transport It is interesting that this enhancement of the activity of

tobramycin by magnetic field application was not observed in the 60-day-old biofilms

More work is necessary to determine whether magnetic field application alone and what

rangeranges of field strengthduration interfere with biofilm growth

625 Motility Testing Results

Biofilm bacteria (3-days and older) tested positive for motility while the liquid cultures

(grown overnight) appeared to have minimal if any motile individuals This is a testimony

to the large genetic diversity of the bacteria composing a biofilm

Figure 68 Results of

motility test for P

aeruginosa grown in liquid

or biofilm cultures This

image was taken after a total

of 36 hours of growth

626 Comparison of Inhibition in Liquid Cultures

All cultures were inoculated in exactly the same manner with the same volume of bacteria

from the same liquid culture The OD600 of the negative control samples (containing only

133

inoculated broth) was determined to be 022 to 024 This result is slightly higher than the

lowest treatment concentration (8x10-6 mgmL) Since there is no CLSI breakpoint or

standard inhibitory concentration it was necessary to investigate a large range of

concentrations to determine MIC The range used was 1735 mgmL to 8x10-6 mgmL in a

consistent volume determined by serial dilution as the graph in figure 69 illustrates

Figure 69 Shows optical density (OD) at a 600 nm wavelength for liquid

cultures exposed to treatment with iron-oxide NPs zero-valent iron or

tobramycin-conjugated iron-oxide NPs The calculated average error for OD

measurements was plusmn001 Specific errors not the average error were used to

calculate statistical significance

134

Complete inhibition was observed for all materials at concentrations at 175 mgmL (or

higher) and various degrees of inhibition fall off somewhat linearly at concentrations

below 1735 mgmL (Fig 69) The inhibition by zero-valent iron was not surprisingly

higher than iron-oxide NPs and NP-drug conjugates We attribute this to the high reactivity

of zero-valent iron and its ability to increase reactive oxygen species (ROS) in the local

region [Hsueh 2017] Although speculative at this stage it is also possible that high levels

of iron contribute to cellular toxicity More work is necessary to determine toxic and non-

toxic dose ranges

Figure 610 Percent bacterial inhibition vs treatment concentration in liquid

cultures in cuvette All NP samples presented here are alginate capped

135

ANOVA results showed that while there was no statistically significant difference

between the zero-valent iron iron oxide or iron-oxide ndash tobramycin conjugates when

compared to control the results for all three NP treatments were found to be extremely

statistically significant (p lt 00001) The figures show that the inhibition of bacterial cells

was evident even at surprisingly low (8 ngmL) concentrations although the minimum

therapeutic dose would probably be much higher Speculation on a therapeutic dose for

targeted delivery would likely differ from the systemic dose and both will depend on

observed cytotoxicity in mammalian cell cultures at these concentrations Even higher

doses may be required for the treatment of chronic infections involving biofilms that have

been established for several years however more research is necessary to determine this

The MIC for different strains of P aeruginosa may differ as well According to

another report P aeruginosa (MTTC 1034) was not found to be susceptible to iron-oxide

NPs at 50 mgmL whereas our strain exhibited positive susceptibility [Behera 2012] It has

been shown previously that oxygen limitation and metabolic activity can alter MIC of

tobramycin in P aeruginosa [Walters 2003] Differences in zone diameter for

susceptibility testing have also been known to differ with different batches of growth agar

[Reller 1974] [Niemirowicz 2015] reported positive bacterial inhibition for P aeruginosa

PAO1 in agreement with our findings We attribute differences in susceptibilities to

genetic differences among strains in combination with the contribution of environmental

factors such as growth media and the use of different capping agents

The mechanism by which iron-oxide NPs exhibit antibacterial activity remains

unknown However according to the findings of [Musk 2005] iron may very well be the

136

bioactive component Zero-valent iron as predicted had a dramatic antibacterial effect

verifying the findings of [Diao 2009] Although zero-valent iron is too reactive for in vivo

use at present it may be a candidate for incorporation into antibacterial coatings Similarly

iron-oxide NPs having high biocompatibility may be a candidate material for

incorporation into polymer for use as antibacterial coatings on virtually any inert surface

used outside of the body as well as medical devices such as stents catheters and surgical

sutures as a low-cost alternative to silver NPs We anticipate that the combination of

tobramycin or other drugs with iron-oxide NPs incorporated into biodegradable polymers

may hold promise for the long-term control of biofilms and multidrug resistant microbial

strains More work is needed to determine antibacterial properties of these materials on

other microbial species

69 Summary of Sensitivity and Dosage Study Findings

We have shown that both drugs ciprofloxacin and tobramycin are effective against

biofilms and planktonic cells in a dosage-dependent manner Magnetic field application

may in some cases enhance drug susceptibility The drug seems to have exerted action

both in the free form as well as covalently bonded to a crosslinker chain There appears to

be no need for a drug release mechanism since the bound drug remains bioactive

Surprisingly the magnetite NPs alone inhibited bacterial growth and subsequent biofilm

formation We have examined standard models in addition to more accurate models using

inert surfaces for biofilm growth thus allows for purification of the bound colonies from

the planktonic cells Using this method we have also shown that the biofilm colonies

137

contain motile mutants previously undocumented evidence of the complex genetics

implied by such a rapid phenotypic switch

Although it appears that the iron oxide NPs inhibited growth better than drug-

conjugated iron oxide we must use caution in the interpretation of these results Recall that

conjugation was done which may have increased the mass of the non-active ingredients

Further characterization such as drug loading efficiency would allow the calculation of the

percentage by mass of iron oxide tobramycin and inert material Once those calculations

are done these parameters may be further understood as a function of active ingredients It

is apparent however that the iron oxide did inhibit bacterial growth via a presently

uncharacterized mechanism

Zero-valent iron had a dramatic antibacterial effect verifying the findings of [Diao

2009] Although zero-valent iron is too reactive for in vivo use at present it may be a

candidate for incorporation into antibacterial coatings Iron oxide alone may be a candidate

for antibacterial coatings on medical devices such as stents catheters and surgical sutures

as a low-cost alternative to silver NPs The drug tobramycin an aminoglycoside

annihilates bacterial cells in a synergistic manner It electrostatically binds the negatively

charged lipopolysaccharide bacterial membrane compromising membrane integrity and

thus resulting in its degradation [Shakil 2008] Once internalized acting from the inside of

the bacterial cell tobramycin inhibits ribosomal translocation thus interfering with protein

synthesis [Saiman 2004] We anticipate that the combination of tobramycin or other drugs

with iron oxide NPs incorporated into biodegradable polymers may hold promise for the

long-term control of multidrug resistant bacterial strains

138

Chapter 7

CYTOTOXICITY of IRON OXIDE NANOPARTICLEs

Not only is lung toxicity a crucial parameter to investigate due to the nature of our

application but also in acute inhalation exposure the organ system subjected to the

highest initial concentrations is the lungs Therefore a thorough investigation of the acute

toxicity of inhaled nanomaterials must begin with a baseline analysis of human lung cell

toxicity We have investigated the in vitro cytotoxicity of ~16 nm spherical magnetite

nanoparticles capped with succinylated polyethylene glycol on a human lung carcinoma (A

549) cell line at 6 12 and 24-hour exposure periods and at 05 mg mL and 1 mgmL

nanoparticle concentrations We investigated acute toxicity in a comprehensive study by

comparing overall cytotoxicity cell viability and apoptosis profiles against positive

controls We report a dose-dependent decrease in viability at the 12-hour time point

exhibiting a complete cell recovery by 24-hours as well as a dose independent time-

dependent alteration in cell proliferation rate No statistically significant deviation from

control in overall cytotoxicity or apoptosis was observed upon exposure to iron oxide

nanoparticles in this cell-line at the time points or concentrations investigated

Animal models have revealed a link between inhaled particles and murine lung

inflammation [Oberdoumlrster 2000] and lung cancer [Knappen 2004] [Borm 2004]

139

Although the dextran-coated iron oxide NP solution finding application as the IV-

administered MRI contrast agent Feridexreg had received FDA approval for human use in

the United States it was discontinued by the manufacturer [Anselmo 2016 Wei 2016] and

is no longer commercially available To date there still exists a significant lack of

knowledge regarding the effects of NPs in general but more specifically on the effects of

iron oxide (magnetite) NPs on cell viability and normal functionality [Sonen and De

Cuyper 2010] In fact many researchers have reported that the use of these particles can

exert severely detrimental actions on the living cell [Sonen and De Cuyper 2010] [Wei

2016] reports a SPION dosage-dependent iron overload linked to cirrhosis of the liver in a

murine systemic toxicity model Some other negative observations include LDH leakage

and abnormal IL-6 secretion at high (gt50 mgmL) concentrations [Mbeh 2012] significant

reductions in viability in murine and human cell lines [van den Bos 2003 Soto 2007]

[Pisanic 2007] decreased cell proliferation [Berry 2004 van den Bos 2003] and migration

[Berry 2004] Many of these studies reporting increased toxicity attribute toxic effects to

the failure of the dextran coating to remain bound to the cell Because of this we have

engineered the terminal hydroxyl group (OH-) on the FDA approved polymer polyethylene

glycol (PEG) to terminate in a more electronegative carboxyl group (COOH-) by

succinylation increasing binding efficiency to the metal oxide (M+) NP Due to the

association of uncapped iron oxide NP and toxicity in some cell types increased binding

efficiency is expected to reduce cytotoxicity of the iron oxide NPs

Regarding human inhalation exposure the occupational health literature abounds

with illustrations of aerosol-associated respiratory hazards and related lung pathologies

140

dating back many decades However the context of this prior research pertains specifically

to occupational exposure to nanoscale particulates formed accidentally as by-products

from processes such as welding smelting and combustion [Maynard and Kuempel 2005]

as opposed to engineered nanomaterials It is crucial that toxicity data on nanomaterials

having the potential to expose workers via the inhalation aerosol route be communicated

quickly to researchers so that they may cater future engineering design to reduce toxicity

At present there is limited data on the toxicity of these methodically engineered nanoscale

materials in the human respiratory tract Due to the exponential growth in the manufacture

and utilization of such nanomaterials which still remains largely unregulated we

anticipate an exponential increase in their presence in both the natural environment as well

as the workplace This rapid increased in commercialization of such novel materials

having unknown toxicity will merit an accurate determination of a safe exposure range

not only for a patient receiving nanomedical treatment but also for the employees

engineering transporting administering and disposing of these materials Toxicity profiles

are crucial for the determination of proper engineering controls proper personal protective

equipment (PPE) and emergency procedures for employees administering transporting

and manufacturing the material Dosage-dependent cytotoxicity will also be an important

parameter for determining the feasibility of purposely administering this material to the

lungs and determining and balancing dosages that are both safe and effective

71 Experimental Procedure

141

Succinylated PEG-capped iron oxide NPs were prepared as described in Chapters 2 and

capped using the methodology described in Chapter 5

711 Materials and Reagents

Iron(III) chloride hexahydrate (97) m-PEG 5000 (methyl-terminated PEG) powder

succinic anhydride (gt99) phosphate buffered saline (PBS) powder TRIS hydrochloride

(PharmaGrade) digitonin ionomycin and staurospirine were purchased from Sigma-

Aldrich n-docosane (99) was purchased from Alfa Aesar sodium oleate (gt97) was

purchased from Tokyo Chemical Industry Co hexanes (95) ethanol (99) and acetone

(99) chloroform (999 ) hexane (99) pyridine (99) methanol (99) were

purchased from EMD Chemicals Inc the ApoTox-Glotrade triplex assay (Catalog No

G6320) was purchased from Promegareg A 549 human alveolar epithelial carcinoma cells

(ATCCreg No CLL-185) and 025 Trypsin053 mM EDTA (ATCCreg No 30-2101) were

purchased from ATCCreg Hamrsquos F-12 Kaignrsquos modification (Catalog No 21127-022)

10 fetal bovine serum heat-inactivated (Catalog No 10082-147) and 100 unitmL pen-

strep (Catalog No 15140-122) were purchased from Invitrogen All chemicals were used

as received without purification

712 Dynamic Light Scattering (DLS)

Titan DLS module from Wyatt Technology Corporation In order to reduce aggregation

and maximize the accuracy of the measurement samples were prepared for analysis by

142

diluting the NP stock solution to 50 microgmL in pure chloroform The 1 mL sample was

vortexed then sonicated at 40 Hz for 5 minutes prior to analysis in order to separate

agglomerates and ensure that a more homogeneous solution was analyzed

713 UV-vis-NIR Spectroscopy

Light absorbance of iron oxide nanoparticles and succinylated PEG was characterized

using the Cary 5000 UV-vis-NIR Spectrometer Many published assay results fail to

report or even consider doing these measurements Nanomaterials or quantum dot are

known to have highly sought-after interactions with light It is important that we consider

these interactions when designing experiments using these kinds of assays that were not

developed with such considerations in mind By determining light absorbance we are able

to determine any possible interaction or interference of these materials with the assays

which are dependent on total light detection via the plate reader

714 Human Lung Adenocarcinoma Cell Growth

Cells were stored in liquid nitrogen in a cryostat until their use To initiate growth the

sample was thawed and centrifuged and then the culture medium was removed After that

the cells were rinsed with 025 Trypsin053 mM EDTA solution to remove any

remaining serum that may contain trypsin inhibitor Next 25 mL of Trypsin-EDTA

solution was added After 15 minutes the cells had dispersed into the solution and 7 mL of

complete growth medium (F-12K medium with 10 FBS) was combined with the cells by

gentile aspiration Cultures were incubated at 370 degC under 5 carbon dioxide weighted

with HEPA-filtered air

143

715 Cytotoxicity Assay

Bis-alanylalanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110) is a fluorogenic cell-

impermeant peptide substrate marker for dead-cell protease activity This is used to

measure protease enzyme which has been released from cells that have lost membrane

integrity No signal from this marker is generated from viable (intact) cells because bis-

AAF-R110 is not cell-permeant and cannot cross the cell membrane Dead cells release

protease enzymes that will cleave the rhodamine 110 (R110) from the rest of the molecule

causing it to fluoresce R110 has an excitation peak at 498 nm and an emission peak at 520

nm

In growth medium 05 and 1 mgmL concentrations of NPs were incubated with

the cells for 12 or 24-hours exposure time Digitonin ionomycin and staurosporine are

known to elicit cytotoxic necrotic and apoptotic damage upon cells respectively and were

used as positive controls For the twelve (12) hour exposures cells in positive control wells

were treated with either 30 microgmL of digitonin for an incubation period of 15 minutes 100

microM of ionomycin or 10 microM of staurosporine both applied for 6-hour incubation periods

For the 24-hour measurements cells in positive control wells were treated with either 45

microgmL of digitonin for an incubation period of 30 minutes 150 microM of ionomycin or 15

microM for staurosporine for 6-hour incubation periods A 96 well-plate was used except for

background control wells which contained growth media alone each individual well was

seeded with 10000 A 549 cells dispersed in growth media Each well was filled to a

volume of 100 microL and cells were cultured for the respective time periods The background

readings from the wells containing no cells were averaged and subtracted from the

144

obtained averaged readings After the 6 12 or 24-hour period 20 μL of the

viabilitycytotoxicity reagent containing both GF-AFC substrate and bis-AAF-R110

substrate was added to all the wells Immediately after that the solutions were mixed by

orbital shaking at 300-500 rpm for ~30 sec The plate was incubated for 30 minutes at 37

degC Finally the samples were exposed to 485 nm light with a 20 nm bandwidth for

excitation fluorescence measurements were taken at 528 nm with a 20 nm bandwidth

Measurements were taken with a BioTech Flx800 Microplate Reader measuring

fluorescence from the bottom of the 96-well plate

716 Viability Assay

Glycylphenylalanyl-aminofluorocoumarin (GF-AFC) is a florigenic cell-permeant peptide

substrate which is used as a marker for live-cells Since live-cell proteases must be

detected from within the living cell having an intact membrane this substrate must cross

the cell-membrane and enter the cell Once inside the cell protease enzymes cleave the

AFC from the substrate triggering the fluorescence signal The AFC has an excitation

peak at 370 nm and a fluorescence emission peak at 490 nm Should the membrane rupture

while the substrate is inside the cell the fluorescence is quenched and the signal ceases

Therefore this substrate is able to give an accurate measure of viable cells

the cells for 6 12 or 24-hours exposure time Digitonin ionomycin and staurosporine are

used as positive controls For the six (6) and twelve (12) hour exposures cells in positive

145

control wells were treated with either 30 microgmL of digitonin for an incubation period of 15

minutes 100 microM of ionomycin or 10 microM of staurosporine both applied for 6-hour

incubation periods For the 24-hour measurements cells in positive control wells were

treated with either 45 microgmL of digitonin for an incubation period of 30 minutes 150 microM

of ionomycin or 15 microM for staurosporine for 6-hour incubation periods A 96 well-plate

was used except for background control wells which contained growth media alone each

individual well was seeded with 10000 A 549 cells dispersed in growth media Each well

was filled to a volume of 100 microL and cells were cultured for the respective time periods

The background readings from the wells containing no cells were averaged and subtracted

from the obtained averaged readings After the 6 12 or 24-hour period 20μL of the

717 Apoptosis Assay

In this assay cell apoptosis is measured by detecting the apoptosis biomarkers

caspase 3 and caspase 7 Cell lysis is followed by caspase cleavage of the substrate and

results in generation of a luminescent signal The fluorophore in this assay is luciferase

146

(aminoluciferin) a natural luminescent molecule borrowed from the firefly [Gould 1988]

Luminescence is proportional to the amount of caspase activity and thus apoptosis The

luminogenic caspase-37 substrate which contains the tetrapeptide sequence DEVD (Asp-

Glu-Val-Asp) in an optimized reagent (Caspase-Gloreg 37 Reagent Promegareg) optimized

for caspase activity luciferase activity and cell lysis

the cells for 6 12 or 24-hours exposure time Ionomycin is known to induce necrosis and

staurosporine is known to induce apoptosis therefore these compounds were used as

controls for this assay For the six (6) and twelve (12) hour exposures positive control

cells were treated with either 100 microM of ionomycin or 10 microM of staurosporine both

applied for 6-hour incubation periods For the 24-hour measurements cells in positive

control wells were treated with either 150 microM of ionomycin or 15 microM for staurosporine for

6-hour incubation periods A 96 well-plate was used except for background control wells

which contained growth media alone each individual well was seeded with 10000 A 549

cells dispersed in growth media Each well was filled to a volume of 100 microL and cells were

cultured for the respective time periods The background readings from the wells

containing no cells were averaged and subtracted from the obtained averaged readings

After the 6 12 or 24-hour period 20μL of the viabilitycytotoxicity reagent containing

both GF-AFC substrate and bis-AAF-R110 substrate was added to all the wells

Immediately after that the solutions were mixed by orbital shaking at 300-500 rpm for ~30

sec The plate was incubated for 30 minutes at 37 degC After fluorescence measurements

were taken 100μL of Caspase-Gloreg 37 Reagent was added to all wells and briefly mixed

147

by orbital shaking at 300ndash500 rpm for ~30 sec Measurements were taken with a BioTech

Flx800 Microplate Reader measuring luminescence from the bottom of the 96-well plate

718 Statistical Analysis Correction Factor and Mathematical Methods

This experiment was done in triplicate the median values presented and standard

deviations were calculated For comparison of the mean values for each test both a two-

way analysis of variance (ANOVA) was run for grouped values and to analyze trends over

time and a double-tailed t-test was run to compare single values to control All statistical

analyses were run in GraphPad Prismreg Values of plt05 (95 confidence interval) were

considered significant plt 001 (99 confidence interval) were considered very

significant and values of plt 0001 (999 confidence interval) were considered extremely

significant

In order to correct for the signal reduction caused fluorescence absorption by the

colloidal NPs a general correction was applied as follows The experimental findings of

[Doak 2009] in which fluorescence quenching by iron oxide NPs was measured at different

concentrations were plotted as a function of percent signal reduction The maximum

concentration of fluorescent dye used in the assay assuming 100 fluorophore activation

as calculated from the stock solution concentration and dilution factor is 5 microM Although

there is a slight difference in the percent reduction based on the difference between the 2

microM and 4 microM fluorophore concentrations we analyzed the mean collected values at each

NP concentration compared between the dye concentrations did not find them to be

statistically significant Although it is unlikely that 100 of the fluorophores were

148

activated in the assay the maximum concentration which is not likely to exceed 1

variance from the calculated value and should be encompassed by the correction factor

and corrected error The plot (Fig 71) demonstrates a nearly identical trend for both

concentrations of fluorescent dye which suggest that signal reduction is consistent over a

range of fluorophore concentrations and the values can be fit to the same trend line

Reduction of Fluorescence Intensity by Magnetite NPs

NP Concentration (gmL)

Perc

en

t In

ten

sit

y R

ed

ucti

on

co

ntr

ol

1x

10

-3

1x

10

-2

1x

10

-1 1

10

0

20

40

60

80 reduction 4 M dye

reduction 2 M dye

Figure 71 Reduction of fluorescence signal by magnetite NPs at two fluorescent dye

concentrations Based on the findings of [Doak 2009]

Extrapolating out one data point encompasses the two concentrations used for this study

The log transforms and linear curve calculations were run on GraphPad Prismreg The data

was fit to an exponential trend line in Microsoft Excelreg The exponential trend line

149

equations for the 4 microM and 2 microM concentrations of fluorescent dye were y=43311e03718x

and y=69758e03062x respectively According to this model the next data point

corresponding to a 1 mgmL concentration of magnetite NPs is between 585-595 At

this range the variation between the two fluorophore concentrations was found to be only

~1 Since the concentration of the fluorophores does contribute minimally to the

measurement this range was incorporated to the error margin Based on this model the

measured fluorescence values were reduced by ~59 for the 1 mgmL concentration and

~51 for the 05 mgmL concentration Since simply taking an increase by the percentage

of the measured value will not return the original value the measured values must be

adjusted according to

measured value

(100 minus reduction)

The collected values were included in the standard deviation for comprehensiveness

72 Results

721 Dynamic Light Scattering (DLS) Size Distribution

DLS results are shown in Figure 72 where the particles were measured for size in

chloroform solution Because polymer coating increases the NP hydrodynamic size this

measurement was done prior to polymer capping in order to verify NP sizes observed in

TEM measurements The colloidal NPs demonstrate some very minor aggregation

150

(responsible for the peaks at 30 and 35 nm) The average hydrodynamic diameter is 15946

nm with a standard deviation of 4393 nm in agreement with the TEM observations

Figure 72 DLS size distribution of colloidal magnetite nanoparticles

This graph shows an average hydrodynamic diameter of ~16 nm

722 UV-vis-NIR Spectroscopy Absorbance Measurements

Absorbance spectrum (Figure 73) of iron oxide NPs in colloidal suspension with

chloroform shows a strong absorbance peak at ~375 nm in the UV portion of the

spectrum The spectrum shows minimal absorption (05 AU) consistently throughout the

rest of the visible and near-infrared range These findings are in agreement with previously

demonstrated absorbance results for iron oxide NPs [Wang 2005] [Shi 2007] [Awwad

151

2012] and [Sathyanarayanan 2013] The succinylated PEG (capping agent) shows a strong

peak in the UV portion of the spectrum and no absorption throughout the visible range

Figure 73 Absorbance spectrum for magnetite NPs

Figure 74 Absorbance spectrum for succinylated polyethylene glycol (PEG)

152

723 Cytotoxicity Assay Results

Figure 75 Cytotoxicity results dead-cell marker fluorescence at 12- and 24-hours

exposure denotes statistical significance where plt05

A double-tailed t-test was conducted on the measured values compared to controls for

each time point None of the reagents applied to the cells demonstrated any statistically

significant effects at the 12-hour time point including digitonin the cytotoxicity positive

control These findings are important for future use of this assay in this cell type The

concentrations or periods of exposure for all three positive controls may need to be

153

increased in order to elicit a noteworthy response in this cell line The NPs did not exhibit

a statistically significant cytotoxic effect at this time point for either of the concentrations

investigated At the 24-hour time point the cytotoxicity positive control digitonin as well

as the necrosis positive control staurosporine demonstrated statistically significant

cytotoxic effects compared to untreated cells The magnetite NPs did not exhibit any

statistically significant cytotoxicity at various concentrations or time points

724 Viability Assay Results

A two-way ANOVA was run on the treatment results compared to control at all three time

points investigated The ANOVA showed a statistically significant time factor in all treated

and untreated cells attributed to the normal doubling time as expected a linear increase

was observed The normal doubling time of A549 cells is approximately 22 hours (ATCC)

this rate corresponds to our observed rate Statistical reductions in viability were found to

be very significant for the staurosporine positive control the 1 mgmL concentration of

magnetite NPs and the ionomycin positive control having p-values of 00083 00027 and

0001 respectively An initial increase in live-cells was observed in the magnetite NP

treated wells at the six-hour time point However a significant concentration-dependent

decrease in viable cells was observed at the 12-hour time point By the time the 24-hour

measurement was taken the magnetite NP-treated cells had more than recovered and the

viable-cell count was in the range of the untreated cells Taking into consideration the

range of measured values and the slope of the line it appears that the 05 mgmL NP

154

concentration may not have had lethal effects but simply inhibited cell differentiation over

the 6 to 12-hour time points

Viability

Time Point

Flu

ore

scen

ce (

RF

U)

6 hou

rs

12 h

ours

24 h

ours

0

20000

40000

60000

Digitonin

Staurosporine

Ionomycin

Magnetite NPs (1 mgmL)

Untreated Cells

Figure 76 Cell viability over exposure time Image shows overall increase in live-cell

fluorescence over time for all exposures The ionomycin positive control and 01 mgmL

concentration of magnetite NPs demonstrate a statistically significant reduction in cell

viability

The 1 mgmL NP concentration seems to have had cytotoxic effects as demonstrated by

the negative slope between the 6- and 12-hour time points In both cases the growth rate of

the NP-treated cells seems to have demonstrated an overall increase compared to control

and the slope of the line corresponding to growth rate is identical for both NP treatment

concentrations A double-tailed t-test was performed on the individual treatments

compared to control at the 24-hour time point The ionomycin positive control was the

155

only treatment that exhibited an extremely statistically significant deviation from control at

this time point No statistically significant reduction in viability was observed in the NP

treated cells at the 24-hour time point

725 Apoptosis Assay Results

The apoptosis positive control staurosporine showed a statistically significant deviation

from control at all time points The 05 mgmL NP concentration exhibited nearly identical

results to the untreated cells at the 12 and 24-hour time points No statistically significant

deviation from control was noted in the NP treated cells at any time point The higher than

average mean values observed at the 6-hour time point were not only found to be not

statistically significant due to the range of values in which low values are very close to

those of control cells but are not verified by the results of the viability assay The

ionomycin (necrosis) marker showed even further reduced apoptosis signal than the other

four treatments The apoptosis observed in the ionomycin positive control wells were

reduced compared to controls this reduction was found to be statistically significant and

very statistically significant at the 6 and 12-hour time points respectively It is important

to note that cell death is occurring in the ionomycin treated wells as evidenced by the

reduced viability of cells exposed to this compound Ionomycin is known to induce cell

death by necrosis as opposed to apoptosis Therefore the low levels of caspase an enzyme

biomarker for apoptosis specifically are to be expected No statistically significant

increase (or decrease) in apoptosis was observed for NP-treated wells compared to control

at any of the time points investigated

156

Figure 77 Apoptosis luminescence The apoptosis positive control demonstrated

statistically significant results as expected The necrosis positive control ionomycin

demonstrates statistically significant results lower than the value for untreated cells

denotes significance in which plt001 and denotes significance in which plt0001

denotes extreme statistical significance (plt00001)

The apoptosis time curve shows a linear increase in all treatments and untreated

cells except the ionomycin-treated cells which show a slight increase from 6 to 12 hours

followed by a plateau from 12 to 24 hours The apoptosis rate over time is not sufficient to

exhibit a decrease or even steady plateau in cell differentiation as evidenced by the

viability over time The apoptosis increase likely demonstrates a percent of the total

number of cells as opposed to an increased incidence of apoptosis over time

157

Apoptosis Time Curve

Time Point

Lu

min

escen

ce (

RL

U)

6 hou

r

12 h

our

24 h

our

0

2100 5

4100 5

6100 5

Staurosporine

NPs (1 mgmL)

NPs (05 mgmL)

Untreated Cells

Ionomycin

Figure 78 Apoptosis time curve Shows relatively linear increase in apoptosis over all

time points for all exposures to include untreated cells No statistically significant

differences among iron oxide NPs treatment concentrations or untreated cells were

observed

This is because we observed the normal doubling rate in the viability studies for all treated

and untreated cells Therefore there are more cells at the later time points and apoptosis in

a constant percent of the population would be expected to follow the same linear increase

that the viability graph demonstrated Although it appears from the graph that the 05

mgmL concentration exhibited increased incidence of apoptosis the overlap of the error

bars reveals a similar range and thus no statistically significant difference between the

two concentrations Only the apoptosis positive control staruosporine was found to

exhibit a statistically significant deviation from the control cells in the apoptosis assay

158

73 Discussion

This study investigated the acute in vitro cytotoxicity of two concentrations of colloidal

magnetite NPs in a human lung carcinoma cell line (A549) by comparing cytotoxicity

viability and apoptosis profiles over time Although the reliability of assays used to

investigate nanomaterial toxicity has been called into question due to the potential for

fluorescent NPs to enhance the fluorescent signal or for other metal and metal oxide NPs

to absorb the fluorescent signal [Doak 2009 2012] [Monteiro-Riviere 2009] [Han 2011]

[Love 2012] [Darolles 2013] at this time the fluorescence signal reduction by both

magnetite and maghemite NPs has been well characterized [Doak 2009] We have

accounted for the fluorescence signal absorption by the magnetite NPs at the

concentrations investigated by producing a mathematical model and correction factor using

experimental data The absorbance data demonstrates that the absorption of visible light is

consistent throughout all the frequencies detected by the assay thereby affirming the

reliability of the method The combination of assays has previously been proposed to

verify findings of a single assay in the investigation of NP toxicity [Han 2011] [Alinovi

2015] therefore our use of three assays was justified as the comparison of viability and

cytotoxicity data add another dimension of quality control to the interpretation of the data

This investigation has revealed an overall low-toxicity of magnetite NPs when

capped with PEG Despite the dose-dependent decreZase in viability at the 12-hour time

point all other parameters did not show a statistically significant deviation from the control

values The measured viability decrease at the 12-hour time point may need to be

investigated further as the viability was nearly identical to controls by the 24-hour time

159

point In addition the cytotoxicity and apoptosis profiles at 12-hours do not demonstrate a

corresponding increase in apoptotoic or necrotic (lysed) cells this time point Although the

mean apoptosis values for the NP solutions were slightly higher than the control cells the

overlap of the error bars demonstrating the range of measured values makes this not

significant The slightly elevated apoptosis signal at the 6-hour time point is not

corroborated by the viability and cytotoxicity findings as the viability was slightly higher

than the control cells at the 6-hour time point and a low number of dead cells were

measured Taking into account the increased live cell count at 6-hours and the slope of the

viability over time line in NP-treated wells it appears that the doubling time (growth rate)

of the cells was altered initially being increased from 0-6 hours halted from 6-12 hours

then increased again from 12-24 hours If the iron oxide NP solution did in fact increase

the growth rate of the cells then the slightly increased apoptosis detection as a percentage

of the total number of viable cells as demonstrated by the viability results at 6-hours still

results in a low ratio comparable to the control wells The low viability measured at 12-

hours was not observed as apoptosis by this assay therefore either cell death having been

induced by necrosis cell proliferation was impaired or both Typically we would expect

viability and cytotoxicity to be inversely proportional However it has been well-

established that prototypical anticancer agents can exhibit antiproliferative effects

(specifically a reduction in cell division) for a prolonged period of time prior to

membrane rupture Until membrane rupture occurs it is difficult to detect cell stress using

this assay Further investigation such as detection of pro-inflammatory cytokines

characteristic of necrosis and present prior to cell death could be used to determine whether

160

this pathway has been activated and when Cell cycle arrest in A549 cells in response to

potential toxin exposure has been reported previously in G-1 phase (Chang et al 2004)

G-2M cell cycle arrest (Wu et al 2005 Lee et al 2011 Wu et al 2013) S-phase arrest

(Chairuangkittiet al 2013) by nanoparticles (Choudhury et al 2013 Wu et al 2013

Kansara et al 2015) and DNA breaks have been discovered along with cell cycle arrest

(Kansara et al 2015) in a nanoparticle toxicity study Since this cell cycle (growth) arrest

is commonly observed in this cell type we must at least take into consideration the

evidence which seems to suggest that the decrease in viability is due to cell cycle arrest

The time period of cell cycle arrest is observed as a decline in viability with no

concomitant increase in cytotoxic biomarker which is exactly what was observed Caspase

activation which would have been detectable by the apoptosis marker may or may not be

measurable during this period Conversely a measurable decline in apparent viability may

be paired with a substantially reduced or unmeasurable cytotoxicity biomarker if cells died

early (typically by primary necrosis) in the exposure period (Niles et al 2008)

Considering the lack of evidence of cytotoxicity of the NPs revealed by the

cytotoxicity assay in combination with the low level of observed apoptosis in addition to

the numerous evidence suggesting cell cycle arrest as a response to toxicity in A549 cells

the main contributing factor to the observation of reduced viability (a low measurement of

viable cells) at the 12- hour time point is probably reduced proliferation both preceded and

followed by increased proliferation as opposed to cell death Another study into

nanoparticle toxicity in this cell line showed that Ag NPs reduced cell viability and

modulated cell cycle distribution with an accumulation of cells at G2M and sub-G1 phases

161

(cell death) leading to a decrease in cells at G1 (Lee et al 2011) Results suggest that Ag

NPs induce strong toxicity and G2M cell cycle arrest by a mechanism involving PKCζ

downregulation in A549 cells (Lee et al 2011) It appears that the iron oxide NPs may

also be causing a cell cycle arrest as evidenced by the decrease in viability at 12-hours

More work is needed to investigate whether or not this is the case Superparamagnetic iron

oxide NPs have shown promote cell proliferation by effecting cyclins and cyclin-

dependent kinases in human stem-cells (Huang 2009) The effect on proliferation is

probably dosage-dependent and more dosages and time-points should be investigated in

the future Therefore such NPs may very likely have a complex effect on the proliferation

cycle in certain human cell lines This effect and the mechanism(s) thereof merit

significant further research

Since PEG is an FDA-approved polymer and it has been shown to cause no

significant adverse effects [Working 1997] we do not attribute any cytotoxicity or

alterations in cell proliferation to the PEG NP coating Previous studies on dextran-coated

NPs have shown that detrimental effects of magnetite NPs may be facilitated by the

biochemical modifications to dextran by biological systems as well as the weak interaction

between the dextran coating and the nanoparticle Dextran undergoes conformational

changes and may completely desorb from the nanoparticle surface [Sonen and De Cuyper

2010] Cellular uptake of magnetite NPs coated with dextran have been degraded in acidic

lysosomes leaving a rapidly degraded iron core This iron can then induce toxic reactive

oxygen species (ROS) intermediates by the Fenton reaction [Arbab 2003] [Idee 2007]

One of the causes for the weak interaction between dextran and the NP stems from the

162

functional groups binding to the hydrocarbon polymer to the metal oxide NP Dextran uses

a hydroxyl (OH-) functional group to bind the NP (M+) As a potential solution to this

problem we succinylated our PEG cap which changes the terminal functional group from a

hydroxyl to a carbonyl (COOH-) group increasing the negative character and thus

strengthening the bond between the polymer and the NP This capping method of

incorporation of a stronger bonding FDA-approved polymer is anticipated to reduce ROS-

mediated cytotoxicity

Comprehensive toxicity profiles should include data on toxicity in multiple cell

lines in addition to animal models to include investigations on developmental effects It is

important to translate cytotoxic effects revealed by exposure to a concentration in cell

culture to a no observed adverse effects level (NOAEL) systemic dose administration

which is not necessarily straightforward Even in cases where in vivo studies have

demonstrated a NOAEL localization in specific organ systems and subsequent toxicity to

those specific cell types may not yet have been identified In vitro cytotoxicity of NP

systems in specific cell types is also useful for identifying mechanisms of toxicity after

systemic toxicity is observed Also higher concentrations of the investigational

nanomaterial than could be feasibly systemically administered may be investigated in cell

culture This is important for materials that will be targeted to a specific cell type or

administered as inhalation aerosols as in our application

163

CHapter 8

Conclusions and future work

Nanotechnology being often described as an emerging technology brings with it what we

call ldquothe promise of nanotechnologyrdquo This promise of nanotechnology hopes to realize

novel batteries magnetic and semiconductor materials individualized medicine faster

computers in vivo genetic alterations non-invasive medical procedures and countless

others A recent article was published in the magazine ldquoRisk Managementrdquo entitled ldquoThe

big risk of small particleshelliprdquo I believe this title says it all [Piper 2013] We must use

caution when embarking on such exciting new scientific ventures The excitement over our

ability to make these materials must not take precedent over the fact that our knowledge of

the toxicity of both the nanomaterials as well as the procedures for engineering them is at

present still limited Nikola Tesla the famous electrical engineer proclaimed that there

was a difference between Progress and Technology ldquoProgress benefits mankind

Technology does not necessarily do that If you have a technology that is polluting the

planet thats not progress [Tesla 1891]rdquo Therefore in the interest of progress let us

examine our methodology

164

81 Importance of Green Methodology

In 1857 Michael Faraday discovered and demonstrating that nanostructured colloidal gold

under certain lighting conditions produces different-colored solutions [Thompson 2007] It

is not until 2005 that the EPA begins reviewing new chemical notices under the Toxic

Substance Control Act (TSCA) for nanoscale materials This nearly 150-year gap in

regulation of such materials is hardly surprising During this time it was the responsibility

of the researchers to ensure safety and environmental soundness Now despite the EPA

regulation and some minimal oversight we as researchers maintain much of the

responsibility for ensuring immediate safety while tailoring our methods for the long-term

benefit of humanity Now more than ever with the population of the planet well on its

way to reaching 8 billion people we must maintain consciousness regarding the long-term

effects of our work Green chemistry standards along with our adherence to them will

undoubtedly facilitate true progress Iron of course exists naturally in the environment in

a few phases the dissolved phase as ferric (Fe3+) or ferrous (Fe2+) salts (as in our

precursor) or in the solid phase iron oxides such as goethite magnetite or Wuumlstite (as in

our product) and hematite [Ponnamperuma 1972] [Klaine 2008] [Ševců 2011] In the

case of this work I can genuinely affirm that we have done our finest to ensure safety

while minimizing long-term risk to our delicate environment As described in Chapter 2

we have replaced harsh metal nitrate precursors with chloride salts in all cases We have

used as a hydrocarbon carrier for the facilitation of epitaxial (layer-by-layer) crystal

growth as well as our stabilizing agents with constituents of vegetable oil rather than

petroleum products We have committed to the use of natural and biodegradable polymers

165

not only due to the positive results realized by their utilization Additionally we have

redistilled our waste solvents for multiple uses reducing waste from 4 L per 200 mg of

product to less than 05 L By using paraffin wax as an alternative to high purity long chain

hydrocarbons we are able to reuse this wax solvent at least five times reducing waste and

cost It is also important to note that due to the use of these environmentally friendly

solvents and precursors that the biocompatibility of our engineered materials is

intrinsically amplified

82 Bacterial Sensitivity Discussion

Despite the fact that we have demonstrated the antibacterial properties of NP-drug or NPs

alone one issue that resists prediction is the cellular permeability of P aeruginosa by the

tobramycin-loaded Fe3O4 complexes Future work may overcome the realization that very

little is known regarding uptake NPs by the individual Pseudomonas aeruginosa cells It is

known that aminoglycoside antibiotics such as tobramycin enter the cell through porin

channels along with water and electrolytes It is also known that they are not only

somewhat actively transported but may also cause nonspecific membrane toxicity even to

the point of bacterial cell lysis [Frasier 1986] The outer membrane of gram-negative

microbes is composed of lipopolysaccharide which differs from the cell membrane of other

microbes The outer membranes produce something called periplasmic protrusions under

stress conditions or upon virulence requirements while encountering a host target cell and

thus such protrusions function as virulence organelles [Yash Roy 1999] It has been

demonstrated that P aeruginosa has a comparatively large exclusion limit the actual

166

molecular weight cutoff is 3 kDa allowing the passage low molecular weight organic acids

(amino acids) carbohydrates alcohols aliphatic molecules aromatics and nitrogenous

compounds used as nutrient sources [Nikaido 2012]

As presented in Chapter 6 it appears that a destructive consequence appears under

of greater concentrations of zero-valent iron and consequently reactive oxygen species

(ROS) induced by the presence of iron Experimentation regarding the bactericidal effects

of zero-valent iron and the theoretical mechanisms leading to cell death has been

thoroughly investigated and the established findings may be referenced in any of the

following notable publications The first of which reports significant disruption of the

Escherichia coli cell membrane by zero-valent iron NPs suggesting inactivation or

enhanced the biocidal effects of dissolved iron as well as oxidative stress as mechanisms of

cell death [Lee 2008] Another report [Chen 2011] investigated the use of zero-valent iron

NPs against gram negative Escherichia coli and gram-positive Bacillus subtilis showing

that B subtilis was more tolerant to zero-valent iron NPs than E coli but states that the

bactericidal mechanism has not yet been elucidated Lastly another report claims that zero-

valent iron had no deleterious effect on total bacterial abundance in the microcosms

Surprisingly zero-valent iron with a biodegradable polyaspartate cap actually increased

bacterial populations by an order of magnitude relative to controls [Kirschling 2010]

Perhaps once naturally oxidized this material will benefit symbiotic bacterial populations

in the environment by providing beneficial doses of iron

It is possible that iron NPs may indirectly generate ROS which subsequently damage

ironndashsulfur clusters located in an assortment of metalloproteins examples are the well-

167

known NADH dehydrogenase ferredoxins hydrogenases nitrogenase coenzyme Q and

succinate dehydrogenase [Lippard 1994] This combination leads to Fentonrsquos reagent a

solution of hydrogen peroxide and iron in which iron is a catalyst that is used to oxidize

contaminants In industrial applications Fentons reagent can be used to destroy organic

compounds by catalyzing the production of additional ROS ROS generated via this

reaction can easily diffuse into the cell cytoplasm triggering ROS-induced ROS release in

the mitochondria triggering death A known mechanism of bacterial cell death induced by

zero-valent iron NPs as we demonstrated in Chapter 6 and is illustrated in Figure 81 In

our case it also may occur that initial disruption of the outside membrane of bacteria by

tobramycin assists the subsequent penetration of NP-tobramycin complexes and or iron

ions into the bacterial cell via simple diffusion since it is known that one mechanism of

action of aminoglycoside antibiotics is cell wall disruption

It may also be possible that initial disruption of the outside membrane of bacteria

by tobramycin assists the subsequent penetration of NP-tobramycin complexes and or iron

action of aminoglycoside antibiotics is interference with protein synthesis leading to cell

membrane disruption However if this is occurring it is not happening on a large scale

since no statistically significant difference in the MIC or susceptibility was noted in

tobramycin conjugated iron-oxide NPs compared to unconjugated NPs However more

work is needed to clarify the antibacterial mechanism(s) of action of iron-oxide NPs alone

and in combination with the aminoglycoside or other antibiotic drugs and to clarify the

overall role of the capping agent

168

Figure 81 Mechanisms of cell damage and response after exposure to iron-

containing NPs Iron ions released from NPs can cross the membrane via either

active cellular uptake or leakage through sites with reduced membrane integrity

Highly reactive hydroxyl radicals resulting from Fe2+ reaction with hydrogen

peroxide primarily cause oxidative damage Fe3+ could be reduced by NADH and

thus regenerating Fe2+ OHmiddot radicals could also cause damage to DNA proteins and

lipids Fe2+ may also directly damage DNA

It is apparent that the composition of the capping agent and possibly the interactions of

the capping agent with the NP surface the ROS and the cell surfaces are primarily

responsible for facilitating or negating the antimicrobial effects Since uncapped iron-oxide

NPs (~16 nm) had similar antibacterial effects as the alginate capped and alginate capped-

169

tobramycin conjugated NPs (~200 nm) whereas the PEG-capped NPs (~40 nm) were

ineffective we do not attribute these findings to size effects At least at this size range

83 Conclusions

We have presented an alternative method for the treatment of P aeruginosa biofilms in

cystic fibrosis potentially to be administered via the inhalation aerosol route Positive

inhibition of bacterial growth was observed for uncapped and alginate-capped iron-oxide

NPs and the corresponding MICs have been presented We have observed zero

susceptibility to iron-oxide NPs capped with polyethylene glycol (PEG) suggesting that

the capping agent plays a major role in enabling bactericidal ability in of the

nanocomposite Our findings suggest that the alginate-coated nanocomposites investigated

in this study have the potential to overcome the bacterial biofilm barrier possibly by

simple diffusion due to the favorable solubility of the alginate-coated NPs within the

alginate biofilm Magnetic field application increases the action likely via enhanced

diffusion of the iron-oxide NPs and NP-drug conjugates through mucin and alginate

barriers which are characteristic of CF respiratory infections We have demonstrated that

iron-oxide NPs coated with alginate as well as alginate-coated magnetite ndash tobramycin

conjugates inhibit P aeruginosa growth and biofilm formation in established colonies

which are often the most difficult to treat We have also determined that susceptibility to

tobramycin decreases for longer culture times as the colonies are allowed to differentiate

for longer periods of time However susceptibility to the iron-oxide NP compounds did

not demonstrate any comparable decrease with increasing culture time In addition these

170

findings imply that iron-oxide NPs are promising lower-cost alternatives to silver NPs in

antibacterial coatings solutions and drugs as well as other applications in which

microbial abolition or infestation prevention is sought

We report on alteration of a basic repeatable solvothermal green chemistry

synthesis method that can be used to produce iron oxide nanoparticles in various

monodispersed size ranges from 10-100 nm and in a variety of shapes (spherical

polymorphous cube wire) Taking the iron oxide NPs produced by these methods we can

convert them into zero-valent iron or iron nitride We have investigated several capping

agent compositions and provided proven methods for application of the cap We have also

demonstrated the importance of the capping agent in functionalization and antibacterial

properties of the nanocomposites

We have investigated the cytotoxicity of iron oxide NPs on a lung adenocarcinoma

cell line We have shown that in general the NPs did not exhibit a statistically significant

cytotoxic effect at the concentrations investigated We did observe a slight decrease in

viability at the 12-hour time point which was not observed at the 24-hour time point

84 Future Work

If future experiments do determine that the NPs do in fact enter the cell further issues

requiring clarification remain As discussed in Chapter 5 there are five amine groups

present on the tobramycin molecule representing the functional groups bound to the NP in

practice one of these functional groups must also bind to the molecule of the ribosomal

RNA stopping protein synthesis This property is one responsible for the bactericidal

171

effects of the drug Our drug conjugation does not allow for determination of which of the

amine groups bind to the NP capping agent however may pose an additional problem It

has been demonstrated that the loss of only one of these sterically unhindered functional

groups reduce binding affinity for RNA 10-fold [Wong 1998] Although we did not

directly witness a reduction in drug activity the antibacterial effects of the iron-containing

NP may have balanced out the loss Tobramycin also binds to a site on the bacterial 30S

and 50S ribosome preventing formation of the 70S complex inhibiting protein synthesis

in this manner Whether or not the entire drug conjugated nanocomposite remains attached

when the drug binds the binding to a site on the bacterial RNA or ribosome despite

significant steric hindrance remains to be uncovered

Despite this we have shown that iron oxide NPs zero-valent iron NPs and

tobramycin-coupled iron oxide NPs exhibit a marked antibacterial result against P

aeruginosa bacteria in planktonic and biofilm mode There is no need to ldquodetachrdquo the

drug from the NP in order to observe a bactericidal effect These findings imply that at

least a certain percentage of the bound tobramycin molecules remain active after delivery

In fact there does not appear to be a need to conjugate any drug at all as the iron oxide

NPs with biodegradable alginate coating or no coating also exhibit a significant

bactericidal effect

Some minor issues remain to be investigated regarding this work investigation into

the theory that iron oxide NPs produce ROS clarification regarding what a therapeutic

dose would be investigation regarding the feasibility of the use of these materials as

preventative medicine for CF patients and of course characterization in vivo An

172

additional more in-depth investigation into the cytotoxicity of all these materials would be

beneficial More work is needed to determine the dose-dependent cytotoxicity over a larger

range of concentrations and cell types A more straightforward method such as individual

livedead cell counting might work better due to the potential of iron oxide to absorb light

and potentially interfere with the fluorescence assay Largely our investigations into the

use of SPIONS for the treatment of chronic biofilm infections in cystic fibrosis shows

promising results for drug-susceptible as well as drug-resistant strains of Pseudomonas

aeruginosa and may in the future help to extend the life expectancy of cystic fibrosis in

both developing countries and the developed world

173

REFERENCES

[Afreen 2011] Afreen R V and E Ranganath ldquoSynthesis of monodispersed silver

nanoparticles by Rhizopus stolonifer and its antibacterial activity against MDR strains of

Pseudomonas aeruginosa from burnt patientsrdquo International Journal of Environmental

Sciences 1 no 7 (2011) 1583ndash92

[Agnihotri 2014] Agnihotri S S Mukherji S Mukherji ldquoSize-controlled silver

nanoparticles synthesized over the range 5ndash100 nm using the same protocol and their

antibacterial efficacyrdquo RSC Advances 4 no 8 (2014) 3974ndash83

[Alexiou 2006] Alexiou C R Jurgons C Seliger and H Iro ldquoMedical applications of

magnetic nanoparticlesrdquo Journal of Nanoscience and Nanotechnology 6 no 9-10 (2006)

2762-2768

[Alinovi 2015] Alinovi R M Goldoni S Pinelli M Campanini I Aliatis D Bersani P

Paolo Lottici S Iavicoli M Petyx P Mozzoni and Mutti A ldquoOxidative and pro-

inflammatory effects of cobalt and titanium oxide nanoparticles on aortic and venous

endothelial cellsrdquo Toxicology in Vitro 29 no 3 (2015) 426-437

[Allan 1973] Allan J D A Mason and A D Moss ldquoNutritional supplementation in

venous endothelial cellsrdquo Toxicology in Vitro 29 no 3 (2015) 426-437

[An 2007] An D and M R Parsek ldquoThe promise and peril of transcriptional profiling in

biofilm communitiesrdquo Current Opinion in Microbiology 10 no 3 (2007) 292-296

[Anderson 2016] Anderson C and C Flask ldquoID 63 rapid 3D preclinical quantitative

lung imaging with ultrashort-echo time (UTE) MRI in a mouse model of cystic fibrosis

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[Andrauml 2007] Andrauml W and H Nowak eds Magnetism in Medicine A Handbook John

Wiley amp Sons 2007

[Annereau 2003] Annereau J Y Ko and P Pedersen ldquoCystic fibrosis transmembrane

conductance regulator the NBF1+ R (nucleotide-binding fold 1 and regulatory domain)

segment acting alone catalyses a Co2+Mn2+Mg2+-ATPase activity markedly inhibited by

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[Ansari 2014] Ansari M A H M Khan A A Khan S S Cameotra Q Saquib and J

Musarrat ldquoGum arabic capped‐silver nanoparticles inhibit biofilm formation by multi‐drug

174

resistant strains of Pseudomonas aeruginosardquo Journal of Basic Microbiology 54 no 7

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[Anselmo 2016] Anselmo A C and S Mitragotri ldquoNanoparticles in the clinicrdquo

Bioengineering amp Translational Medicine 1 no 1 (2016) 10-29

[Anwar 1989] Anwar H T Van Biesen M Dasgupta K Lam and J W Costerton

ldquoInteraction of biofilm bacteria with antibiotics in a novel in vitro chemostat systemrdquo

Antimicrobial Agents and Chemotherapy 33 no 10 (1989) 1824-1826

[Arakha 2015] Arakha M S Pal D Samantarrai T K Panigrahi B C Mallick K

Pramanik B Mallick and S Jha ldquoAntimicrobial activity of iron oxide nanoparticle upon

modulation of nanoparticle-bacteria interfacerdquo Scientific Reports 5 (2015) 14813 (12+4

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[Arbab 2003] Arbab A S L A Bashaw B R Miller E K Jordan B K Lewis H

Kalish and J A Frank ldquoCharacterization of biophysical and metabolic properties of cells

labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular

MR imagingrdquo Radiology 229 no 3 (2003) 838-846

[Armijo 2012a] Armijo L M Y I Brandt D Mathew S Yadav S Maestas A C

Rivera N C Cook N J Withers G A Smolyakov N Adolphi T C Monson D L

Huber H D C Smyth and M Osiński ldquoIron oxide nanocrystals for magnetic

hyperthermia applicationsrdquo Nanomaterials 2 no 2 (2012) 134-146

[Armijo 2012b] Armijo L M Y I Brandt N J Withers J B Plumley N C Cook J B

Plumley M Kopciuch A C Rivera S Yadav G A Smolyakov D L Huber H D C

Smyth and M Osiński ldquoMultifunctional superparamagnetic nanocrystals for imaging and

targeted drug delivery to the lungrdquo Colloidal Nanocrystals for Biomedical Applications

VII (W J Parak M Osiński and K Yamamoto Eds) SPIE International Symposium on

Biomedical Optics BiOS 2012 San Francisco California 21-23 January 2012

Proceedings of SPIE Vol 8232 Paper 82320M (11 pp) doi10111712913577

[Armijo 2014] Armijo L M M Kopciuch Z Olszoacutewka and S J Wawrzyniec A C

Rivera J B Plumley N C Cook Y I Brandt G A Smolyakov D L Huber H D C

Smyth and M Osinski ldquoDelivery of tobramycin coupled to iron oxide nanoparticles

across the biofilm of mucoidal Pseudonomas aeruginosa and investigation of its efficacyrdquo

Colloidal Nanoparticles for Biomedical Applications IX (W J Parak M Osiński and K

Yamamoto Eds) SPIE International Symposium on Biomedical Optics BiOS 2014 San

Francisco California 1-3 February 2014 Proceedings of SPIE Vol 8955 Paper 9550I

(12 pp) doi101117122043340

175

[Asharani 2008] Asharani P V Y L Wu Z Y Gong and S Valiyaveettil ldquoToxicity of

silver nanoparticles in zebrafish modelsrdquo Nanotechnology 19 no 25 (2008) 255102 (8

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[Auffan 2008] Auffan M W Achouak J Rose M-A Roncato C Chaneacuteac D T Waite

A Masion J C Woicik M R Wiesner and J-Y Bottero ldquoRelation between the redox

state of iron-based nanoparticles and their cytotoxicity toward Escherichia colirdquo

Environmental Science amp Technology 42 no 17 (2008) 6730-6735

[Awwad 2012] Awwad A M and N M Salem ldquoA green and facile approach for

synthesis of magnetite nanoparticlesrdquo Nanoscience and Nanotechnology 2 no 6 (2012)

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[Baltch 1994] Baltch A L and R P Smith ldquoPseudomonas aeruginosa infections and

treatmentrdquo Infectious Disease and Therapy Series 12 (1994)

[Bao 1994] Bao X H R M Metzger and M Carbucicchio ldquoSynthesis and properties of

αrdquo‐Fe16N2 in magnetic particlesrdquo Journal of Applied Physics 75 no 10 (1994) 5870 5872

[Bao 2005] Bao Y A B Pakhomov and K M Krishnan ldquoA general approach to

synthesis of nanoparticles with controlled morphologies and magnetic propertiesrdquo Journal

of Applied Physics 97 no 10 (2005) 10J317

[Baronzio 2006] Baronzio G F and E D Hager eds Hyperthermia in Cancer

Treatment A Primer Landes Bioscience and Springer Science amp Business Media 2006

[Basak 2007] Basak S D R Chen and P Biswas ldquoElectrospray of ionic precursor

solutions to synthesize iron oxide nanoparticles Modified scaling lawrdquo Chemical

Engineering Science 62 no 4 (2007) 1263-1268

[Batten 1965] Batten John ldquoCystic fibrosis A reviewrdquo British Journal of Diseases of the

Chest 59 no 1 (1965) 9-8

[Bauernfeind 1987] Bauernfeind A K Rotter and C H Weisslein-Pfister ldquoSelective

procedure to isolate Haemophilus influenzae from sputa with large quantities of

Pseudomonas aeruginosardquo Infection 15 no 4 (1987) 278-280

[Beer 2012] Beer C R Foldbjerg Y Hayashi D S Sutherland and H Autrup ldquoToxicity

of silver nanoparticlesmdashnanoparticle or silver ionrdquo Toxicology Letters 208 no 3 (2012)

286ndash92

[Behera 2012] Behera S S J K Patra K Pramanik N Panda and H Thatoi

ldquoCharacterization and evaluation of antibacterial activities of chemically synthesized iron

176

oxide nanoparticlesrdquo World Journal of Nano Science and Engineering 2 no 4 (2012)

196ndash200

[Benamara 2014] Benamara H C Rihouey I Mohamed AB Mlouka J Hardouin T

Jouenne and S Alexandre ldquoCharacterization of Membrane Lipidome Changes in

Pseudomonas aeruginosa during Biofilm Growth on Glass Woolrdquo (2014) e108478

[Berlyne 2000] Berlyne G S K Parameswaran D Kamada A Efthimiadis and F E

Hargreave ldquoA comparison of exhaled nitric oxide and induced sputum as markers of

airway inflammationrdquo Journal of Allergy and Clinical Immunology 106 no 4 (2000)

638-644

[Berry 2004] Berry C C S Wells S Charles G Aitchison and A S Curtis ldquoCell

response to dextran-derivatised iron oxide nanoparticles post internalizationrdquo Biomaterials

25 no 23 (2004) 5405-5413

[Bezeljak 2012] Bezeljak U A Golob M Jerala L Kandunc Z Lužnik F Pavlovec B

Pirš M Somrak M Stražar D Vucko U Zupancic M Bencina V Forstneric T Lebar

A Majerle A Oblak R Gaber J Lonzarić M Mraz M Moškon A Smole and R

Jerala Switch IT Inducible Therapeutics iGEM 2012 Synthetic Biology Jamboree

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Degradationampoldid=290454 accessed 12 Jan 2014

[Bilberg 2011] Bilberg K K B Doslashving K Beedholm and E Baatrup ldquoSilver

nanoparticles disrupt olfaction in Crucian carp (Carassius carassius) and Eurasian perch

(Perca fluviatilis)rdquo Aquatic Toxicology 104 no 1 (2011) 145ndash52

[Borm 2004] Borm P J R P Schins and C Albrecht ldquoInhaled particles and lung cancer

part B paradigms and risk assessmentrdquo International Journal of Cancer 110 no 1 (2004)

3-14

[Bossi 2004] Bossi A G Casazza R Padoan and S Milani ldquoWhat is the incidence of

cystic fibrosis in Italy Data from the National Registry (1988-2001)rdquo Human Biology 76

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[Boucher 2009] Boucher H W G H Talbot J S Bradley J E Edwards D Gilbert L

B Rice M Scheld B Spellberg and J Bartlett ldquoBad bugs no drugs no ESKAPE An

update from the Infectious Diseases Society of Americardquo Clinical Infectious Diseases 48

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Bioconjugate Chemistry 4 no 4 (1993) 262-267

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J J Rahal S Brooks S Cebular and J Quale ldquoEmergence of carbapenem-resistant

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resistant TEM-30 β-lactamases in New York Cityrdquo Clinical Infectious Diseases 39 no 1

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[Brandt 2013] Brandt Y I L M Armijo A C Rivera J B Plumley N C Cook G A

Smolyakov H D C Smyth and M Osiński ldquoEffectiveness of tobramycin conjugated to

iron oxide nanoparticles in treating infection in cystic fibrosisrdquo Colloidal Nanoparticles for

Biomedical Applications VIII (W J Parak M Osiński and K Yamamoto Eds) SPIE

International Symposium on Biomedical Optics BiOS 2013 San Francisco California 2-4

February 2013 Proceedings of SPIE Vol 8595 Paper 85951C (9 pp)

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Stein and B Dragnea ldquoInfluence of iron oleate complex structure on iron oxide

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[Brown 2012] Brown W H S Foote B L Iverson E V Anslyn Organic Chemistry 6th

ed BrooksCole Cengage Learning 2012

[Buckley 2006] Buckley P R G H McKinley T S Wilson W Small IV W J Benett

J P Bearinger M W McElfresh and D J Maitland ldquoInductively heated shape memory

polymer for the magnetic actuation of medical devicesrdquo IEEE Transactions on Biomedical

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[Burney 2012] Burney T J and J C Davies ldquoGene therapy for the treatment of cystic

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[Busch 1866] Busch W ldquoUumlber den Einfluss welchen heftigere Erysipelen zuweilen auf

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[Busch 1989] Busch R ldquoOn the history of cystic fibrosisrdquo Acta Universitatis Carolinae

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[Cadogan 1997] Cadogan J M ldquoAre there giant magnetic moments in Fe-nitridesrdquo

Australian Journal of Physics 50 no 6 (1997) 1093-1102

[Cai 2007] Cai W and J Q Wan ldquoFacile synthesis of superparamagnetic magnetite

nanoparticles in liquid polyolsrdquo Journal of Colloid and Interface Science 305 no 2

(2007) 366-370

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an Opportunistic Pathogen Plenum Press New York 1993

[Cardo 2004] Cardo D T Horan M Andrus M Dembinski J Edwards G Peavy J

Tolson and D Wagner ldquoNational Nosocomial Infections Surveillance (NNIS) System

Report data summary from January 1992 through June 2004 issued October 2004rdquo

American Journal of Infection Control 32 no 8 (2004) 470ndash85

[Casula 2006] Casula M F Y-W Jun D J Zaziski E M Chan A Corrias A P

Alivisatos ldquoThe concept of delayed nucleation in nanocrystal growth demonstrated for the

case of iron oxide nanodisksrdquo Journal of the American Chemical Society 128 no 5

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[Cavaliere 2015] Cavaliere E S De Cesari G Landini E Riccobono L Pallecchi G M

Rossolini and L Gavioli ldquoHighly bactericidal Ag nanoparticle films obtained by cluster

beam depositionrdquo Nanomedicine 11 no 6 (2015) 1417ndash23

[Chairuangkitti 2013] Chairuangkitti P S Lawanprasert S Roytrakul S Aueviriyavit

D Phummiratch K Kulthong P Chanvorachote and R Maniratanachote ldquoSilver

nanoparticles induce toxicity in A549 cells via ROS-dependent and ROS-independent

pathwaysrdquo Toxicology in Vitro 27 no 1 (2013) 330-338

[Chang 2004] Chang GC SL Hsu JR Tsai FP Liang SY Lin GT Sheu and CY

Chen ldquoMolecular mechanisms of ZD1839-induced G1-cell cycle arrest and apoptosis in

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[Chase 1979] Chase H P M A Long and M H Lavin ldquoCystic fibrosis and

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[Chen 2011] Chen J W Z M Xiu G V Lowry and P J J Alvarez nrdquo Water

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secretions in cystic fibrosis of the pancreas and bronchiectasisrdquo Pediatrics 24 no 5

(1959) 739-745

[Childers 2007] Childers M G Eckel A Himmel and J Caldwell ldquoA new model of

cystic fibrosis pathology Lack of transport of glutathione and its thiocyanate conjugatesrdquo

Medical Hypotheses 68 no 1 (2007) 101-112

[Chin 2007] Chin A B and I I Yaacob ldquoSynthesis and characterization of magnetic

iron oxide nanoparticles via wo microemulsion and Massarts procedurerdquo Journal of

Materials Processing Technology 191 no 1-3 (2007) 235-237

[Cho 2005] Cho K-H J-E Park T Osaka and S-G Park ldquoThe study of antimicrobial

activity and preservative effects of nanosilver ingredientrdquo Electrochimica Acta 51 no 5

(2005) 956ndash60

[Choudhury 2013] Choudhury D P L Xavier K Chaudhari R John AK Dasgupta T

Pradeep and G Chakrabarti ldquoUnprecedented inhibition of tubulin polymerization directed

by gold nanoparticles inducing cell cycle arrest and apoptosisrdquo Nanoscale 5 no 10

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[Chow 2007] Chow A H L H H Y Tong P Chattopadhyay and B Y Shekunov

ldquoParticle engineering for pulmonary drug deliveryrdquo Pharmaceutical Research 24 no 3

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[Chudasama 2010] Chudasama B A K Vala N Andhariya N R V Mehta and R V

Upadhyay ldquoHighly bacterial resistant silver nanoparticles synthesis and antibacterial

activitiesrdquo Journal of Nanoparticle Research 12 no 5 (2010) 1677ndash85

[Chung 2002] Chung F N Barnes M Allen R Angus P Corris A Knox J Miles A

Morice J OReilly and M Richardson ldquoAssessing the burden of respiratory disease in the

UKrdquo Respiratory Medicine 96 no 12 (2002) 963-975

[Clancy 2012] Clancy J P S M Rowe F J Accurso M L Aitken R S Amin M A

Ashlock M Ballmann M P Boyle I Bronsveld P W Campbell K De Boeck S H

Donaldson H L Dorkin J M Dunitz P R Durie M Jain A Leonard K S McCoy R

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Botfield C L Ordontildeez G T Spencer-Green L Vernillet S Wisseh K Yen and M W

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Konstan ldquoResults of a phase IIa study of VX-809 an investigational CFTR corrector

compound in subjects with cystic fibrosis homozygous for the F508del-CFTR mutationrdquo

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[Corey 1988] Corey M F J McLaughlin M Williams and H Levison ldquoA comparison

of survival growth and pulmonary function in patients with cystic fibrosis in Boston and

Torontordquo Journal of Clinical Epidemiology 41 no 6 (1988) 583-591

[Cornell 2006] Cornell R M and U Schwertmann The Iron Oxides Structure

Properties Reactions Occurrences and Uses 2nd ed John Wiley amp Sons 2006

[Coyne 2009] Coyne D W ldquoFerumoxytol for treatment of iron deficiency anemia in

patients with chronic kidney diseaserdquo Expert Opinion on Pharmacotherapy 10 no 15

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[Crozier 1974] Crozier D N ldquoCystic fibrosis a not-so-fatal diseaserdquo Pediatric Clinics of

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[Danes 1968] Danes B S and A G Bearn A genetic cell marker in cystic fibrosis of the

pancreasrdquo The Lancet 291 no 7551 (1968) 1061-1063

[Dang 2006] Dang J M and K W Leong ldquoNatural polymers for gene delivery and

tissue engineering Advanced Drug Delivery Reviews 58 no 4 (2006) 487-499

[Darrolles 2013] Darolles C N Sage J Armengaud and V Malard V ldquoIn vitro

assessment of cobalt oxide particle toxicity identifying and circumventing interferencerdquo

Toxicology in Vitro 27 no 6 (2013) 1699-1710

[Darwish 2015] Darwish M S A N H A Nguyen A Ševců and I Stibor

ldquoFunctionalized magnetic nanoparticles and their effect on Escherichia coli and

Staphylococcus aureusrdquo Journal of Nanomaterials (2015)416012 (10 pp)

[Davey 2003] Davey M E N C Caiazza and G A OToole ldquoRhamnolipid surfactant

production affects biofilm architecture in Pseudomonas aeruginosa PAO1rdquo Journal of

Bacteriology 185 no 3 (2003) 1027-1036

[Davis 2006] Davis P B ldquoCystic fibrosis since 1938rdquo American Journal of Respiratory

and Critical Care Medicine 173 no 5 (2006) 475-482

[De Boeck and Amaral 2016] De Boeck K and M D Amaral ldquoProgress in therapies for

cystic fibrosisrdquo The Lancet Respiratory Medicine 4 no 8 (2016) 662-674

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[Denning 1968] Denning C R S C Sommers and H J Quigley ldquoInfertility in male

patients with cystic fibrosisrdquo Pediatrics 41 no 1 (1968) 7-17

[Diao 2009] Diao M H and M S Yao ldquoUse of zero-valent iron nanoparticles in

inactivating microbesrdquo Water Research 43 no 20 (2009) 5243-5251

[Doak 2009] Doak S H S M Griffiths Bella Manshian N Singh P M Williams A P

Brown and G J S Jenkins ldquoConfounding experimental considerations in

nanogenotoxicologyrdquo Mutagenesis 24 no 4 (2009) 285-293

[Dobson 2006] Dobson J ldquoMagnetic nanoparticles for drug deliveryrdquo Drug Development

Research 67 no 1 (2006) 55-60

[Dodge 2007] Dodge J A P A Lewis M Stanton and J Wilsher ldquoCystic fibrosis

mortality and survival in the UK 1947ndash2003rdquo European Respiratory Journal 29 no 3

(2007) 522-526

[Dong 2012] Dong P V C H Ha L T Binh and J Kasbohm ldquoChemical synthesis and

antibacterial activity of novel-shaped silver nanoparticlesrdquo International Nano Letters 2

no 1 (2012) 9 (9 pp)

[Dupuis 2005] Dupuis A D Hamilton D E C Cole and M Corey ldquoCystic fibrosis

birth rates in Canada a decreasing trend since the onset of genetic testingrdquo The Journal of

Pediatrics 147 no 3 (2005) 312-315

[Duraacuten 2007] Duraacuten N PD Marcato G I H De Souza O L Alves and E Esposito

ldquoAntibacterial effect of silver nanoparticles produced by fungal process on textile fabrics

and their effluent treatmentrdquo Journal of Biomedical Nanotechnology 3 no 2 (2007) 203ndash

8

[Eck 1999] Eck B R Dronskowski M Takahashi and S Kikkawa ldquoTheoretical

calculations on the structures electronic and magnetic properties of binary 3d transition

metal nitridesrdquo Journal of Materials Chemistry 9 no 7 (1999) 1527-1537

[Eid 2013] Eid M and E Araby ldquoBactericidal effect of poly(acrylamideitaconic acid)-

silver nanoparticles synthesized by gamma irradiation against Pseudomonas aeruginosardquo

Applied Biochemistry and Biotechnology 171 no 2 (2013) 469ndash87

[Elborn 1991] Elborn J Stuart D J Shale and J R Britton ldquoCystic fibrosis current

survival and population estimates to the year 2000rdquo Thorax 46 no 12 (1991) 881-885

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[El-Kheshen 2012] El-Kheshen A A and S F G El-Rab ldquoEffect of reducing and

protecting agents on size of silver nanoparticles and their anti-bacterial activityrdquo Der

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[Emeka 2014] Emeka E E O C Ojiefoh C Aleruchi L A Hassan O M Christiana

M Rebecca E O Dare and A E Temitope ldquoEvaluation of antibacterial activities of

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[EPA 2015] EPA ldquoBasics of Green Chemistryrdquo Accessed February 18 2015

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[Falgas 2005] Falagas M E S K Kasiakou and L D Saravolatz ldquoColistin the revival

of polymyxins for the management of multidrug-resistant gram-negative bacterial

infectionsrdquo Clinical Infectious Diseases 40 no 9 (2005) 1333ndash41

[Falgas 2007] Falagas M E and I A Bliziotis ldquoPandrug-resistant Gram-negative

bacteria the dawn of the post-antibiotic erardquo International Journal of Antimicrobial

Agents 29 no 6 (2007) 630ndash6

[Fannin 1989] Fannin P C and S W Charles ldquoThe study of a ferrofluid exhibiting both

Brownian and Neacuteel relaxationrdquo Journal of Physics D Applied Physics 22 no 1 (1989)

187-191

[Fannin 1994] Fannin P C Y P Kalmykov and S W Charles ldquoOn the use of

frequency-domain measurements to investigate time-domain magnetization decay in a

ferrofluidrdquo Journal of Physics D Applied Physics 27 no 2 (1994) 194-197

[Farrell 2007] Farrell P S Joffe L Foley G J Canny P Mayne and M Rosenberg

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Medical Journal 100 no 8 (2007) 557-560

[Farrell 2008] Farrell P M ldquoThe prevalence of cystic fibrosis in the European Unionrdquo

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[Feuchtbaum 2012] Feuchtbaum L J Carter S Dowray R J Currier and F Lorey

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[Fick 1992] Fick Jr R B F Sonoda and D B Hornick ldquoEmergence and persistence of

Pseudomonas aeruginosa in the cystic fibrosis airwayrdquo In Seminars in Respiratory

Infections 7 no 3(1992)168-178

183

[Fishkum 1985] Fiskum G ldquoIntracellular levels and distribution of Ca2+ in digitonin-

permeabilized cellsrdquo Cell Calcium 6 no 1-2 (1985) 25-37

[FitzSimmons 1993] FitzSimmons S C ldquoThe changing epidemiology of cystic fibrosisrdquo

The Journal of Pediatrics 122 no 1 (1993) 1-9

[Foldbjerg 2011] Foldbjerg R D A Dang and H Autrup ldquoCytotoxicity and genotoxicity

of silver nanoparticles in the human lung cancer cell line A549rdquo Archives of Toxicology

85 no 7 (2011) 743ndash50

[Fourmy 1996] Fourmy D M I Recht S C Blanchard J D Puglisi ldquoStructure of the A-

site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside

antibioticrdquo Science 274 (1996) 1367ndash1371

[Fourmy 1998] Fourmy D M I Recht and J D Puglisi ldquoBinding of neomycin-class

aminoglycoside antibiotics to the A-site of 16 S rRNArdquo Journal of Molecular Biology

277 no 2 (1998) 347-362

[Franci 2015] Franci G A Falanga S Galdiero L Palomba M Rai G Morelli M

Galdiero ldquoSilver nanoparticles as potential antibacterial agentsrdquo Molecules 20 no 5

(2015) 8856ndash74

[Fraser 1986] Fraser C M ed The Merck Veterinary Manual Sixth Edition Merck amp Co

1986

[Frederiksen 1996] Frederiksen B S Lanng C Koch and N Hoslashlby ldquoImproved survival

in the Danish center‐treated cystic fibrosis patients Results of aggressive

treatmentrdquo Pediatric Pulmonology 21 no 3 (1996) 153-158

[Frizzel 2012] Frizzell R A and J W Hanrahan ldquoPhysiology of epithelial chloride and

fluid secretionrdquo Cold Spring Harbor Perspectives in Medicine 2 no 6 (2012) a009563

[Gabriel 1994] Gabriel S E K N Brigman B H Koller R C Boucher and M J

Stutts ldquoCystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse

modelrdquo Science 266 no 5182 (1994) 107-109

[Gacesa 1990] Gacesa P and N J Russell ldquoThe Structure and Properties of Alginaterdquo

Pseudonomas Infection and Alginates Biochemistry Genetics and Pathology (P Gacesa

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[Gash 2001] Gash A E T M Tillotson J H Satcher J F Poco L W Hrubesh and R

L Simpson ldquoUse of epoxides in the sol-gel synthesis of porous iron (III) oxide monoliths

from Fe (III) saltsrdquo Chemistry of Materials 13 no 3 (2001) 999-1007

[Ge 2009] Ge S X Y Shi K Sun C P Li C Uher J R Baker Jr M M Banaszak

Holl and B G Orr ldquoFacile hydrothermal synthesis of iron oxide nanoparticles with

tunable magnetic propertiesrdquo The Journal of Physical Chemistry C 113 no 31 (2009)

13593-13599

[Geelen 2005] Geelen Math JH ldquoThe use of digitonin-permeabilized mammalian cells for

measuring enzyme activities in the course of studies on lipid metabolismrdquo Analytical

Biochemistry 347 no 1 (2005) 1ndash9 doi101016jab200503032 PMID 16291302

[Giessen 2016] Giessen T W and P A Silver PA ldquoConverting a natural protein

compartment into a nanofactory for the size-constrained synthesis of antimicrobial silver

nanoparticlesrdquo ACS Synthetic Biology 5 no 12 (2016) 1497ndash504

[Gilani 2005] Gilani K A R Najafabadi M Barghi and M Rafiee‐Tehrani ldquoThe effect

of water to ethanol feed ratio on physical properties and aerosolization behavior of spray

dried cromolyn sodium particlesrdquo Journal of Pharmaceutical Sciences 94 no 5 (2005)

1048-1059

[Gilligan 1991] P H Gilligan ldquoMicrobiology of airway disease in patients with cystic

fibrosisrdquo Clinical Microbiology Reviews vol 4 (1) pp 35-51 Jan 1991

[Gould 1988] Gould S J and S Subramani ldquoFirefly luciferase as a tool in molecular and

cell biologyrdquo Analytical Biochemistry 175 no 1 (1988) 5-13

[Govan 1996] Govan J R and V Deretic ldquoMicrobial pathogenesis in cystic fibrosis

Mucoid Pseudomonas aeruginosa and Burkholderia cepaciardquo Microbiological Reviews

60 no 3 (1996) 539-574

[Gracey 1969] Gracey M V Burke and C M Anderson ldquoTreatment of abdominal pain

in cystic fibrosis by oral administration of n-acetyl cysteinerdquo Archives of Disease in

Childhood 44 no 235 (1969) 404-405

[Grachev 2001] Grachev S D M Borsa S Vongtragool and D O Boerma ldquoThe

growth of epitaxial iron nitrides by gas flow assisted MBErdquo Surface Science 482 (2001)

802-808

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[Grassmeacute 2000] Grassmeacute H S Kirschnek J Riethmueller A Riehle G von Kuumlrthy F

Lang M Weller and E Gulbins ldquoCD95CD95 ligand interactions on epithelial cells in

host defense to Pseudomonas aeruginosardquo Science 290 no 5491 (2000) 527-530

[Greenwood 1999] Greenwood R and K Kendall ldquoSelection of suitable dispersants for

aqueous suspensions of zirconia and titania powders using acoustophoresisrdquo Journal of the

European Ceramic Society 19 no 4 (1999) 479ndash88

[Griesenbach 2006] Griesenbach U D M Geddes and E W F W Alton ldquoGene therapy

progress and prospects cystic fibrosisrdquo Gene Therapy 13 no 14 (2006) 1061-1067

[Grottone 2014] Grottone G T R R Loureiro J Covre E B Rodrigues J Aacute Pereira

Gomes ldquoARPE-19 cell uptake of small and ultrasmall superparamagnetic iron oxiderdquo

Current Eye Research 39 no 4 (2014) 403ndash10

[Gupta 2005] Gupta A K and M Gupta ldquoSynthesis and surface engineering of iron

oxide nanoparticles for biomedical applicationsrdquo Biomaterials 26 no 18 (2005) 3995-

4021

[Hacein-Bey-Albina 2008] Hacein-Bey-Abina S A Garrigue G P Wang J Soulier A

Lim E Morillon E Clappier L Caccavelli E Delabesse K Beldjord and V Asnafi

Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1rdquo

The Journal of Clinical Investigation 118 no 9 (2008) 3132-3142

[Haumlfeli 1998] Haumlfeli U ldquoThe history of magnetism in medicinerdquo Magnetism in Medicine

A Handbook Second Edition (1998) 1-25

[Haghighi 2016] Haghighi Pak Z H Abbaspour N Karimi and A Fattahi ldquoEco-friendly

synthesis and antimicrobial activity of silver nanoparticles using Dracocephalum

moldavica seed extractrdquo Applied Sciences 6 no 3 (2016) 69 (10 pp)

[Hamishehkar 2012] Hamishehkar H Y Rahimpour and Y Javadzadeh ldquoThe role of

carrier in dry powder inhalerrdquo INTECH Open Access Publisher 2012

[Han 2011] Han X R Gelein N Corson P Wade-Mercer J Jiang P Biswas J N

Finkelstein A Elder and G Oberdoumlrster G ldquoValidation of an LDH assay for assessing

nanoparticle toxicityrdquo Toxicology 287 no 1 (2011) 99-104

[Hanoar 2012] Hanaor D M Michelazzi C Leonelli and C C Sorrell ldquoThe effects of

carboxylic acids on the aqueous dispersion and electrophoretic deposition of ZrO2rdquo

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[Hattori 2001] Hattori T N Kamiya and Y Kato ldquoMagnetic properties of Fe16N2 fine

particlesrdquo Journal of the Magnetics Society of Japan 25 (2001) 927-930

[Hauser 2003] Hauser A R and J Rello Severe Infections Caused by Pseudomonas

aeruginosa Springer London March 2003

[Hays 1945] Hays EE IC Wells PA Katzman CK Cain FA Jacobs SA Thayer

EA Doisy WL Gaby EC Roberts RD Muir CJ Carroll ldquoAntibiotic Substances

produced by Pseudomonas aeruginosardquo Biological Chemistry 159 no 3 (1945) 725ndash50

[Hearst 1995] Hearst J E and K E Elliott ldquoIdentifying the killer in cystic fibrosisrdquo

Nature Medicine 1 no 7 (1995) 626

[Henderson 1908a] Henderson L J ldquoConcerning the relationship between the strength of

acids and their capacity to preserve neutralityrdquo American Journal of Physiology 21 no 2

(1908) 173ndash9

[Henderson 1908b] Henderson L J ldquoThe theory of neutrality regulation in the animal

organismrdquo American Journal of Physiology 21 no 4 (1908) 427ndash48

[Henle 1997] Henle E S and S Linn ldquoFormation prevention and repair of DNA

damage by ironhydrogen peroxiderdquo Journal of Biological Chemistry 272 no 31 (1997)

19095-19098

[Hergt 1998] Hergt R W Andra C G dAmbly I Hilger W A Kaiser U Richter and

H-G Schmidt ldquoPhysical limits of hyperthermia using magnetite fine particlesrdquo IEEE

Transactions on Magnetics 34 no 5 (1998) 3745-3754

[Hergt 2006] Hergt R S Dutz R Muumlller and M Zeisberger ldquoMagnetic particle

hyperthermia Nanoparticle magnetism and materials development for cancer therapyrdquo

Journal of Physics Condensed Matter 18 no 38 (2006) S2919-S2934

[Hermanson 2013] Hermanson G T Bioconjugate Techniques Second ed Academic

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[Hickey 2003] Hickey A J ed Pharmaceutical Inhalation Aerosol Technology CRC

Press 2003

[Hickey 2007] Hickey A J H M Mansour M J Telko Z Xu H D C Smyth T

Mulder R McLean J Langridge D Papadopoulos ldquoPhysical characterization of

component particles included in dry powder inhalers II Dynamic characteristicsrdquo Journal

of Pharmaceutical Sciences 571 no 96 (2007) 1302-1319

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[Hide 1969] Hide D W and D Burman ldquoAn infant with both cystic fibrosis and coeliac

diseaserdquo Archives of Disease in Childhood 44 no 236 (1969) 533

[Hirsch 2003] Hirsch L R R J Stafford J A Bankson S R Sershen B Rivera R E

Price J D Hazle N J Halas and J L West ldquoNanoshell-mediated near-infrared thermal

therapy of tumors under magnetic resonance guidancerdquo Proceedings of the National

Academy of Sciences 100 no 23 (2003) 13549-13554

[Hsueh 2017] Hsueh Y-H P-H Tsai K-S Lin W-J Ke C-L Chiang ldquoAntimicrobial

effects of zero-valent iron nanoparticles on gram-positive Bacillus strains and gram-

negative Escherichia coli strainsrdquo Journal of Nanobiotechnology 3 no 15 (2017)77 (12

pp)

[Hu 2007] Hu X L J C Yu J M Gong Q Li and G S Li ldquoα‐Fe2O3 nanorings

prepared by a microwave‐assisted hydrothermal process and their sensing

propertiesrdquo Advanced Materials 19 no 17 (2007) 2324-2329

[Huang 2009] Huang D-M J-K Hsiao Y-C Chen L-Y Chien M Yao Y-K Chen

B-S Ko S-C Hsu L-A Tai and H-Y Cheng ldquoThe promotion of human mesenchymal

stem cell proliferation by superparamagnetic iron oxide nanoparticlesrdquo Biomaterials 30

no 22 (2009) 3645-3651

[Hyeon 2003] Hyeon T G ldquoChemical synthesis of magnetic nanoparticlesrdquo Chemical

Communications 8 (2003) 927-934

[Idee 2007] Idee J M M Port I Raynal M Schaefer B Bonnemain P Prigent P

Robert C Robic and C Corot C ldquoSuperparamagnetic nanoparticles of iron oxides for

magnetic resonance imaging applicationsrdquo Nanotechnologies for the Life Sciences 10

(2007) 51-84

[Iida 2007] Iida H K Takayanagi T Nakanishi and T Osaka ldquoSynthesis of Fe3O4

nanoparticles with various sizes and magnetic properties by controlled

hydrolysisrdquo Journal of Colloid and Interface Science 314 no 1 (2007) 274-280

[Indira 2010] Indira T K and P K Lakshmi ldquoMagnetic nanoparticlesmdashA

reviewrdquo International Journal of Pharmarmaceutical Sciences and Nanotechnology 3 no

3 (2010) 1035-1042

[Jack 1951] Jack K H ldquoThe iron-nitrogen system The preparation and the crystal

structures of nitrogen-austenite (γ) and nitrogen-martensite (αrsquo)rdquo Proceedings of the Royal

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[Javanbakht 2016] Javanbakht T S Laurent D Stanicki and K J Wilkinson ldquoRelating

the surface properties of superparamagnetic iron oxide nanoparticles (SPIONs) to their

bactericidal effect towards a biofilm of Streptococcus mutansrdquo PLoS ONE 11 no 4

(2016) e0154445 (13 pp)

[Jensen 1987] Jensen T S S Pedersen S Garne C Heilmann N Hoslashiby and C Koch

ldquoColistin inhalation therapy in cystic fibrosis patients with chronic Pseudomonas

aeruginosa lung infectionrdquo Journal of Antimicrobial Chemotherapy 19 no 6 (1987) 831-

838

[Ji 2010] Ji N X Liu and J-P Wang ldquoTheory of giant saturation magnetization in α-

Fe16N2 Role of partial localization in ferromagnetism of 3d transition metalsrdquo New

Journal of Physics 12 no 6 (2010) 063032

[Johannsen 2007] Johannsen M U Gneveckow B Thiesen K Taymoorian C H Cho

N Waldoumlfner R Scholz A Jordan S A Loening and P Wust ldquoThermotherapy of

prostate cancer using magnetic nanoparticles Feasibility imaging and three-dimensional

temperature distributionrdquo European Urology 52 no 6 (2007) 1653-1662

[Johnson 1984] Johnson Sir R ldquoHistory of the Cystic Fibrosis Research Trustrdquo 20th

Anniversary Meeting Brighton (1984) pp 3-6

[Kadasi 1997] Kadasi L H Polakova A Zatkova and H Kayserova ldquoDistribution of 9

common mutations in the CFTR gene in Slovak cystic fibrosis patientsrdquo Gene Geography

11 (1997) 51-56

[Kaialy 2012] Kaialy W G P Martin H Larhrib M D Ticehurst E Kolosionek and

A Nokhodchi ldquoThe influence of physical properties and morphology of crystallised

lactose on delivery of salbutamol sulphate from dry powder inhalersrdquo Colloids and

Surfaces B Biointerfaces 89 (2012) 29-39

[Kanicky 2002] Kanicky J R and D O Shah ldquoEffect of degree type and position of

unsaturation on the pka of long-chain fatty acidsrdquo Journal of Colloid and Interface

Science 256 no 1 (2002) 201ndash7

[Kansara 2015] Kansara K P Patel D Shah R K Shukla S Singh A Kumar and

Dhawan ldquoTiO2 nanoparticles induce DNA double strand breaks and cell cycle arrest in

human alveolar cellsrdquo Environmental and Molecular Mutagenesis 56 no 2 (2015) 204-

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S Holsclaw ldquoReproductive failure in males with cystic fibrosisrdquo New England Journal of

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[Kasithevar 2017] Kasithevar M P Periakaruppan S Muthupandian and M Mohan

ldquoAntibacterial efficacy of silver nanoparticles against multi-drug resistant clinical isolates

from post-surgical wound infectionsrdquo Microbial Pathogenesis 107 (2017) 327ndash34

[Kawata 2009] Kawata K M Osawa S Okabe ldquoIn vitro toxicity of silver nanoparticles

at noncytotoxic doses to HepG2 human hepatoma cellsrdquo Environmental Science amp

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[Kere 1994] Kere J X Estivill M Chilloacuten N Morral V Numes R Norio E Savilahti

and A de la Chapelle ldquoCystic fibrosis in a low-incidence population two major mutations

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[Kim 1972] Kim T K and M Takahashi ldquoNew magnetic material having ultrahigh

magnetic momentrdquo Applied Physics Letters 20 no 12 (1972) 492-494

[Kim 2005] Kim E H H S Lee B K Kwak and B-K Kim ldquoSynthesis of ferrofluid

with magnetic nanoparticles by sonochemical method for MRI contrast agentrdquo Journal of

Magnetism and Magnetic Materials 289 (2005) 328-330

[Kim 2007] Kim J S E Kuk K N Yu J-H Kim S J Park H J Lee S H Kim Y K

Park Y H Park C-Y Hwang Y-K Kim Y-S Lee D H Jeong and M-H Cho

ldquoAntimicrobial effects of silver nanoparticlesrdquo Nanomedicine Nanotechnology Biology

and Medicine 3 no 1 (2007) 95ndash101

[Kim 2012] Kim D-J S-G Chung S-H Lee and J-W Choi ldquoRelation of microbial

biomass to counting units for Pseudomonas aeruginosardquo African Journal of Microbiology

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[Kirby 2010] Kirby Brian J Micro-and nanoscale fluid mechanics transport in

microfluidic devices Cambridge university press 2010

[Kirschling 2010] Kirschling T L K B Gregory E G Minkley Jr G V Lowry and R

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[Klaine 2008] Klaine S J P J J Alvarez G E Batley T F Fernandes R D Handy D

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[Klassen 1998] Klaassen T M Teder M Viikmaa and A Metspalu ldquoNeonatal

screening for the cystic fibrosis main mutation ΔF508 in Estoniardquo Journal of Medical

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[Klausen 2003] Klausen M A Heydorn P Ragas L Lambertsen A Aaes‐Joslashrgensen S

Molin and T Tolker‐Nielsen ldquoBiofilm formation by Pseudomonas aeruginosa wild type

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[Knappen 2004] Knaapen A M P J Borm C Albrecht and R P Schins (2004)

ldquoInhaled particles and lung cancer Part A Mechanismsrdquo International Journal of Cancer

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Hagen ldquoHyperthermia and thermosensitive liposomes for improved delivery of

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[Konstan 2004] Konstan M W P B Davis J S Wagener K A Hilliard R C Stern L

J H Milgram T H Kowalczyk S L Hyatt T L Flink C R Gedeon and S M Oette

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[Kosorok 1996] Kosorok M R W‐H Wei and P M Farrell ldquoThe incidence of cystic

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[Krishnan 2010] Krishnan S P Diagaradjane and S H Cho ldquoNanoparticle-mediated

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[Kumon 1994] Kumon H K‐I Tomochika T Matunaga M Ogawa and H Ohmori ldquoA

sandwich cup method for the penetration assay of antimicrobial agents through

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[Lannefors 2002] Lannefors Louise and Anna Lindgren ldquoDemographic transition of the

Swedish cystic fibrosis communitymdashresults of modern carerdquo Respiratory medicine 96 no

9 (2002) 681-685

[Lara 2011] Lara H H E N Garza-Trevintildeo L Ixtepan-Turrent and D K Singh ldquoSilver

nanoparticles are broad-spectrum bactericidal and virucidal compoundsrdquo Journal of

Nanobiotechnology 9 (2011) 30 (8 pp)

[Lara 2015] Lara H H D G Romero-Urbina C Pierce J L Lopez-Ribot M J

Arellano-Jimeacutenez and M Jose-Yacaman ldquoEffect of silver nanoparticles on Candida

albicans biofilms an ultrastructural studyrdquo Journal of Nanobiotechnology 13 (2015) 91

(12 pp)

[Laurent 2008] Laurent S D Forge M Port A Roch C Robic L Vander Elst and R

N Muller ldquoMagnetic iron oxide nanoparticles Synthesis stabilization vectorization

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[Le 2012] Le A-T T T Le V Q Nguyen H H Tran D A Dang Q H Tran and D L

Vu ldquoPowereful silver nanoparticles for the prevention of gastrointestinal bacterial

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[Lee 2007] Lee Y C B J Ahn J S Jin J U Kim S H Lee D Y Song W K Lee

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all antimicrobial agents but susceptible to colistin in Daegu Koreardquo Journal of

Microbiology (Seoul Korea) 45 no 4 (2007) 358-363

[Lee 2008] Lee C J Y Kim W I Lee K L Nelson J Yoon and D L Sedlak

ldquoBactericidal effect of zero-valent iron nanoparticles on Escherichia colirdquo Environmental

Science amp Technology 42 no 13 (2008) 4927-4933

[Lee 2011] Lee YS DW Kim YH Lee JH Oh S Yoon MS Choi SK Lee JW

Kim K Lee and CW Song ldquoSilver nanoparticles induce apoptosis and G2M arrest via

PKCζ-dependent signaling in A549 lung cellsrdquo Archives of Toxicology 85 no 12 (2011)

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[Lehr 1992] Lehr C-M J A Bouwstra E H Schacht and H E Junginger ldquoIn vitro

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[Lev 1965] Lev R S S Spicer ldquoAn historical chemical comparison of human epithelial

mucins in normal and hypersecretory states including pancreatic cystic fibrosisrdquo American

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[Levine 2011] Levine B N Mizushima and H W Virgin ldquoAutophagy in immunity and

inflammationrdquo Nature 469 no 7330 (2011) 323-335

[Li 2011] Li W L Sun M Corey F Zou S Lee A L Cojocaru C Taylor S M

Blackman A Stephenson A J Sandford R Dorfman M L Drumm G R Cutting M

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[Liao 2015] Liao S H C H Liu B P Bastakoti N Suzuki Y Chang Y Yamauchi F

H Lin K C Wu ldquoFunctionalized magnetic iron oxidealginate core-shell nanoparticles

for targeting hyperthermiardquo International Journal of Nanomedicine 10 (2015) 3315ndash28

[Linsdell 2001] Linsdell P ldquoDirect block of the cystic fibrosis transmembrane

conductance regulator Clminus channel by butyrate and phenylbutyraterdquo European Journal of

Pharmacology 411 no 3 (2001) 255-260

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[Lippard 1994] Lippard S J and J M Berg Principles of Bioinorganic Chemistry Mill

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[Liu 2009] Liu T Y K H Liu D M Liu S Y Chen and I W Chen ldquoTemperature‐

sensitive nanocapsules for controlled drug release caused by magnetically triggered

structural disruptionrdquo Advanced Functional Materials 19 no 4 (2009) 616-623

[Liu 2012] Liu Y L K L Ai J H Liu Q H Yuan Y Y He and L H Lu ldquoA high‐performance ytterbium‐based nanoparticulate contrast agent for in vivo x‐ray computed

tomography imagingrdquo AngewandteChemie International Edition 51 no 6 (2012) 1437-

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Savage and J C Sylvester ldquoAssay of streptomycin by the paper-disc plate methodrdquo

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[Loacutepez Peacuterez 1997] Loacutepez Peacuterez J A M A Loacutepez Quintela J Mira J Rivas and S W

Charles ldquoAdvances in the preparation of magnetic nanoparticles by the microemulsion

methodrdquo The Journal of Physical Chemistry B 101 no 41 (1997) 8045-8047

[Losasso 2014] Losasso C S Belluco V Cibin P Zavagnin I Micetić F Gallocchio

M Zanella L Bregoli G Biancotto and A Riccirdquo Antibacterial activity of silver

nanoparticles sensitivity of different Salmonella serovarsrdquo Frontiers in Microbiology 5

(2014) 227 (9 pp)

[Love 2012] Love S A M A Maurer-Jones J W Thompson Y-S Lin and C L

Haynes ldquoAssessing nanoparticle toxicityrdquo Annual Review of Analytical Chemistry 5

(2012) 181-205

[Lu 2002] Lu Y Y D Yin B T Mayers and Y N Xia ldquoModifying the surface

properties of superparamagnetic iron oxide nanoparticles through a sol-gel

approachrdquo Nano Letters 2 no 3 (2002) 183-186

[Lu 2010] Lu M M H Cohen D Rieves and R Pazdur ldquoFDA report Ferumoxytol for

intravenous iron therapy in adult patients with chronic kidney diseaserdquo American Journal

of Hematology 85 no 5 (2010) 315ndash9

[Luciani 2010] Luciani A V R Villella S Esposito N Brunetti-Pierri D Medina C

Settembre M Gavina L Pulze I Giardino M Poettoello-Mantovani M DrsquoApolito S

Guido E Masliah B Spencer S Quaratino V Raia A Ballabio and L Maiuri

ldquoDefective CFTR induces aggresome formation and lung inflammation in cystic fibrosis

through ROS-mediated autophagy inhibitionrdquo Nature Cell Biology 12 no 9 (2010) 863-

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[Lucotte 1995] Lucotte G S Hazout and M De Braekeleer ldquoComplete map of cystic

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[Ludwig 2005] Ludwig F S Maumluselein E Heim and M Schilling

ldquoMagnetorelaxometry of magnetic nanoparticles in magnetically unshielded environment

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[Lukanov 2011] Lukanov P V K Anuganti Y Krupskaya A‐M Galibert B Soula C

Tilmaciu A H Velders R Klingeler B Buumlchner and E Flahaut ldquoCCVD synthesis of

carbon‐encapsulated cobalt nanoparticles for biomedical applicationsrdquo Advanced

Functional Materials 21 no 18 (2011) 3583-3588

[Maek 1997] Macek M Jr Mackova A Hamosh A Hilman BC Selden RF

Lucotte G Friedman KJ Knowles MR Rosenstein BJ and GR Cutting

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(1997) 1122-1127

[Maier-Hauff 2011] Maier-Hauff K F Ulrich D Nestler H Niehoff P Wust B

Thiesen H Orawa V Budach and A Jordan ldquoEfficacy and safety of intratumoral

thermotherapy using magnetic iron-oxide nanoparticles combined with external beam

radiotherapy on patients with recurrent glioblastoma multiformerdquo Journal of Neuro-

oncology 103 no 2 (2011) 317-324

[Majewski 2007] Majewski P and B Thierry ldquoFunctionalized magnetite nanoparticlesmdash

Synthesis properties and bio-applicationsrdquo Critical Reviews in Solid State and Materials

Sciences 32 no 3-4 (2007) 203-215

[Mandelbaum 1995] Mandelbaum R T D L Allan and L P Wackett ldquoIsolation and

characterization of a Pseudomonas sp that mineralizes the s-triazine herbicide atrazinerdquo

Applied and Environmental Microbiology 61 no 4 (1995) 1451-1457

[Mapara 2015] Mapara N M Sharma V Shriram R Bharadwaj K C Mohite and V

Kumar ldquoAntimicrobial potentials of Helicteres isora silver nanoparticles against

extensively drug-resistant (XDR) clinical isolates of Pseudomonas aeruginosardquo Applied

Microbiology and Biotechnology 99 no 24 (2015) 10655ndash67

[Martiacutenez-Castantildeoacuten 2008] Martiacutenez-Castantildeoacuten G A N Nintildeo-Martiacutenez F Martiacutenez-

Gutierrez J R Martiacutenez-Mendoza and F Ruiz ldquoSynthesis and antibacterial activity of

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silver nanoparticles with different sizesrdquo Journal of Nanoparticle Research 10 no 8

(2008) 1343ndash8

[Massie 2010] Massie J L Curnow L Gaffney J Carlin and I Francis ldquoDeclining

prevalence of cystic fibrosis since the introduction of newborn screeningrdquo Archives of

Disease in Childhood 95 no 7 (2010) 531-533

[Mateu 2002] Mateu Eva Francesc Calafell Maria Dolors Ramos Teresa Casals and

Jaume Bertranpetit ldquoCan a place of origin of the main cystic fibrosis mutations be

identifiedrdquo The American Journal of Human Genetics 70 no 1 (2002) 257-264

[Maynard 2005] Maynard A D and E D Kuempel ldquoAirborne nanostructured particles

and occupational healthrdquo Journal of Nanoparticle Research 7 no 6 (2005) 587-614

[Mbeh 2012] Mbeh D R Franccedila Y Merhi X Zhang X T Veres E Sacher and L

Yahia ldquoIn vitro biocompatibility assessment of functionalized magnetite nanoparticles

Biological and cytotoxicological effectsrdquo Journal of Biomedical Materials Research Part

A 100 no 6 (2012) 1637-1646

[McGill 2009a] McGill S L C Cuylear N L Adolphi M Osiński and H D C Smyth

ldquoEnhanced drug transport through alginate biofilms using magnetic nanoparticlesrdquo

Colloidal Quantum Dots for Biomedical Applications IV (M Osiński T M Jovin and K

Yamamoto eds) SPIE BiOS Biomedical Optics International Society for Optics and

Photonics Proceedings of SPIE (2009) 7189

[McGill 2009b] McGill S L C L Cuylear N L Adolphi M Osiński and H D C

Smyth ldquoMagnetically responsive nanoparticles for drug delivery applications using low

magnetic field strengthsrdquo IEEE Transactions on NanoBioscience 8 no 1 (2009) 33-42

[McNeil 2011] McNeil Scott E ed Characterization of nanoparticles intended for drug

delivery Vol 697 New York NY Humana press 2011

[Mearns 1974] Mearns M B ldquoCystic fibrosisrdquo British Journal of Hospital Medicine

(1974) 497-506

[Meenach 2013] Meenach S A K W Anderson J Z Hilt R C McGarry and H M

Mansour ldquoCharacterization and aerosol dispersion performance of advanced spray-dried

chemotherapeutic PEGylated phospholipid particles for dry powder inhalation delivery in

lung cancerrdquo European Journal of Pharmaceutical Sciences 49 no 4 (2013) 699-711

[Mehdaoui 2011] Mehdaoui B A Meffre J Carrey S Lachaize L‐M Lacroix M

Gougeon B Chaudret and M Respaud ldquoOptimal size of nanoparticles for magnetic

196

hyperthermia A combined theoretical and experimental studyrdquo Advanced Functional

Materials 21 no 23 (2011) 4573-4581

[Meiser 2004] Meiser F C Cortez and F Caruso ldquoBiofunctionalization of fluorescent

rare‐earth‐doped lanthanum phosphate colloidal nanoparticlesrdquo Angewandte Chemie

International Edition 43 no 44 (2004) 5954-5957

[Minev 2011] Minev B R ed Cancer Management in Man Chemotherapy Biological

Therapy Hyperthermia and Supporting Measures Springer 2011

[Mirtajani 2017] Mirtajani S B Poopak Farnia Maryam Hassanzad Jalaledin Ghanavi

Parissa Farnia and Ali Akbar Velayati Geographical distribution of cystic fibrosis The

past 70 years of data analyzis Biomedical and Biotechnology Research Journal (BBRJ) 1

no 2 (2017) 105

[Monteiro-Riviere 2009] Monteiro-Riviere N A Inman L and Zhang ldquoLimitations and

relative utility of screening assays to assess engineered nanoparticle toxicity in a human

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[Moreau-Marquis 2010] Moreau-Marquis S C V Redelman B A Stanton and G G

Anderson Co-culture models of Pseudomonas aeruginosa biofilms grown on live human

airway cells Journal of visualized experiments JoVE 44 (2010)

[Moritz 2010] Moritz M M H-C Flemming and J Wingender ldquoIntegration of

Pseudomonas aeruginosa and Legionella pneumophila in drinking water biofilms grown on

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Health 213 no 3 (2010) 190-197

[Moritz 2013] Moritz M and M Geszke-Moritz ldquoThe newest achievements in synthesis

immobilization and practical applications of antibacterial nanoparticlesrdquo Chemical

Engineering Journal 228 (2013) 596-613

[Mornet 2004] Mornet S S Vasseur F Grasset and E Duguet ldquoMagnetic nanoparticle

design for medical diagnosis and therapyrdquo Journal of Materials Chemistry 14 no 14

(2004) 2161-2175

[Morones 2005] Morones J R J L Elechiguerra A Camacho K Holt J B Kouri J T

Ramiacuterez M J Yacaman ldquoThe bactericidal effect of silver nanoparticlesrdquo Nanotechnology

16 no 10 (2005) 2346ndash53

[Moros 2013] Moros E G ed Physics of Thermal Therapy Fundamentals and Clinical

Applications CRC Press 2013

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[Morral 1994] Morral N J Bertranpetit X Estivill V Nunes T Casals J Gimenez A

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Desgeorges M Schwartz M Schwarz B Dallapiccola G Novelli C Ferec M de Arce

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[Mushin 2014] Muhsin T M and A K Hachim ldquoMycosynthesis and characterization of

silver nanoparticles and their activity against some human pathogenic bacteriardquo World

Journal of Microbiology and Biotechnology 30 no 7 (2014) 2081ndash90

[Musk 2005] Musk D J D A Banko and P J Hergenrother ldquoIron salts perturb biofilm

formation and disrupt existing biofilms of Pseudomonas aeruginosardquo Chemistry amp

Biology 12 no 7 (2005) 789-796

[Nasiri 2016] Nasiri A R Afsar Gharebagh S A Nojoumi SA M Akbarizadeh S

Harirchi M Arefnezhad S Sahraei M Hesaraki M Afshari F Javadian M Sheykhzade

Asadi Z Shahi and A Sargazi ldquoEvaluation of the antimicrobial activity of silver

nanoparticles on antibiotic-resistant Pseudomonas aeruginosardquo International Journal of

Basic Science in Medicine 1 no 1 (2016) 25ndash8

[Neacuteel 1949] Neacuteel L Originally published in 1949 as ldquoTheacuteorie du traicircnage magneacutetique des

ferromagneacutetiques en grains fins avec application aux terres cuitesrdquo Annales de

Geacuteophysique 5 99-136 Nicholas Kurti ed Selected Works of Louis Neacuteel Gordon and

Breach Science Publishers 1988 pp 405ndash427ISBN 2-88124-300-2

[Nehara 2018] Nehra P R P Chauhan N Garg K Verma ldquoAntibacterial and antifungal

activity of chitosan coated iron oxide nanoparticlesrdquo British Journal of Biomedical

Science 75 no 1 (2018) 13-18

[Nichols 1998] Nichols W W M J Evans M P E Slack and H L Walmsley ldquoThe

penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas

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[Nickel 1985] Nickel J C I Ruseska J B Wright and J W Costerton ldquoTobramycin

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[Nielsen 1988] Nielsen O H B L Thomsen A Green P K Andersen M Hauge and

P O Schioslashtz ldquoCystic fibrosis in Denmark 1945 to 1985rdquo Acta Paediatrica 77 no 6

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[Nielsen 2002] Nielsen R and D Gyrd‐Hansen ldquoPrenatal screening for cystic fibrosis an

economic analysisrdquo Health Economics 11 no 4 (2002) 285-299

[Niemirowicz 2015] Niemirowicz K U Surel A Z Wilczewska J Mystkowska E

Piktel X Gu Z Namiot A Kułakowska P B Savage and R Bucki ldquoBactericidal

activity and biocompatibility of ceragenin-coated magnetic nanoparticlesrdquo Journal of

Nanobiotechnology 13 no 1 (2015) 32 (11 pp)

[NIH 2016] ldquoHow Do Geneticists Indicate the Location of a Gene - Genetics Home

Referencerdquo US National Library of Medicine November 22 2016 Accessed November

28 2016 httpsghrnlmnihgovprimerhowgenesworkgenelocation

[Nikaido 1986] Nikaido H and R E W Hancock ldquoOuter membrane permeability of

Pseudomonas aeruginosardquo The Bacteria A treatise on structure and function Orlando

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[Niles 2007] Niles AL RA Moravec PE Hesselberth MA Scurria WJ Daily and

TL Riss ldquoA homogeneous assay to measure live and dead cells in the same sample by

detecting different protease markersrdquo Analytical Biochemistry 366 no 2 (2007) 197-206

[Noblett 1969] Noblett H R ldquoTreatment of uncomplicated meconium ileus by

Gastrografin enema a preliminary reportrdquo Journal of Pediatric Surgery 4 no 2 (1969)

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Applications New York Wiley 2000

[Oberdoumlrster 2000] Oberdoumlrster G ldquoPulmonary effects of inhaled ultrafine particlesrdquo

International Archives of Occupational and Environmental Health 74 no 1 (2000) 1-8

[Pal 2007] Pal S Y K Tak and J M Song ldquoDoes the antibacterial activity of silver

nanoparticles depend on the shape of the nanoparticle A study of the gram-negative

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[Palanisamy 2014] Palanisamy N K N Ferina A N Amirulhusni Z Mohd-Zain J

Hussaini L J Ping and R Durairaj ldquoAntibiofilm properties of chemically synthesized

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silver nanoparticles found against Pseudomonas aeruginosardquo Journal of

[Palchoudhury 2011] Palchoudhury S W An Y L Xu Y Qin Z T Zhang N Chopra

R A Holler C H Turner and Y P Bao ldquoSynthesis and growth mechanism of iron oxide

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[Palomaki 2004] Palomaki G E S C Fitzsimmons and J E Haddow ldquoClinical

sensitivity of prenatal screening for cystic fibrosis via CFTR carrier testing in a United

States panethnic populationrdquo Genetics in Medicine 6 no 5 (2004) 405-414

[Panaacutecek 2006] Panaacutecek A L Kviacutetek R Prucek M Kolaacuter R Veceřovaacute N Pizuacuterovaacute V

K Sharma T Nevĕcnaacute and R Zbořil ldquoSilver colloid nanoparticles Synthesis

characterization and their antibacterial activityrdquo The Journal of Physical Chemistry B 110

no 33 (2006) 16248-16253

[Park 2004] Park J K An Y Hwang J-G Park H-J Noh J-Y Kim J-H Park N-M

Hwang and T Hyeon ldquoUltra-large-scale syntheses of monodisperse nanocrystalsrdquo Nature

Materials 3 no 12 (2004) 891-895

[Patra 2017] Patra J K and K-H Baek ldquoAntibacterial activity and synergistic antibacterial

potential of biosynthesized silver nanoparticles against foodborne pathogenic bacteria

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[Peebles 2005] Peebles A and J Maddison eds Cystic Fibrosis Care A Practical

Guide Elsevier Health Sciences 2005

[Pier 1998] Pier G B M Grout T Zaidi G Meluleni S S Mueschenborn G Banting

R Ratcliff M J Evans and W H Colledge ldquoSalmonella typhi uses CFTR to enter

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[Piper 2013] Piper A ldquoThe big risk of small particles The threats and promise of

nanotechnologyrdquo Risk Management April 9 2013

[Pisanic 2007] Pisanic T R J D Blackwell V I Shubayev R R Fintildeones and S Jin

ldquoNanotoxicity of iron oxide nanoparticle internalization in growing neuronsrdquo Biomaterials

28 no 16 (2007) 2572-2581

[Ponnamperuma 1972] Ponnamperuma F N ldquoThe chemistry of submerged soilsrdquo

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[Popa 1997] Popa I L Pop Z Popa M J Schwarz G Hambleton G M Malone A

Haworth and M Super ldquoCystic fibrosis mutations in Romaniardquo European Journal of

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[Prabhu 2012] Prabhu S and E K Poulose ldquoSilver nanoparticles mechanism of

antimicrobial action synthesis medical applications and toxicity effectsrdquo International

Nano Letters 2 no 1 (2012) 32 (10 pp)

[Prabhu 2015] Prabhu Y T K V Rao B S Kumari V S S Kumar and T Pavani

ldquoSynthesis of Fe3O4 nanoparticles and its antibacterial applicationrdquo International Nano

Letters 5 (2015) 85ndash92

[Praetorius 2007] Praetorius N P and T K Mandal ldquoEngineered nanoparticles in cancer

therapyrdquo Recent Patents on Drug Delivery amp Formulation 1 no 1 (2007) 37-51

[Prencipe 2009] Prencipe G S M Tabakman K Welsher Z Liu A P Goodwin L

Zhang J Henry and H J Dai ldquoPEG branched polymer for functionalization of

nanomaterials with ultralong blood circulationrdquo Journal of the American Chemical Society

131 no 13 (2009) 4783-4787

[Preacutevot 2001] Preacutevot M and D Dunlop ldquoLouis Neacuteel Forty years of magnetismrdquo Physics

of the Earth and Planetary Interiors 126 (2001) 3-6

[Prodan 2013] Prodan A M S L Iconaru C S Ciobanu M C Chifiriuc M Stoicea

and D Predoi ldquoIron oxide magnetic nanoparticles characterization and toxicity evaluation

by in vitro and in vivo assaysrdquo Journal of Nanomaterials (2013) 587021 (10 pp)

[Prodan 2013] Prodan A M S L Iconaru C M Chifiriuc C Bleotu C S Ciobanu M

Motelica-Heino S Sizaret D Predoi ldquoMagnetic properties and biological activity

evaluation of iron oxide nanoparticlesrdquo Journal of Nanomaterials (2013)893970 (7 pp)

[Provenzano 2009] Provenzano R B Schiller M Rao D Coyne L Brenner and B J

Pereira ldquoFerumoxytol as an intravenous iron replacement therapy in hemodialysis

patientsrdquo Clinical Journal of the American Society of Nephrology 4 no 2 (2009) 386ndash93

[Qiang 2006] Qiang Y J Antony A Sharma J Nutting D Sikes and D Meyer

ldquoIroniron oxide core-shell nanoclusters for biomedical applicationsrdquo Journal of

Nanoparticle Research 8 no 3-4 (2006) 489-496

[Rai 2009] Rai M A Yadav and A Gade ldquoSilver nanoparticles as a new generation of

antimicrobialsrdquo Biotechnology Advances 27 no 1 (2009) 76ndash83

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[Rai 2012] Rai M K S D Deshmukh A P Ingle and A K Gade ldquoSilver nanoparticles

the powerful nanoweapon against multidrug-resistant bacteriardquo Journal of Applied

Microbiology 112 no 5 (2012) 841ndash52

[Ramirez 2003] Ramirez LP and K Landfester ldquoMagnetic polystyrene nanoparticles

with a high magnetite content obtained by miniemulsion processesrdquo Macromolecular

Chemistry and Physics 204 (2003) 22ndash31

[Raymond 2010] Raymond K General Organic and Biological Chemistry An

Intergrated Approach Wiley 2010

[Raza 2016] Raza M A Z Kanwal A Rauf A N Sabri S Riaz and S Naseem ldquoSize-

and shape-dependent antibacterial studies of silver nanoparticles synthesized by wet

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susceptibility testing of Pseudomonas aeruginosa selection of a control strain and criteria

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[Rice 2008] Rice L B ldquoFederal funding for the study of antimicrobial resistance in

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[Riordan 2008] Riordan J R ldquoCFTR function and prospects for therapyrdquo Annual Reviews

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[Romeo 1989] Romeo G M Devoto and L J V Galietta ldquoWhy is the cystic fibrosis

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[Rosan 1962] Rosan R C H Shwachman and L L Kulczycki ldquoDiabetes mellitus and

cystic fibrosis of the pancreas Laboratory and clinical observationsrdquo American Journal of

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[Rusol 2017] Rusol Al-Bahrani R J Raman H Lakshmanan A A Hassan and V

Sabaratnam ldquoGreen synthesis of silver nanoparticles using tree oyster mushroom

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Pleurotus ostreatus and its inhibitory activity against pathogenic bacteriardquo Materials

[Sabath 1976] Sabath LD ldquoThe assay of antimicrobial compoundsrdquo Human Pathology 7

no 3 (1976) 287ndash95

[Sadeghi 2012] Sadeghi B F S Garmaroudi M Hashemi H R Nezhad A Nasrollahi

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[Sahoo 2003] Sahoo S K and V Labhasetwar ldquoNanotech approaches to drug delivery

and imagingrdquo Drug Discovery Today 8 no 24 (2003) 1112-1120

[Saiman 2004] Saiman L ldquoThe use of macrolide antibiotics in patients with cystic

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[Salazar-Alvarez 2006] Salazar-Alvarez G M Muhammed and A A Zagorodni ldquoNovel

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[Samanta 2008] Samanta B H Yan N O Fischer J Shi D J Jerry V M Rotello

ldquoProtein-passivated Fe3O4 nanoparticles low toxicity and rapid heating for thermal

therapyrdquo Journal of Materials Chemistry 18 no 11 (2008) 1204ndash8

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[Santra 2001] Santra S R Tapec N Theodoropoulou J Dobson A Hebard and W

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[Sathyanarayanan 2013] Sathyanarayanan M B R Balachandranath Y Genji

Srinivasulu S K Kannaiyan and G Subbiahdoss ldquoThe effect of gold and iron-oxide

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[Scheank 2013] Schwank G Koo B-K Sasselli V Dekkers J F Heo I Demircan T

Sasaki N Boymans S Cuppen E van der Ent CK and E E Nieuwenhuis ldquoFunctional

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[Schmidt 2008] Schmidt A L K Hughes Z Cai F Mendes H Li D N Sheppard and

M D Amaral ldquoProlonged treatment of cells with genistein modulates the expression and

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[Schmitt 1986] Schmitt D D D F Bandyk A J Pequet and J B Towne ldquoBacterial

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N Sasaki S Boymans E Cuppen E C K van der Ent and E E Nieuwenhuis

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[Setua 2010] Setua S D Menon A Asok S Nair and M Koyakutty ldquoFolate receptor

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[Ševců 2011] Ševců A Y S El-Temsah E J Joner and M Černiacutek ldquoOxidative stress

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Environments 26 no 4 (2011) 271-281

[Shafi 2001] Shafi K V P M A Ulman X Z Yan N-L Yang C Estournes H White

and M Rafailovich ldquoSonochemical synthesis of functionalized amorphous iron oxide

nanoparticlesrdquo Langmuir 17 no 16 (2001) 5093-5097

[Shaker 2017] Shaker M A and M I Shaaban MI ldquoSynthesis of silver nanoparticles with

antimicrobial and anti-adherence activities against multidrug-resistant isolates from

Acinetobacter baumanniirdquo Journal of Taibah University Medical Sciences 12 no 4

(2017) 291ndash7

[Shakil 2008] Shakil S R Khan R Zarrilli and A U Khan ldquoAminoglycosides versus

bacteriandasha description of the action resistance mechanism and nosocomial battlegroundrdquo

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[Shawar 1999] Shawar RM DL MacLeod RL Garber JL Burns JR Stapp CR

Clausen SK Tanaka ldquoActivities of tobramycin and six other antibiotics against

Pseudomonas aeruginosa isolates from patients with cystic fibrosisrdquo Antimicrobial Agents

and Chemotherapy 34 no 12 (1999) 2877ndash80

[Shete 2015] Shete P B R M Patil B M Tiwale and S H Pawar Water dispersible

oleic acid-coated Fe3 O4 nanoparticles for biomedical applicationsrdquo Journal of Magnetism

and Magnetic Materials 377 (2015) 406-410

[Shi 2007] Shi X T P Thomas L A Myc A Kotlyar and J R Baker Jr ldquoSynthesis

characterization and intracellular uptake of carboxyl-terminated poly (amidoamine)

dendrimer-stabilized iron oxide nanoparticlesrdquo Physical Chemistry Chemical Physics 9

no 42 (2007) 5712-5720

[Shi 2016] Shi S-F J-F Jia X-K Guo Y-P Zhao D-S Chen Y-Y Guo and X-L

Zhang ldquoReduced Staphylococcus aureus biofilm formation in the presence of chitosan-

coated iron oxide nanoparticlesrdquo International Journal of Nanomedicine 11 (2016) 6499ndash

506

[Shieh 2005] Shieh D-B F-Y Cheng C-H Su C-S Yeh M-T Wu Y-N Wu C-Y

Tsai C-L Wu D-H Chen and C-H Chou ldquoAqueous dispersions of magnetite

nanoparticles with NH3+ surfaces for magnetic manipulations of biomolecules and MRI

contrast agentsrdquo Biomaterials 26 no 34 (2005) 7183-7191

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[Shin 2017] Shin H Y Wang C Lee H K Yoo K H Zeng X Kuhns T Yang C M

Mohr T Liu C and L Hennighausen ldquoCRISPRCas9 targeting events cause complex

deletions and insertions at 17 sites in the mouse genomerdquo Nature Communications 8

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[Shliomis 1974] Shliomis M I ldquoMagnetic fluidsrdquo Soviet Physics Uspekhi 17 no 2

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[Shliomis 1993] Shliomis M I and V I Stepanov ldquoFrequency dependence and long-

time relaxation of the susceptibility of the magnetic fluidsrdquo Journal of Magnetism and

Magnetic Materials 122 no 1 (1993) 176-181

[Shoshani 1992] Shoshani T A Augarten E Gazit N Bashan Y Yahav Y Rivlin A

Tal H Seret L Yaar E Kerem and B Kerem ldquoAssociation of a nonsense mutation

(W1282X) the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in

Israel with presentation of severe diseaserdquo American Journal of Human Genetics 50 no 1

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[Shrestha 2009] Shrestha A S-W Fong B-C Khoo and A Kishen ldquoDelivery of

antibacterial nanoparticles into dentinal tubules using high-intensity focused ultrasoundrdquo

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[Shrivastava 2007] Shrivastava S T Bera A Roy G Singh P Ramachandrarao D

Dash ldquoCharacterization of enhanced antibacterial effects of novel silver nanoparticlesrdquo

Nanotechnology 18 no 22 (2007) 225103 (9 pp)

[Shtykova 2007] Shtykova E V X Huang N Remmes D Baxter B Stein B Dragnea

D I Svergun and L M Bronstein ldquoStructure and properties of iron oxide nanoparticles

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[Shwachman 1965] Shwachman H L L Kulczycki and K-T Khaw ldquoStudies in cystic

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689-699

[Siegel 1960] Siegel B and S Siegel ldquoPregnancy and delivery in a patient with cystic

fibrosis of the pancreas Report of a caserdquo Obstetrics amp Gynecology 16 no 4 (1960) 438-

440

[Simon 1970] Simon HJ EJ Yin ldquoMicrobioassay of antimicrobial agentsrdquo Applied

Microbiology 1970 Apr 119(4)573ndash9

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[Singh 2014a] Singh K M Panghal S Kadyan U Chaudhary and J P Yadav

ldquoAntibacterial activity of synthesized silver nanoparticles from Tinospora cordifolia

against multi drug resistant strains of Pseudomonas aeruginosa isolated from burn

patientsrdquo Journal of Nanomedicine amp Nanotechnology 5 no 2 (2014) 192 (6 pp)

[Singh 2014b] Singh K M Panghal S Kadyan U Chaudhary and J P Yadav ldquoGreen

silver nanoparticles of Phyllanthus amarus as an antibacterial agent against multi drug

resistant clinical isolates of Pseudomonas aeruginosardquo Journal of Nanobiotechnology 12

(2014) 40 (9 pp)

[Sinn 2011] Sinn P L R M Anthony and P B McCray ldquoGenetic therapies for cystic

fibrosis lung diseaserdquo Human molecular genetics 20 no R1 (2011) R79-R86

[Sio 2006] Sio C F L G Otten R H Cool S P Diggle P G Braun R Bos M

Daykin M Caacutemara P Williams and W J Quax ldquoQuorum quenching by an N-acyl-

homoserine lactone acylase from Pseudonomas aeruginosa PAO1rdquo Infection and

Immunology 74 no 3 (2006) 1673-1682

[Slieker 2005] Slieker M G C S P M Uiterwaal M Sinaasappel H G M Heijerman

J van der Laag and C K van der Ent ldquoBirth prevalence and survival in cystic fibrosis a

national cohort study in the Netherlandsrdquo Chest Journal 128 no 4 (2005) 2309-2315

[Smith 2002] Smith R S S G Harris R Phipps and B Iglewski ldquoThe Pseudonomas

aeruginosa quorum-sensing molecule N-(3-oxododecanoyl) homoserine lactone

contributes to virulence and induces inflammation in vivordquo Journal of Bacteriology 184

no 4 (2002) 1132-1139

[Smyth 2008] Smyth H D Marek Osinski and Shayna L McGill ldquoActive nanoparticles

and method of usingrdquo US Patent Application 12313847 filed November 25 2008

[Soenen 2010] Soenen S J H and M De Cuyper ldquoAssessing iron oxide nanoparticle

toxicity in vitro current status and future prospectsrdquo Nanomedicine 5 no 8 (2010) 1261-

1275

[Soenen 2011] Soenen S J H U Himmelreich N Nuytten and M De Cuyper

ldquoCytotoxic effects of iron oxide nanoparticles and implications for safety in cell labellingrdquo

Biomaterials 32 no 1 (2011) 195-205

[Sondi 2004] Sondi I B Salopek-Sondi ldquoSilver nanoparticles as antimicrobial agent a

case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and

Interface Science 275 no 1 (2004) 177ndash82

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[Song 2009] Song Y H H Lou J L Boyer M P Limberis L H Vandenberghe N R

Hackett P L Leopold J M Wilson and R G Crystal ldquoFunctional cystic fibrosis

transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells

by AAV6 2-mediated segmental trans-splicingrdquo Human Gene Therapy 20 no 3 (2009)

267-281

[Soto 2007] Soto K K Garza and L Murr ldquoCytotoxic effects of aggregated

nanomaterialsrdquo Acta Biomaterialia 3 no 3 (2007) 351-358

[Southern 2007] Southern K W A Munck R Pollitt G Travert L Zanolla J Dankert-

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newborn screening for cystic fibrosis in Europerdquo Journal of Cystic Fibrosis 6 no 1

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[Speert 1990] Speert DP SW Farmer ME Campbell JM Musser RK Selander S

Kuo ldquoConversion of Pseudomonas aeruginosa to the phenotype characteristic of strains

from patients with cystic fibrosisrdquo Journal of Clinical Microbiology 28 no 2 (1990)

188ndash94

[Spock 1967] Spock A H M C Heick H Cress and W S Logan ldquoAbnormal serum

factor in patients with cystic fibrosis of the pancreasrdquo Pediatric Research 1 no 3 (1967)

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[Staab 1998] Staab D K Wenninger N Gebert K Rupprath S Bisson M Trettin K

D Paul K M Keller and U Wahn ldquoQuality of life in patients with cystic fibrosis and

their parents what is important besides disease severityrdquo Thorax 53 no 9 (1998) 727-

731

[Streffer 2012] Streffer G ed Hyperthermia and the Therapy of Milignant Tumors

Volume 104 Springer 2012

[Strohbehn 1984] Strohbehn J W and Douple E B ldquoHyperthermia and cancer therapy

A review of biomedical engineering contributions and challengesrdquo IEEE Transactions on

Biomedical Engineering BME-31 no 12 (1984) 779-787

[Stutman 2002] Stutman H R J M Lieberman E Nussbaum M I Marks and the

Antibiotic Prophylaxis in Cystic Fibrosis Study Group ldquoAntibiotic prophylaxis in infants

and young children with cystic fibrosis a randomized controlled trialrdquo The Journal of

Pediatrics 140 no 3 (2002) 299-305

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[Sugita 1991] Sugita Y K Mitsuoka M Komuro H Hoshiya Y Kozono and M

Hanazono ldquoGiant magnetic moment and other magnetic properties of epitaxially grown

Fe16N2 single‐crystal filmsrdquo Journal of Applied Physics 70 no 10 (1991) 5977-5982

[Sun 2010] Sun C K Du C Fang N Bhattarai O Veiseh F Kievit Z Stephen D Lee

R G Ellenbogen B Ratner and M Zhang ldquoPEG-mediated synthesis of highly dispersive

multifunctional superparamagnetic nanoparticles their physicochemical properties and

function in vivordquo ACS Nano 4 no 4 (2010) 2402ndash10

[Suzuki 1973] Suzuki T Y Ichihara M Yamada and K Tonomura ldquoSome

characteristics of Pseudomonas 0ndash3 which utilizes polyvinyl alcoholrdquo Agricultural and

Biological Chemistry 37 no 4 (1973) 747-756

[Szaff 1983] Szaff M N Hoslashiby and E W Flensborg ldquoFrequent antibiotic therapy

improves survival of cystic fibrosis patients with chronic Pseudomonas aeruginosa

infectionrdquo Acta Paediatrica 72 no 5 (1983) 651-657

[Takami 2007] Takami S T Sato T Mousavand S Ohara M Umetsu and T Adschiri

ldquoHydrothermal synthesis of surface-modified iron oxide nanoparticlesrdquo Materials

Letters 61 no 26 (2007) 4769-4772

[Tan 2015] Tan S Y and Y Tatsumura ldquoAlexander Fleming (1881ndash1955) discoverer of

penicillinrdquo Singapore Medical Journal 56 no 7 (2015) 366-367

[Tang 2010] Tang B C J Fu D N Watkins and J Hanes ldquoEnhanced efficacy of local

etoposide delivery by poly (ether-anhydride) particles against small cell lung cancer in

vivordquo Biomaterials 31 no 2 (2010) 339-344

[Teja 2009] Teja A S and P-Y Koh ldquoSynthesis properties and applications of

magnetic iron oxide nanoparticlesrdquo Progress in Crystal Growth and Characterization of

Materials 55 no 1 (2009) 22-45

[Tendencia 2004] Tendencia E A ldquoDisk diffusion methodrdquo In Laboratory Manual of

Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from

Aquatic Animals and Environment pp 13-29 SEAFDEC Aquaculture Department 2004

[Tesla 1891] Tesla N ldquoThe secrets behind the geniusrdquo Ancient Code no 8211 March 10

1891 Accessed February 19 2015 httpwwwancient-codecomnikola-tesla-secrets-

behind-genius

[Thiesen 2008] Thiesen B and A Jordan ldquoClinical applications of magnetic

nanoparticles for hyperthermiardquo International Journal of Hyperthermia 24 no 6 (2008)

467-474

209

[Thompson 2007] Thompson D ldquoMichael Faradays recognition of ruby gold The birth of

modern nanotechnologyrdquo Gold Bulletin 40 no 4 (2007) 267-269

[Thukkaram 2014] Thukkaram M S Sitaram S K Kannaiyan and G Subbiahdoss

ldquoAntibacterial efficacy of iron-oxide nanoparticles against biofilms on different

biomaterial surfacesrdquo International Journal of Biomaterials (2014) 716080 (6 pp)

[Thuret 2003] Thuret G C Chiquet S Herrag J M Dumollard D Boudard J Bednarz

L Campos and P Gain ldquoMechanisms of staurosporine induced apoptosis in a human

corneal endothelial cell linerdquo British Journal of Ophthalmology 87 no 3 (2003) 346-352

[Tomoda 2009] Tomoda K T Ohkoshi K Hirota G S Sonavane T Nakajima H

Terada M Komuro K Kitazato and K Makino ldquoPreparation and properties of inhalable

nanocomposite particles for treatment of lung cancerrdquo Colloids and Surfaces B

Biointerfaces 71 no 2 (2009) 177-182

[Torres 1990] Torres A R Aznar J M Gatell P Jimeacutenez J Gonzaacutelez A Ferrer R

Celis and R Rodriguez-Roisin ldquoIncidence risk and prognosis factors of nosocomial

pneumonia in mechanically ventilated patientsrdquo American Review of Respiratory Disease

142 no 3 (1990) 523-528

[Tosi 2004] Tosi M F A Van Heeckeren T W Ferkol D Askew C V Harding and J

M Kaplan ldquoEffect of Pseudomonas-induced chronic lung inflammation on specific

cytotoxic T-cell responses to adenoviral vectors in micerdquo Gene Therapy 11 no 19 (2004)

1427-1433

[Tran 2010] Tran N A Mir D Mallik A Sinha S Nayar T J Webster ldquoBactericidal

effect of iron oxide nanoparticles on Staphylococcus aureusrdquo International Journal of

Nanomedicine 5 (2010) 277ndash83

[Urban 2008] Urban C P A Bradford M Tuckman S Segal-Maurer W Wehbeh L

Grenner R Colon-Urban N Mariano J J Rahal ldquoCarbapenem-resistant Escherichia coli

harboring Klebsiella pneumoniae carbapenemase β-lactamases associated with long-term

care facilitiesrdquo Clinical Infectious Diseases 46 NO 11 (2008) e127ndash30

[US Food and Drug Administration 2008] US Food and Drug Administration

Information for healthcare professionals fluoroquinolone antimicrobial drugs

[ciprofloxacin (marketed as Cipro and generic ciprofloxacin) ciprofloxacin extended-

release (marketed as Cipro XR and Proquin XR) gemifloxacin (marketed as Factive)

levofloxacin (marketed as Levaquin) moxifloxacin (marketed as Avelox) norfloxacin

210

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warning (2008)

[Van Bijsterveld 1969] Van Bijsterveld O P ldquoDiagnostic tests in the sicca syndromerdquo

Archives of Ophthalmology 82 no 1 (1969) 10-14

[van den Bos 2003] van den Bos E J A Wagner H Mahrholdt R B Thompson

Morimoto Y Sutton B S Judd R M and D A Taylor ldquoImproved efficacy of stem

cell labeling for magnetic resonance imaging studies by the use of cationic liposomesrdquo

Cell Transplantation 12 no 7 (2003) 743-756

[Vandevivere 1993] Vandevivere P and D L Kirchman ldquoAttachment stimulates

exopolysaccharide synthesis by a bacteriumrdquo Applied and Environmental Microbiology

59 no 10 (1993) 3280-3286

[Vehring 2007] Vehring R W R Foss and D Lechuga-Ballesteros ldquoParticle formation

in spray dryingrdquo Journal of Aerosol Science 38 no 7 (2007) 728-746

[Veiseh 2005] Veiseh O C Sun J Gunn N Kohler P Gabikian D Lee N Bhattarai

R Ellenbogen R Sze A Hallahan J Olson and Miqin Zhang ldquoOptical and MRI

multifunctional nanoprobe for targeting gliomasrdquo Nano Letters 5 no 6 (2005) 1003-

1008

[Veiseh 2010] Veiseh O J W Gunn and M Q Zhang ldquoDesign and fabrication of

magnetic nanoparticles for targeted drug delivery and imagingrdquo Advanced Drug Delivery

Reviews 62 no 3 (2010) 284-304

[Wainwright 1985] Wainwright B J P J Scambler J Schmidtke E A Watson H-Y

Law M Farrall H J Cooke H Eiberg and R Williamson ldquoLocalization of cystic

fibrosis locus to human chromosome 7cenndashq22rdquo Nature 318 no 6044 (1985) 384-385

[Walters 2003] Walters M C F Roe A Bugnicourt M J Franklin and P S Stewart

ldquoContributions of antibiotic penetration oxygen limitation and low metabolic activity to

tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycinrdquo

[Wang 2003] Wang X W T Zheng H W Tian S S Yu W Xu S H Meng X D He

J C Han C Q Sun and B K Tay ldquoGrowth structural and magnetic properties of iron

nitride thin films deposited by dc magnetron sputteringrdquo Applied Surface Science 220 no

1 (2003) 30-39

211

[Weaver 1994] Weaver L T M R Green K Nicholson J Mills M E Heeley J A

Kuzemko S Austin G A Gregory A E Dux and J A Davis ldquoPrognosis in cystic

fibrosis treated with continuous flucloxacillin from the neonatal periodrdquo Archives of

[Wei 2016] Wei Y M Zhao F Yang Y Mao H Xie and Q Zhou ldquoIron overload by

superparamagnetic iron oxide nanoparticles is a high risk factor in cirrhosis by a systems

toxicology assessmentrdquo Scientific Reports 6 (2016) 29110 (11 pp)

[Weissleder 1989] Weissleder R D D Stark B L Engelstad B R Bacon C C

Compton D L White P Jacobs J Lewis ldquoSuperparamagnetic iron oxide

pharmacokinetics and toxicityrdquo American Journal of Roentgenology 152 no 1 (1989)

167ndash73

[White 1985] White R S Woodward M Leppert P OConnell M Holf J Herbstl J-M

Lalouel M Deanri and G V Woudei ldquoA closely linked genetic marker for cystic

fibrosisrdquo Nature 318 no 6044 (1985) 382-384

[Whiteley 2001] Whiteley M M G Bangera R E Bumgarner M R Parsek G M

Teitzel S Lory and E P Greenberg ldquoGene expression in Pseudomonas aeruginosa

biofilmsrdquo Nature 413 no 6858 (2001) 860-864

[Witkamp 2001] Witkamp A J E de Bree R Van Goethem and F A N Zoetmulder

ldquoRationale and techniques of intra-operative hyperthermic intraperitoneal chemotherapyrdquo

Cancer Treatment Reviews 27 no 6 (2001) 365-374

[Wong 1998] Wong C-H M Hendrix E S Priestley and W A Greenberg ldquoSpecificity

of aminoglycoside antibiotics for the A-site of the decoding region of ribosomal RNArdquo

Chemistry amp Biology 5 no 7 (1998) 397-406

[Wood 1976] Wood R E Boat T F Doershuk C F ldquoCystic fibrosis state of the artrdquo

American Review of Respiratory Disease 113 (1976) 833-878

[Working 1997] Working P K MS Newman J Johnson and J B Cornacoff Safety of

poly (ethylene glycol) and poly (ethylene glycol) derivatives ACS Publications 1997

[Worlitzsch 2002] Worlitzsch D R Tarran M Ulrich U Schwab A Cekici K C

Meyer P Birrer G Bellon J Berger T Weiss K Botzenhart J R Yankaskas S

Randell R C Boucher and G Doumlring ldquoEffects of reduced mucus oxygen concentration

in airway Pseudomonas infections of cystic fibrosis patientsrdquo The Journal of Clinical

Investigation 109 no 3 (2002) 317-325

212

[Wu 2005] Wu XJ F Kassie and V Mersch-Sundermann ldquoThe role of reactive oxygen

species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle

arrest in human A549 lung carcinoma cellsrdquo Mutation ResearchFundamental and

Molecular Mechanisms of Mutagenesis 579 no 1-2 (2005) 115-124

[Wu 2008] Wu W Q G He and C Z Jiang ldquoMagnetic iron oxide nanoparticles

Synthesis and surface functionalization strategiesrdquo Nanoscale Research Letters 3 no 11

(2009) 397-415

[Wu 2013] Wu H H Zhu X Li Z Liu W Zheng T Chen B Yu and KH Wong

ldquoInduction of apoptosis and cell cycle arrest in A549 human lung adenocarcinoma cells by

surface-capping selenium nanoparticles an effect enhanced by polysaccharidendashprotein

complexes from Polyporus rhinocerosrdquo Journal of Agricultural and Food Chemistry 61

no 41 (2013) 9859-9866

[Wust 2006] Wust P C H Cho B Hildebrandt and J Gellermann ldquoThermal

monitoring Invasive minimal-invasive and non-invasive approachesrdquo International

Journal of Hyperthermia 22 no 3 (2006) 255-262

[Xie 2009] Xie J J Huang X Li S Sun and X Chen ldquoIron oxide nanoparticle platform

for biomedical applicationsrdquo Current Medicinal Chemistry 16 no 10 (2009) 1278-1294

[Xie 2010] Xie J K Chen J Huang S K Lee J H Wang J H Gao X G Li and X

Y Chen ldquoPETNIRFMRI triple functional iron oxide nanoparticlesrdquo Biomaterials 31 no

11 (2010) 3016-3022

[Xu 2007] Xu C J and S H Sun ldquoMonodisperse magnetic nanoparticles for biomedical

applicationsrdquo Polymer International 56 no 7 (2007) 821-826

[Yash Roy 1999] Yash Roy R C ldquoA structural Model for virulence organellae of gram-

negative organisms with reference to Salmonella pathogenicity in chicken ileumrdquo Indian

Journal of Poultry Science 34 no 2 (1999) 213-219

[You 2005] You Y W J Han P C Chiu and Y Jin ldquoRemoval and inactivation of

waterborne viruses using zerovalent ironrdquo Environmental Science amp Technology 39 no

23 (2005) 9263-9269

[Zabner 1996] Zabner J B W Ramsey D P Meeker M L Aitken R P Balfour R L

Gibson J Launspach R A Moscicki S M Richards and T A Standaert ldquoRepeat

administration of an adenovirus vector encoding cystic fibrosis transmembrane

213

conductance regulator to the nasal epithelium of patients with cystic fibrosisrdquo Journal of

Clinical Investigation 97 no 6 (1996) 1504

[Zelenski 2000] Zielenski J ldquoGenotype and phenotype in cystic fibrosisrdquo Respiration 67

no 2 (2000) 117-133

[Zhang 2010] Zhang X F S W Chen H-M Wang S-L Hsieh C-H Wu H-H Chou

and S C Hsieh ldquoRole of Neacuteel and Brownian relaxation mechanisms for water-based

Fe3O4 nanoparticle ferrofluids in hyperthermiardquo Biomedical Engineering Applications

Basis and Communications 22 no 05 (2010) 393-399

[Zielenski 1995] Zielenski J and L-C Tsui ldquoCystic fibrosis Genotypic and phenotypic

variationsrdquo Annual Review of Genetics 29 no 1 (1995) 777-807

214

PUBLICATIONS BY LEISHA MARIE MARTIN (ARMIJO)

Journal Papers

1 Savage D D J Chavez L Armijo and M Rosenberg ldquoPrenatal ethanol exposure

alters histamine H-3 receptor-mediated neurotransmission in adult offspringrdquo Alcoholism-

Clinical and Experimental Research 33 no 6 (2009) 133A Impact factor 3392

2 Wilkerson J L K R Gentry E C Dengler J A Wallace A A Kerwin L M

Armijo M N Kuhn G A Thakur A Makriyannis and E D Milligan ldquoIntrathecal

cannabilactone CB2R agonist AM1710 controls pathological pain and restores basal

cytokine levelsrdquo Pain 153 no 5 (2012) 1091-106 Impact factor 5836

2 Armijo L M Y I Brandt D Mathew S Yadav S Maestas A C Rivera N C

Cook N J Withers G A Smolyakov N L Adolphi T C Monson D L Huber H D

C Smyth and M Osiński ldquoIron oxide nanocrystals for magnetic hyperthermia

applicationsrdquo Nanomaterials 2 no 2 (2012) 134-146 Impact factor 3553

3 Dengler E C J Liu A Kerwin S Torres C M Olcott B N Bowman L Armijo

K Gentry J Wilkerson J Wallace X M Jiang E C Carnes C J Brinker and E D

Milligan ldquoMesoporous silica-supported lipid bilayers (protocells) for DNA cargo delivery

to the spinal cordrdquo Journal of Controlled Release 168 no 2 (2013) 209-224

Impact factor 7877

4 Rivera A C N N Glazener N C Cook B A Akins L M Armijo J B Plumley

N J Withers K Carpenter G A Smolyakov R D Busch and M Osiński

ldquoCharacterization of potassium bromide loaded with dysprosium fluoride nanocrystals for

neutron detectionrdquo International Journal of Nanotechnology 11 no 5678 (2014) 529-

538 Impact factor 1114

5 Armijo L M L A Ahureacute-Powell and N M Wereley ldquoRheological characterization

of a magnetorheological ferrofluid using iron nitride nanoparticles Journal of Applied

Physics 117 no 17 (2015) 17C747 Impact factor 2176

6 Armijo L M S J Wawrzyniec M Kopciuch Y I Brandt

A C Rivera N J Withers N C Cook D L Huber T C Monson H DC Smyth and

M Osiński ldquoAntibacterial activity of iron-oxide nanoparticles and tobramycin

nanoconjugates against Pseudomonas aeruginosa biofilmsrdquo Submitted to Journal of

Nanobiotechnology Spring 2019 Impact factor 5294

7 Armijo L M Y Brandt N J Withers J B Plumley P Jain A C Rivera N C

Cook H D C Smyth and M Osinski ldquoIn vitro Cytotoxicity of magnetite nanoparticles

215

in a human lung cell linerdquo Submitted to Toxicology in Vitro Spring 2019 Impact factor

3105

Conference Proceedings

1 M Osiński L M Armijo Y Brandt S R Maestas A C Rivera N C Cook J B

Plumley B A Akins G A Smolyakov N L Adolphi D L Huber S L McGill L

Gong and H D C Smyth ldquoMultifunctional nanoparticles for drug delivery in cystic

fibrosis (Invited Paper)rdquo Zing Nanomaterials Conference Xcaret Quintana Roo Mexico

28 Nov ndash 2 Dec 2011

2 Armijo L M Y Brandt D Mathew S Yadav S Maestas A C Rivera N C Cook

N J Withers G A Smolyakov N L Adolphi T C Monson H D C Smyth and M

Osiński ldquoIron oxide nanocrystals for magnetic hyperthermia applicationsrdquo Technical

Digest Zing Nanomaterials Conference Xcaret Quintana Roo Mexico 28 Nov ndash 2 Dec

2011 p 56

3 Rivera A C N N Glazener N C Cook L M Armijo J B Plumley B A Akins

K Carpenter G A Smolyakov R D Busch and M Osiński ldquoDysprosium-containing

nanocrystals for use as a neutron detector in a solvent suspensionrdquo Technical Digest Zing

Nanomaterials Conference Xcaret Quintana Roo Mexico 28 November ndash 2 December

2011 p 62

4 Armijo L M Y I Brandt N J Withers J B Plumley N C Cook A C Rivera S

Yadav G A Smolyakov T Monson D L Huber H D C Smyth and M Osiński

ldquoMultifunctional superparamagnetic nanocrystals for imaging and targeted drug delivery to

the lungrdquo Colloidal Nanocrystals for Biomedical Applications VII (W J Parak M

Osiński and K Yamamoto eds) SPIE International Symposium on Biomedical Optics

BiOS 2012 San Francisco CA 21-23 Jan 2012 Proceedings of SPIE Vol 8232 Paper

82320M (11 pp)

5 N J Withers Y I Brandt A C Rivera N C Cook L M Armijo G A Smolyakov

and M Osiński ldquoEffects of La02Ce06Eu02F3 nanoparticles capped with polyethylene

glycol on human astrocytoma cells in vitrordquo Colloidal Nanocrystals for Biomedical

Applications VII (W J Parak M Osiński and K Yamamoto eds) SPIE International

Symposium on Biomedical Optics BiOS 2012 San Francisco CA 21-23 Jan 2012

Proceedings of SPIE Vol 8232 Paper 82320R (9 pp)

6 Rivera A C N N Glazener N C Cook S Maestas B A Akins L M Armijo J B

Plumley N J Withers K Carpenter G A Smolyakov R D Busch and M Osiński

ldquoThermal neutron detection with PMMA nanocomposites containing dysprosium fluoride

nanocrystalsrdquo Chemical Biological Radiological Nuclear and Explosives (CBRNE)

Sensing XIII (A W Fountain III ed) SPIE Defense Security and Sensing Symposium

Baltimore MD 23-27 Apr 2012 Proceedings of SPIE Vol 8358 Paper 83581S (9 pp)

216

7 Cook N C A C Rivera N N Glazener B A Akins L M Armijo J B Plumley

N J Withers K Carpenter G A Smolyakov R D Busch and M Osiński ldquoPolyvinyl

tolueneGd2O310Ce scintillating nanocomposites for thermal neutron detectionrdquo

Technical Digest 7th International Conference on Quantum Dots Santa Fe New Mexico

13-18 May 2012 Paper Th-73

8 Armijo L M Y I Brandt S R Maestas A C Rivera N C Cook N J Withers G

A Smolyakov N L Adolphi T C Monson D L Huber H D C Smyth and M

Osiński ldquoMultifunctional nanocrystals for drug delivery in cystic fibrosisrdquo Technical

Digest 7th International Conference on Quantum Dots Santa Fe NM 13-18 May 2012

Paper Th-74

9 Rivera A C N N Glazener N C Cook S R Maestas B A Akins L M Armijo J

B Plumley N J Withers K Carpenter G A Smolyakov R D Busch and M Osiński

ldquoThermal neutron detection with Gd2O310Ce nanocrystals loaded into a polyvinyl

toluene matrixrdquo IEEE Symposium on Radiation Measurements and Applications SORMA

WEST 2012 Oakland CA 14-17 May 2012

10 Armijo L M Y I Brandt A C Rivera N C Cook J B Plumley N J Withers

M Kopciuch G A Smolyakov D L Huber H D C Smyth and M Osiński

ldquoMultifunctional superparamagnetic nanoparticles for enhanced drug transport in cystic

fibrosisrdquo Nanosystems in Engineering and Medicine (S H Choi J-H Choy U Lee and

V K Varadan eds) Incheon Korea 10-12 September 2012 Proceedings of SPIE Vol

8548 Paper 85480E (12 pp)

11 Armijo L M B A Akins J B Plumley A C Rivera N J Withers N C Cook G

A Smolyakov D L Huber H D C Smyth and M Osiński ldquoHighly efficient

multifunctional MnSeZnSeS quantum dots for biomedical applicationsrdquo Colloidal

Nanoparticles for Biomedical Applications VIII (W J Parak M Osiński and K

Yamamoto eds) SPIE International Symposium on Biomedical Optics BiOS 2013 San

Francisco California 2-4 Feb 2013 Proceedings of SPIE Vol 8595 Paper 859517 (7

pp)

12 Brandt Y I L M Armijo A C Rivera J B Plumley N C Cook G A

iron oxide nanoparticles in treating infection in cystic fibrosisrdquo Colloidal Nanoparticles

for Biomedical Applications VIII (W J Parak M Osiński and K Yamamoto Eds) SPIE

International Symposium on Biomedical Optics BiOS 2013 San Francisco CA 2-4 Feb

2013 Proceedings of SPIE Vol 8595 Paper 85951C (9 pp)

13 Withers N J N N Glazener A C Rivera B A Akins L M Armijo J B

Plumley N C Cook J M Sugar R Chan Y I Brandt G A Smolyakov P H Heintz

and M Osiński ldquoEffects of La02Ce06Eu02F3 nanocrystals capped with polyethylene glycol

217

on human pancreatic cancer cells in vitrordquo Colloidal Nanoparticles for Biomedical

Applications VIII (W J Parak M Osiński and K Yamamoto Eds) SPIE International

Symposium on Biomedical Optics BiOS 2013 San Francisco CA 2-4 Feb 2013

Proceedings of SPIE Vol 8595 Paper 85951O (9 pp)

14Osiński M Y I Brandt L M Armijo N C Cook G A Smolyakov and H D C

Smyth ldquoEffectiveness of tobramycin conjugated to superparamagnetic nanoparticles in

treating cystic fibrosis (Invited Paper)rdquo Technical Digest Sixth International Conference

on Advanced Materials and Nanotechnology AMN-6 Auckland New Zealand 11-15 Feb

2013

15 Rivera A C N N Glazener N C Cook L M Armijo J B Plumley N J Withers

K Carpenter G A Smolyakov R D Busch and M Osiński ldquoCharacterization of

potassium bromide loaded with dysprosium fluoride nanocrystals for neutron detectionrdquo

Technical Digest Sixth International Conference on Advanced Materials and

Nanotechnology AMN-6 Auckland New Zealand 11-15 February 2013

16 Armijo L M A C Rivera J B Plumley N C Cook S Maestas G A Smolyakov

T C Monson D L Huber and M Osiński ldquoBasic mechanisms involved in the

magnetization reversal of magnetic single-domain nanoparticlesrdquo Technical Digest Sixth

International Conference on Advanced Materials and Nanotechnology AMN-6 Auckland

New Zealand 11-15 Feb 2013

17 Osiński M Y I Brandt L M Armijo M Kopciuch N J Withers N C Cook G

A Smolyakov and H D C Smyth ldquoHybrid multifunctional nanoparticles for drug

delivery to the lung in cystic fibrosis (Invited Paper)rdquo 21st Annual International

Conference on Composites Nano Engineering ICCE-21 Santa Cruz de Tenerife Spain

July 21-27 2013

18 Rivera A C N N Glazener N C Cook N J Withers L M Armijo D A Huang

J B Wright I Brener K Carpenter R D Busch G A Smolyakov and M Osiński

ldquoSynthesis and characterization of ytterbium-doped dysprosium fluoride nanocrystals for

use as neutron detectorsrdquo 21st Annual International Conference on Composites Nano

Engineering ICCE-21 Tenerife Spain 21-27 Jul 2013

19 Armijo L M Kopciuch B A Akins J B Plumley N J Withers A C Rivera N

C Cook Y I Brandt J M Baca S J Wawrzyniec G A Smolyakov D L Huber and

M Osiński ldquoLow-toxicity magnetic nanomaterials for biomedical applicationsrdquo 21st

Annual International Conference on Composites Nano Engineering ICCE-21 Tenerife

Spain 21-27 Jul 2013

20 Osiński M Y I Brandt L M Armijo M Kopciuch N J Withers N C Cook N

L Adolphi G A Smolyakov and H D C Smyth ldquoEfficacy of tobramycin conjugated to

superparamagnetic iron oxide nanoparticles in treating cystic fibrosis infections (Invited

218

Paper)rdquo Symposium 7E Low-Dimensional Semiconductor Structures (T V Torchynska

L Khomenkova G Polupan and G Burlak Eds) XXII International Material Research

Congress 2013 (IMRC 2013) Cancun Mexico 11-15 Aug 2013 MRS Proceedings Vol

1617 (11 pp) (Available online)

21 Rivera A C N N Glazener N C Cook N J Withers L M Armijo J Wright I

Brener K Carpenter R D Busch G A Smolyakov and M Osiński ldquoThermal neutron

detection using ytterbium-doped dysprosium fluoride nanocrystalsrdquo Zing Nanomaterials

2013 Conference Xcaret Mexico 13-17 Nov 2013

22 Armijo L M M Kopciuch Z Olszoacutewka S J Wawrzyniec A C Rivera J B

Plumley N C Cook Y I Brandt D L Huber G A Smolyakov N L Adolphi H D C

Smyth and M Osiński ldquoDelivery of antibiotics coupled to iron oxide nanoparticles across

the biofilm of mucoid Pseudonomas aeruginosa and investigation of their efficacyrdquo

Francisco CA 1-3 Feb 2014 Proceedings of SPIE Vol 8955 Paper 89550I (12 pp)

23 Osiński M Y I Brandt L M Armijo J B Plumley A C Rivera N C Cook G

A Smolyakov D L Huber and H D C Smyth ldquoSuperparamagnetic iron oxide

nanoparticles conjugated to tobramycin for treating cystic fibrosis infections (Invited

Paper)rdquo Technical Digest 4th Zing Bionanomaterials Conference Nerja Spain 6-9 Apr

2014 p 53

24 Armijo L M A Westphal P Jain A Malagodi F Fornelli A Hayat M French

H D C Smyth and M Osiński ldquoInhibition of bacterial growth by iron oxide

nanoparticles with and without attached drug Have we conquered the antibiotic resistance

problem in cystic fibrosis lung infectionsrdquo Colloidal Nanoparticles for Biomedical

Applications X (W J Parak M Osiński and Xing-Jie Liang eds) SPIE International

Proceedings of SPIE Vol 9338 Paper 1Q (11 pp)

Patent Applications

1 Armijo L M ldquoMethod of making magnetic iron nitride nanoparticlesrdquo US Patent

Application 13987912 filed 16 Sept 2013

2 Osiński M H D C Smyth L M Armijo and H M H Bandara ldquoMethods and

compositions for antimicrobial treatmentrdquo United States Provisional Patent Application

filed on 6 Feb 2015

219

APPENDIX I

List of Chemicals and Physical Properties

1 CAS No 67-64-1

Chemical Name Acetone

Synonyms 2-propanone

Molecular formula C3H6O

Molecular weight 5808

Melting point -94 degC

Boiling point 56 degC

Flash point -1722 degC

Density 0791 gmL

Vapor density 2

Vapor pressure 184 Torr

Refractive index 1359

EPA substance registry system 2-propanone (67-64-1)

Hazard codes F Xi T

2 CAS No 9005-32-7

Chemical name Alginic acid

Synonyms Alginate

Molecular formula (C6H8O6)n

Molecular weight 10000-600000

Melting point 300 degC

EPA substance registry system Alginic acid (9005-32-7)

Hazard codes Xi

3 CAS No 7664-41-7

Chemical name Ammonia

Synonyms Ammonia

Molecular formula NH3

Flash point 1111

Density 1023 gmL

Vapor density 06

Vapor pressure 875 atm

220

EPA substance registry system Ammonia (7664-41-7)

Hazard codes F N T Xn

4 CAS No 67-66-3

Chemical name Chloroform

Synonyms Trichloromethane formyl trichloride

methane trichloride methyl trichloride

Molecular formula CHCl3

Molecular weight 11938 amu

Melting point -63

Boiling point 61

Flash point 1492

Density 075 gmL

Vapor density 41

EPA substance registry system Methane trichloro-(67-66-3)

Hazard codes Xn F T Xi

Hazard codes Xi

5 CAS No 13754-17-1

Chemical name Citrate

Synonyms Citrate

Molecular formula C6H5O7

EPA substance registry system 123-Propanetricarboxylic acid 2-

hydroxy- ion(3-) (13754-17-1)

Hazard codes None

6 CAS No 64-17-5

Chemical name Ethanol

Synonyms Ethyl alcohol thanol grain alcohol

Melting point -114

Boiling point 78

Flash point 12

221

Density 079

EPA substance registry system Ethanol (64-17-5)

Hazard codes Xn F T N

Hazard codes Xi

Hazard codes F T Xn N

CAS No 112-40-3

Chemical name n-dodecane

Synonyms Dodecane

Molecular formula C12H26

Boiling point 215-217 degC

Flash point 83 degC

Density 075 gmL

Vapor density 596

EPA substance registry system Dodecane (112-40-3)

Hazard codes Xn

CAS No 629-97-0

Chemical name n-docosane

Synonyms Docosane

Melting point 42-45 degC

Flash point 95 degC

Density 0778 gmL

Vapor density 108

Hazard codes Xi

CAS No 112-95-8

Chemical name n-eicosane

222

Synonyms Eicosane icosane

Flash point gt113 degC

Density 07886 gmL

Vapor density 98

Hazard codes Xi

7 CAS No 106627-54-7

Chemical name N-hydroxysulfosuccinimide sodium

salt

Synonyms Sulfo-NHS sodium salt NHSS

Molecular formula C4H4NNaO6S

8 CAS No 1333-74-0

Chemical name Hydrogen gas

Synonyms Hydrogen

Molecular formula H2

Boiling point -2528 degC

Flash point lt-150 degC

Density 00899

Vapor density 007

EPA substance registry system Hydrogen (1333-74-0)

Hazard codes F+

9 CAS No 73513-42-5

Chemical name Hexanes

Synonyms Hexane cyclohexane

223

Density 0672 gmL

Vapor density 3

Hazard codes F Xn N

EPA substance registry system Hydrochloric acid (7647-01-0)

Hazard codes T C F Xi F+ Xn

10 CAS No 1317-61-9

Chemical name Iron oxide NPs

Synonyms Black iron oxide magnetite iron(III)

oxide

Molecular formula Fe3O4

Density 48-51 gmL

Hazard codes Xi

11 CAS No 7439-89-6

Chemical name Iron

Synonyms Iron

Molecular formula Fe

Density 105 gmL

EPA substance registry system Iron (7439-89-6)

Hazard codes F Xi

12 CAS No None

Chemical name Phosphate buffered saline

Synonyms PBS

Molecular formula O4P

Hazard codes Xi

224

13 CAS No 25322-68-3

Chemical name Polyethylene glycol

Synonyms PEG poly(oxyethylene)

Molecular formula C2nH4n+2On+1

Molecular weight 1802 + 4405n gmol

Boiling point gt250 degC

Flash point 270 degC

Density 127 gmL

Vapor density gt1

Vapor pressure lt001 Torr

EPA substance registry system Poly(oxy-12-ethanediyl) alpha-hydro-

omega-hydroxy (25322-68-3)

Hazard codes Xi T

14 CAS No 23335-74-2

Chemical name Iron oleate

Synonyms Iron(IIIII) oleate

Molecular formula C54H99FeO6

Hazard codes none

15 CAS No 10025-77-1

Chemical name Iron chloride hexahydrate

Synonyms ferric chloride hyxahydrate iron(III)

chloride

Molecular formula Cl3FeH12O6

Flash point 280-285 degC

Density 182 gmL

Hazard codes Xn C

16 CAS No 110-86-1

Chemical name Pyridine

225

Synonyms Azabenzine

Molecular formula C5H5N

Flash point 20 degC

Density 0983 gmL 20 degC

Vapor density 272

EPA substance registry system Pyridine (110-86-1)

Hazard codes T N F Xn

17 CAS No 1332-37-2

Chemical name Iron oxide

Synonyms Red iron oxide hematite maghemite

iron(III) oxide

Melting point 1539-1565 degC decomposes

EPA substance registry system Iron oxide (1332-37-2)

Hazard codes Xi

18 CAS No 85721-33-1

Chemical name Ciprofloxacin

Synonyms Cipro CPFX

Molecular formula C17H18FN3O3

EPA substance registry system

3-quinolinecarboxylic acid 1-

cyclopentyl-6-fluoro-14-dihydro-4-

oxo-7-(piperazinyl) (85721-33-1

Hazard codes Xi

19 CAS No 77-86-1

Chemical name Trometamol

Synonyms TRIS

Molecular formula C4H11NO3

226

Density 1353 gmL

EPA substance registry system 13-Propanediol 2-amino-2-

(hydroxymethyl)- (77-86-1)

Hazard codes Xi

20 CAS No 143-19-1

Chemical name Sodium oleate

Synonyms Sodium oleate

Molecular formula C18H33NaO2

Molecular weight 30444 degC

Hazard codes None

21 CAS No 108-30-5

Chemical name Succinic anhydride

Synonyms SAA SAN oxolan-25-dione

Density 1572 gmL

Vapor density 358

EPA substance registry system 25-Furandione dihydro-(108-30-5)

Hazard codes Xi Xn

22 CAS No 32986-56-4

Chemical name Tobramycin

Synonyms tobra Tobramax

Molecular formula C18H37N5O9

Melting point 178

Hazard codes Xi

227

23 CAS No 67-56-1

Chemical Name Methanol

Synonyms Methyl alcohol

Molecular formula CH4O

Density 0791 gmL

Vapor density 111

EPA substance registry system Methanol (67-56-1)

Hazard codes Xn T F

24 CAS No 7647-01-0

Chemical Name Hydrochloric acid

Synonyms HCl

Molecular formula HCl

Density 12 gmL

Vapor density 13

Vapor pressure 613 psi

EPA substance registry system hydrochloric acid (7647-01-0)

25 CAS No 1310-73-2

Chemical Name Sodium hydroxide

Synonyms NaOH

Molecular formula HNaO

228

Density 1515 gMl

Vapor density lt1

Refractive index 1473-1475

EPA substance registry system Sodium hydroxide (Na(OH)) (1310-73-

2)

Hazard codes C Xi

Chemical Hazard Codes and Symbols

References for Appendix I

1 Haynes William M ed CRC handbook of chemistry and physics CRC press

2014

2 Globally Harmonized System Hazard symbols Sigma-Aldrich Accessed

July 08 2016 httpwwwsigmaaldrichcomsafety-centerglobally-

harmonizedhtml

229

APPENDIX II

PROCEDURE FOR DRUG CONJUGATION TOBRAMYCIN CONJUGATION TO Fe3O4 HYDROPHILLIC NANOPARTICLES VIA

SULFO-NHS

NOTES Procedure prepared May 21 2012

Approved May 25 2012

Prepared by Kate Brandt and Leisha Armijo

Source

1 Bioconjugate Techniques G Hermanson Second Edition 2008 p 598

A Preparation of coupling (phosphate) buffer (50mM working solution)

i Using weighing paper and spatula weigh out 971 g of Na2HPO4 and add it to the

100 ml graduated cylinder

ii Add distilled water to 684 ml cover with parafilm and mix by inverting to dissolve

completely (makes 1M Na2HPO4 stock solution)

iii Using weighing paper and spatula weigh out 379 g of NaH2PO4 and add it to the

iv Add distilled water to 316 ml cover with parafilm and mix by inverting to dissolve

completely (makes 1M NaH2PO4 stock solution)

v Carefully combine the two solutions together in one of the 100 ml graduated

cylinders Cover the cylinder with parafilm and mix by inverting to mix

completely Pour the solution into a screw top bottle (makes 1 M phosphate buffer

solution) Label date and initial

B Preparation of 50 mM (working) solution of coupling (phosphate) buffer

i Using 500 ml graduated cylinder measure out 190 ml of distilled water

ii Using 10 ml graduated cylinder measure out 10 ml of 1 M phosphate buffer and

add it to the water Pour the solution into a screw top bottle Label date and initial

C Preparation of 50mM (working) solution of coupling phosphate buffer containing

35 mM Tris-OH

230

i Using a spatula weigh 212 mg of Tris-HCl into a 15 ml conical plastic centrifuge

tube

ii Shake the bottle with 50 mM phosphate coupling buffer and add it to 5 ml mark

Mix solution completely by inverting the tube as needed

D Conjugation of NPs to Tobramycin (in fume hood)

i Transfer nanoparticle (NP) solution (citric acid capped Fe3O4) into glass centrifuge

tube Spin 5 min at 4000 rpm to precipitate nanoparticles

ii Using glass pipettor carefully remove as much supernatant as you can without

disturbing the pellet Discard supernatant into the appropriate waste container

iii Carefully turn the tube over on paper towel and blot supernatant on it Position tube

at an angle to allow access of air and leave to dry for 30 - 40 min

iv Transfer pellet into the clean centrifuge tube weigh it and note it down

v Add 5 ml of coupling buffer to the tube and gently mix by pipetting it up and down

vi Spin 5 min at 4000 rpm

vii Using glass pipettor carefully removes as much supernatant as you can without

disturbing the pellet Discard into the appropriate waste container

viii Repeat previous steps (5-7) one more time

ix Finally add 5 ml of coupling buffer per every100 mg of pellet (adjust all the

following numbers accordingly to the weight of the pellet) to the tube and gently

mix by pipetting it up and down

x To make a 10 mgmL Tobramycin solution weigh out 50 mg of Tobramycin sulfate

for each 100 mg of pellet into a 50 ml beaker Add small stir bar to the beaker and

then 5 ml of coupling buffer for each 50 mg of Tobramycin

xi Dissolve Tobramycin by putting the beaker on the stir plate and stirring until its

complete dissolution

xii While stirring add NP solution to the beaker containing Tobramycin solution Stir

for 2 min

xiii Using weighing paper weigh 100 mg of EDC for each 100 mg of pellet weight and

add it to the beaker

xiv Add the entire 5 mM vial of Sulfo-NHS to the beaker

xv Reduce stirring to medium speed and continue stirring to react for 2-4 hours

xvi Wash NPs with 5 ml of coupling buffer as described in steps 5-7

xvii Resuspend NPs in coupling buffer containing 35 mM Tris

xviii Wash NPs twice with coupling buffer as described in steps 5-7

xix Resuspend conjugated NPs in 25 ml of coupling buffer for every 100 mg of pellet

weight (for a 40 mgml concentration) and transfer into a scintillation vial

xx Store the remaining 50 mM (working) solution of coupling (phosphate) buffer

231

APPENDIX III

NanoTherics Magnetherm

Derivation of Working Equation to Determine Potential Frequency and

Magnetic Field Capabilities

When resonance occurs in parallel LC

circuits current circulates between L and C

so source current is at zero or minimum This

Implies impedance of parallel

combination is at maximum

Impedance is determined by

119885 =120596119871lowast

1

120596119862

120596119871+1

120596119862

=120596119871

1205962119871119862+1=

1

120596119862+1

120596119871

=1

0= infin

Where impedance is Z ω is the angular frequency L is inductance (in Henrys) and C is

capacitance (in Farads)

Differentiating to ω

119889

119889120596[120596119862 +

1

120596119871= 119862 minus

1

1205962119871= 0 and

Figure AIII2 Impedance in a

parallel resonance circuit Image by A

Noni 2012 adapted by L Arrmijo

2016

Figure AIII1 LC Circuit diagram Image by A

Noni 2012 adapted by L Armijo 2016

232

Resonance occurs when

119881119871 = minus119881119862 and 119868119871119883119871 = minus119868119862119883119862

where V is voltage L is inductance X is reactance I is current and C is capacitance

When

119883119871 = 119883119862

The reactances of the inductor and the

capacitor are equal so

120596119871 =1

120596119862

And once again we arrive at

At resonance the parallel circuit produces the same equation as for the series resonance

circuit Therefore it makes no difference if the inductor and capacitor are connected in

parallel or series

To calculate the field inside a Solenoid (from Amperersquos Law)

119861119871 = 120583119873119868 rearranging to solve for B we get 119861 = 1205830119873

119897119868

Where B is magnetic flux density within the coil micro0= 4π x7-7 NA2 is the permeability

constant l is length Substituting the relation 119899 =119873

119897 where n is turn density (in turnsm)

we get 119861 = 1205830119899119868 The magnetic flux density in the solenoid is equal to the permeability

times turn density times current

Figure AIII3 Current vs frequency diagram at

resonant frequency Image by A Noni 2012 adapted

by L Armijo2016

233

Total magnetic flux is the product of the average magnetic field times the perpendicular

area that it passes through

Φ=BA

Where Φ is total magnetic flux B is magnetic flux density within the coil and A is the area

of the coil

Substituting for B we get

Φ= 1205830119873119868119860

119897= 120583119899119868119860

Inductance is defined by

119871 =119873120567

119868

Where L is inductance the inductance of a solenoid follows as

119871 = 1205830

1198732119860

119897

Rearranging we get

1205830119873

119897=

119871

119873119860

and 119861 =119871119868

119873119860

so magnetic field is maximum when current is maximum at resonant frequency

V across inductor is proportional to reactance XL=ωL and VL=XLIL

119861 = (119871

119873119860) lowast (

119881

120596119871) =

119881

119873119860120596

234

Thus our working equation is

119913 =119933

119925119912120654

Where N = number of turns in coil = 9 or 17 (2 types of inductors provided by

manufacturer) A = area of coil = 0004045 m2 Since we have 5 available capacitances and

2 available inductances there are 10 possible combinations per B value

Table III1 Tunability Specifications for Magnetherm Inductive Heater

To Achieve Field Strength of 9 mT at Tunable Frequencies Nominal

Frequency

(kHz)

Capacitor

Part No

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 42774

168 A88 88 17 1200 17 65328

176 A198 198 9 800 23 36232

262 A88 88 9 1200 23 53937

335 B22 22 17 2500 17 130267

474 B11 11 17 2500 11 184318

523 B22 22 9 2500 20 107668

633 B62 62 17 2500 9 246146

739 B11 11 9 2500 16 152135

987 B62 62 9 2500 12 203189

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 5228

168 A88 88 17 1200 17 79845

176 A198 198 9 800 23 42284

262 A88 88 9 1200 23 65923

335 B22 22 17 2500 17 159215

474 B11 11 17 2500 11 225278

523 B22 22 9 2500 20 131594

633 B62 62 17 2500 9 300846

739 B11 11 9 2500 16 185942

987 B62 62 9 2500 12 248342

235

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 57032

168 A88 88 17 1200 17 87103

176 A198 198 9 800 23 48309

262 A88 88 9 1200 23 71915

335 B22 22 17 2500 17 173688

474 B11 11 1as7 2500 11 245755

523 B22 22 9 2500 20 143555

633 B62 62 17 2500 9 328192

739 B11 11 9 2500 16 202844

987 B62 62 9 2500 12 270916

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 76042

168 A88 88 17 1200 17 116137

176 A198 198 9 800 23 64412

262 A88 88 9 1200 23 95887

335 B22 22 17 2500 17 231583

474 B11 11 17 2500 11 327673

523 B22 22 9 2500 20 191407

633 B62 62 17 2500 9 437589

739 B11 11 9 2500 16 270459

987 B62 62 9 2500 12 361221

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 80796

168 A88 88 17 1200 17 122339

176 A198 198 9 800 23 68439

262 A88 88 9 1200 23 101881

335 B22 22 17 2500 17 24606

474 B11 11 17 2500 11 348157

523 B22 22 9 2500 20 203372

633 B62 62 17 2500 9 464943

236

739 B11 11 9 2500 16 287365

987 B62 62 9 2500 12 383802

Frequency

(kHz)

Capacitor

Array

TypeVal

ue

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 95054

168 A88 88 17 1200 17 145173

176 A198 198 9 800 23 80516

262 A88 88 9 1200 23 119859

335 B22 22 17 2500 17 289482

474 B11 11 17 2500 11 409595

523 B22 22 9 2500 20 239261

633 B62 62 17 2500 9 546992

739 B11 11 9 2500 16 338077

987 B62 62 9 2500 12 451531

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 109312

168 A88 88 17 1200 17 166949

176 A198 198 9 800 23 92594

262 A88 88 9 1200 23 137838

335 B22 22 17 2500 17 332905

474 B11 11 17 2500 11 471035

523 B22 22 9 2500 20 275151

633 B62 62 17 2500 9 629041

739 B11 11 9 2500 16 388788

987 B62 62 9 2500 12 519261

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 118817

168 A88 88 17 1200 17 181467

176 A198 198 9 800 23 100645

262 A88 88 9 1200 23 149824

335 B22 22 17 2500 17 361853

474 B11 11 17 2500 11 511995

237

523 B22 22 9 2500 20 299077

633 B62 62 17 2500 9 68374

739 B11 11 9 2500 16 422596

987 B62 62 9 2500 16 564414

Tables define the parameters for tuning MagneThermtrade to the desired allowable

frequency and field strengths using different capacitor and inductor combinations These

tables give the voltage that should be applied as well as the maximum peak to peak voltage

(Vp-p) that may be applied without damage to the equipment

Note This information was not provided by the manufacturer and is essential for more in-

depth future work involving hyperthermia characterization studies using the

MagneThermtrade inductive heater These tables were produced using the working equation

Calculations were performed by Leisha Armijo MS Abhyudai Noni and Gennady

Smolyakov PhD (Summer 2012)

University of New Mexico
UNM Digital Repository
- Spring 4-15-2019
- - Iron-containing Nanoparticles for the Treatment of Chrionic Biofilm Infections in Cystic Fibrosis
  - - Leisha M A Martin
    - - Recommended Citation
      - tmp1555356534pdfqbGXI

Page 2: Iron-containing Nanoparticles for the Treatment of ...

i

Leisha Marie Martin Candidate

Department

This dissertation is approved and it is acceptable in quality and form for publication

Approved by the Dissertation Committee

Marek Osiński PhD Chairperson

Terefe Habteyes PhD

Erin Milligan PhD

Pavan Muttil PhD

ii

IRON-CONTAINING

IN CYSTIC FIBROSIS

by

LEISHA MARIE MARTIN

DISSERTATION

May 2019

iii

DEDICATION

사랑해

iv

ACKNOWLEDGEMENTS

v

vi

vii

IN CYSTIC FIBROSIS

by

Leisha Marie Armijo

BS Biology

ABSTRACT

viii

extensive treatment

ix

Table of Contents

x

xi

xii

xiii

xiv

xv

List of Figures

xvi

xvii

xviii

xix

List of Tables

xx

AI auto-inducer

CF cystic fibrosis

DI deionized

hydrochloride

I intermediate

LB Luria-Bertani

xxi

NP nanoparticle

OD optical density

QS quorum sensing

R resistant

r radius

S sensitive

xxii

1

Chapter 1

death

2

3

Table 11

Austria

13500 [Southern 2007]

Belgium

12850 [Lucotte 1995] [Chung 2002]

Bulgaria

12500 [Chung 2002]

Czech Republic

12833 [Romeo 1989] [Lucoette 1995]

Faroe Islands

11775 [Kaplan 1968]

Finland

125000 [Denning 1968]

125000 [Kere 1994] [Klaassen 1998]

France

14700 [Southern 2007]

Germany

4

Italy

14238 [Siegel 1960]

14238 [Bossi 2004]

Italy (Milan)

13170 [Chernick 1959]

Netherlands

14750 [Spock 1967]

14750 [Slieker 2005]

11857 [Noblett 1969]

Norway

16574 [Johnson 1984]

Poland

15000 [Southern 2007]

Republic of Ireland

11353 [Farrell 2007]

Romania

12056 [Popa 1997]

Scotland

11984 [Hide 1969]

Slovakia

11800 [Kadasi 1997]

Spain

13750 [Lucotte 1995] [Chung 2002]

Sweden

12200-4500 [Rosan 1962]

United Kingdom

12415 [Gracey 1969]

12381 [Dodge 2007]

North America

United States

[Bowman 1969]

[Palomaki 2004]

Canada

12500 [Mearns 1974] 13608 [Dupuis 2005]

(Quebec)

1895 [Weaver 1994]

Amish OH USA

1569 [Stutman 2002]

5

Zuni Tribe NM USA

Middle East

14000-18000 [Crozier 1974]

Bahrain

15800 [Corey 1988]

Jordan

12560 [Nielsen 1982]

Oceana

New Zealand

Australia

[Jensen 1987]

Other

Japan

South Africa

12000 (Caucasian) [Allan 1973] 1784-13924

6

Foundation 2015]

in the Population

7

Armijo 2016

8

future

9

10

11

cell results

12

13

of the lungs

Treatments

medicine was built

14

15

16

CF

17

Table 12

Class

Effect on CFTR

Types of Mutation

Therapy Potential

Therapy

(nonsense or frame

shift)

Aminoglycosides

Gene transfer read-

through compounds

Amino acid deletion

mutation)

Correctors

Butyrates

Gene transfer

impaired gating

Missense mutation

(ie G551D)

Potentiators

Genistein

Gene transfer

Missense mutation

(ie R117H or

R347P)

Milrinone

Potentiators

Gene transfer

(ie A445E or 5T)

Aminoglycosides

Antisense

oligonucleotides

Correctors Gene

transfer

transcription

Aminoglycosides

Stabilizers

Gene transfer

Bypass therapies

18

19

20

absence of mRNA

21

Fibrosis Patients

22

disease and illness

23

water

24

25

26

27

28

biofilm

29

30

31

32

33

34

35

36

37

38

Chapter 2

OXIDE NANOPARTICLES

39

40

211 Materials

41

42

43

44

45

46

217 Cost Reduction

47

48

49

50

bar is 5 nm

51

52

53

12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

0

1000

2000

3000

4000

5000

Inte

nsity (

cps)

2-Theta (degree)

[111]

[110]

[311]

[220][422]

[011]

54

55

Chapter 3

NANOPARTICLES AND

56

2013]

57

31 Theory

mp = Msυ (31)

58

2001]

τN = τ0exp(σ) (33)

59

32 Experimental

AB Sweden)

60

0 50 100 150 200 250 300 350

00004

00006

00008

00010

00012

00014

00016

Field-Cooled

Zero Field-Cooled

Mag

ne

tizatio

n (

em

u)

Temperature (K)

2012a]

61

observed

62

63

thermometer

cooling water

64

65

Wire (black)

0 5 10 15 20 25 30 35 40 4515

18

21

24

27

30

33

36

39

42

45

48

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

0 5 10 15 20 25 30 35

24

26

28

30

32

34

36

38

40

42

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

1111 kHz

6292 kHz

Spherical (blue)

Poloymorphus (red)

Figure 37

22 nm spheres

(b) 6292 kHz 9 mT

66

4 degC

67

experiments

68

pronounced

69

0 10 20 30 40 5020

30

40

50

Tem

per

ature

[d

egre

es C

elsi

us]

Time [min]

Fe3O

4 NPs in water

Fe3O

70

71

72

73

Chapter 4

in Chapter 6

74

75

Oe [Hattori 2001]

42 Theory

76

many researchers

2003]

gas

77

431 Materials

in Appendix I

78

solid

79

80

gas for 30 minutes

81

82

chloroform and heat

83

84

magnetometer

hysteresis loops of

iron oxide

-50E4 00 50E4-1

0

1

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

-50E4 00 50E4-80

-60

-40

-20

0

20

40

60

80

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

85

00 20E4 40E40

20

40

60

80

100

(

Am

2k

g)

H (mT)

temperature

86

87

Chapter 5

88

[Prencipe 2009]

51 Experimental

tobramycin molecule

89

511 Materials

90

119901119867 = 119901119870119886 + 11989711990011989210([119860 minus]

[119867119860] (1)

119901119870119886 = minus11989711990011989210([1198673119874+][119860minus]

[119867119860] (2)

91

119870119886 = [119900119897119890119886119905119890 119894119900119899][1198673119874+]

[119900119897119890119894119888 119886119888119894119889] = 1 times 10-5 pKa = 5

92

MarvinSketch

93

513 Citrate Capping

94

95

original synthesis

group

96

(PEG) 5000 powder

97

200 300 400 500 600 700

00

02

04

06

08

10

12

14

Abs

orba

nce

[OD

cm

]

Wavelength [nm]

glycol (PEG) 5000

98

99

Appendix II

MarvinSketch

100

101

102

103

to be ~2

104

105

Chapter 6

SENSITIVITY TESTING

106

positive control

107

108

Figure 61 Pyocyanin

Image showing the

produced by P

aeruginosa cultures

grown on agar for

Image by L Armijo

2014

109

61

611 Materials

110

111

112

strains

113

114

micropipette

115

recorded

growth period

116

117

118

119

62 Results

120

121

time

122

123

a 1 mg biomass

Table 61

Disk diffusion

[mm]

ge 15 13-14 le 12

124

Table 62

Disk

with PEG-OH 17 mm S

with PEG-OH 0 R

tobramycin 17 mm S

125

capped NPs

126

day 3

ZOI (mm)

day 60

5 μg 175S 16S

25 μg 11R 10R

5 μg 0R 0R

25 μg 0R 0R

5 μg 16S 15S

25 μg 10R 8R

5 μg 11R 15I

25 μg 7R 5R

5 μg 21S 22S

25 μg 20S 20S

100 μg 25R 15R

1000 μg 35R 32R

127

inactivate microbes

128

129

130

Table 64

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

4 Iron Oxide NP-

Tobramycin

25S 19S 14I

Table 65

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

9 Iron Oxide NP-

Tobramycin

0R 0R 0R

131

Table 66

Alginate and

Mucin

(No magnet)

Alginate

(magnet)

Mucin

(magnet)

Alginate and

Mucin

(magnet)

DI Water R R R R

Tobramycin R S R S

NP-alginate-

drug

R I S S

NP-citrate-

drug

R I I I

132

motility test for P

133

134

toxic dose ranges

135

136

137

138

Chapter 7

139

140

141

142

143

nm

144

716 Viability Assay

145

717 Apoptosis Assay

146

147

significant

148

Perc

en

t In

ten

sit

y R

ed

ucti

on

co

ntr

ol

1x

10

-3

1x

10

-2

1x

10

-1 1

10

0

20

40

60

reduction 2 M dye

149

measured value

72 Results

150

151

152

153

154

Viability

Time Point

Flu

ore

scen

ce (

RF

U)

6 hou

rs

12 h

ours

24 h

ours

0

20000

40000

60000

Digitonin

Staurosporine

Ionomycin

Untreated Cells

viability

155

156

157

Time Point

Lu

min

escen

ce (

RL

U)

6 hou

r

12 h

our

24 h

our

0

2100 5

4100 5

6100 5

Staurosporine

NPs (1 mgmL)

NPs (05 mgmL)

Untreated Cells

Ionomycin

observed

158

73 Discussion

159

160

161

162

163

CHapter 8

164

165

166

167

168

169

83 Conclusions

170

84 Future Work

171

bactericidal effect

172

173

REFERENCES

2762-2768

Wiley amp Sons 2007

(2003) 451-462

174

(2014) 688ndash99

pp)

(12 pp) doi101117122043340

175

pp)

208-213

Chest 59 no 1 (1965) 9-8

286ndash92

176

196ndash200

638-644

25 no 23 (2004) 5405-5413

3-14

no 3 (2004) 455-467

no 1 (2009)1ndash12

177

(2004) 55ndash60

Engineering 53 no 10 (2006) 2075-2083

Medica 36 no 1-4 (1989) 13-15

178

(2007) 366-370

(2006) 1675-1682

1453-1464

7551-7553

179

Research 45 no 5 (2011) 1995-2001

(1959) 739-745

(2005) 956ndash60

(2013) 4476-4489

(2007) 411-437

180

Thorax 67 no 1 (2012) 12-18

(2009) 2563ndash8

Bacteriology 185 no 3 (2003) 1027-1036

181

Research 67 no 1 (2006) 55-60

(2007) 522-526

no 1 (2012) 9 (9 pp)

Pediatrics 147 no 3 (2005) 312-315

8

182

(2014) 1ndash5

187-191

183

85 no 7 (2011) 743ndash50

277 no 2 (1998) 347-362

(2015) 8856ndash74

1986

184

13593-13599

1048-1059

60 no 3 (1996) 539-574

Childhood 44 no 235 (1969) 404-405

802-808

185

4021

186

(1908) 173ndash9

19095-19098

Press 2013

Press 2003

187

pp)

no 22 (2009) 3645-3651

(2007) 51-84

3 (2010) 1035-1042

200-215

188

(2016) e0154445 (13 pp)

838

11 (1997) 51-56

217

189

Medicine 279 no 2 (1968) 65-69

no 5 (1968) 810-818

Research 6 no 21 (2012) 4620-4622

190

Chemistry 27 no 9 (2008) 1825-1851

Screening 5 no 1 (1998) 16-19

109 no 6 (2004) 799-809

3 Pt 2 (1967) 388-392

1750-1754

191

Science 29 no 5 (1998) 511-535

(2011) 789-808

619

9 (2002) 681-685

(12 pp)

no 6 (2008) 2064-2110

(2012) 045007 (10 pp)

192

1529-1540

(2011) 136-146

Pharmacology 411 no 3 (2001) 255-260

193

Valley 1994

1442

(2014) 227 (9 pp)

(2012) 181-205

875

194

(2005) 106102

(1997) 1122-1127

oncology 103 no 2 (2011) 317-324

Sciences 32 no 3-4 (2007) 203-215

195

(2008) 1343ndash8

A 100 no 6 (2012) 1637-1646

(1974) 497-506

196

Materials 21 no 23 (2011) 4573-4581

no 2 (2017) 105

Health 213 no 3 (2010) 190-197

(2004) 2161-2175

16 no 10 (2005) 2346ndash53

197

175

Biology 12 no 7 (2005) 789-796

Science 75 no 1 (2018) 13-18

198

(1988) 836-841

190-197

1712-1720

199

no 33 (2006) 16248-16253

Materials 3 no 12 (2004) 891-895

167 (14 pp)

28 no 16 (2007) 2572-2581

200

Pediatrics 156 no 3 (1997) 212-213

131 no 13 (2009) 4783-4787

201

(1974)454ndash63

(2008)1079ndash81

4922 (1989) 1066-1073

202

no 3 (1976) 287ndash95

(2012) 22ndash6

(2000) 1905-1911

2906

pp)

203

Pharmacology 153 no 6 (2008) 1311-1323

Surgery 3 no 5 (1986) 732-740

204

(2017) 291ndash7

no 42 (2007) 5712-5720

506

205

(2017) 15464

(1974) 153-169

(1992) 222-228

Chemistry C 111 no 49 (2007) 18078-18086

689-699

440

206

(2014) 40 (9 pp)

Immunology 74 no 3 (2006) 1673-1682

no 4 (2002) 1132-1139

1275

207

267-281

(2007) 57-65

188ndash94

173-177

731

Pediatrics 140 no 3 (2002) 299-305

208

Letters 61 no 26 (2007) 4769-4772

Materials 55 no 1 (2009) 22-45

behind-genius

467-474

209

142 no 3 (1990) 523-528

1427-1433

210

warning (2008)

59 no 10 (1993) 3280-3286

1008

Reviews 62 no 3 (2010) 284-304

1 (2003) 30-39

211

167ndash73

212

(2009) 397-415

no 41 (2013) 9859-9866

11 (2010) 3016-3022

23 (2005) 9263-9269

213

no 2 (2000) 117-133

214

Journal Papers

Impact factor 7877

215

3105

2011 p 56

2011 p 62

82320M (11 pp)

216

13-18 May 2012 Paper Th-73

Paper Th-74

8548 Paper 85480E (12 pp)

pp)

217

2013

July 21-27 2013

218

2014 p 53

Patent Applications

filed on 6 Feb 2015

219

APPENDIX I

1 CAS No 67-64-1

Density 0791 gmL

Vapor density 2

Hazard codes F Xi T

2 CAS No 9005-32-7

Synonyms Alginate

Hazard codes Xi

3 CAS No 7664-41-7

Synonyms Ammonia

Flash point 1111

Density 1023 gmL

Vapor density 06

220

4 CAS No 67-66-3

Melting point -63

Boiling point 61

Flash point 1492

Density 075 gmL

Vapor density 41

Hazard codes Xi

5 CAS No 13754-17-1

Synonyms Citrate

hydroxy- ion(3-) (13754-17-1)

Hazard codes None

6 CAS No 64-17-5

Melting point -114

Boiling point 78

Flash point 12

221

Density 079

Hazard codes Xi

CAS No 112-40-3

Synonyms Dodecane

Flash point 83 degC

Density 075 gmL

Vapor density 596

Hazard codes Xn

CAS No 629-97-0

Synonyms Docosane

Flash point 95 degC

Density 0778 gmL

Vapor density 108

Hazard codes Xi

CAS No 112-95-8

222

Density 07886 gmL

Vapor density 98

Hazard codes Xi

7 CAS No 106627-54-7

salt

8 CAS No 1333-74-0

Synonyms Hydrogen

Density 00899

Vapor density 007

Hazard codes F+

9 CAS No 73513-42-5

223

Density 0672 gmL

Vapor density 3

Hazard codes F Xn N

10 CAS No 1317-61-9

oxide

Density 48-51 gmL

Hazard codes Xi

11 CAS No 7439-89-6

Chemical name Iron

Synonyms Iron

Density 105 gmL

Hazard codes F Xi

12 CAS No None

Synonyms PBS

Hazard codes Xi

224

13 CAS No 25322-68-3

Density 127 gmL

Vapor density gt1

Hazard codes Xi T

14 CAS No 23335-74-2

Hazard codes none

15 CAS No 10025-77-1

chloride

Density 182 gmL

Hazard codes Xn C

16 CAS No 110-86-1

225

Synonyms Azabenzine

Flash point 20 degC

Vapor density 272

17 CAS No 1332-37-2

iron(III) oxide

Hazard codes Xi

18 CAS No 85721-33-1

Synonyms Cipro CPFX

Hazard codes Xi

19 CAS No 77-86-1

Synonyms TRIS

226

Density 1353 gmL

Hazard codes Xi

20 CAS No 143-19-1

Hazard codes None

21 CAS No 108-30-5

Density 1572 gmL

Vapor density 358

Hazard codes Xi Xn

22 CAS No 32986-56-4

Melting point 178

Hazard codes Xi

227

23 CAS No 67-56-1

Density 0791 gmL

Vapor density 111

Hazard codes Xn T F

24 CAS No 7647-01-0

Synonyms HCl

Density 12 gmL

Vapor density 13

25 CAS No 1310-73-2

Synonyms NaOH

228

Density 1515 gMl

Vapor density lt1

2)

Hazard codes C Xi

2014

harmonizedhtml

229

APPENDIX II

SULFO-NHS

Source

35 mM Tris-OH

230

tube

for 2 min

231

APPENDIX III

119885 =120596119871lowast

1

120596119862

120596119871+1

120596119862

=120596119871

1205962119871119862+1=

1

120596119862+1

120596119871

=1

0= infin

119889

119889120596[120596119862 +

1

120596119871= 119862 minus

1

1205962119871= 0 and

2016

232

119881119871 = minus119881119862 and 119868119871119883119871 = minus119868119862119883119862

When

119883119871 = 119883119862

120596119871 =1

120596119862

parallel or series

119897119868

by L Armijo2016

233

Φ=BA

of the coil

Φ= 1205830119873119868119860

119897= 120583119899119868119860

119871 =119873120567

119868

119871 = 1205830

1198732119860

119897

Rearranging we get

1205830119873

119897=

119871

119873119860

and 119861 =119871119868

119873119860

119861 = (119871

119873119860) lowast (

119881

120596119871) =

119881

119873119860120596

234

119913 =119933

119925119912120654

Frequency

(kHz)

Capacitor

Part No

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 42774

168 A88 88 17 1200 17 65328

176 A198 198 9 800 23 36232

262 A88 88 9 1200 23 53937

335 B22 22 17 2500 17 130267

474 B11 11 17 2500 11 184318

523 B22 22 9 2500 20 107668

633 B62 62 17 2500 9 246146

739 B11 11 9 2500 16 152135

987 B62 62 9 2500 12 203189

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 5228

168 A88 88 17 1200 17 79845

176 A198 198 9 800 23 42284

262 A88 88 9 1200 23 65923

335 B22 22 17 2500 17 159215

474 B11 11 17 2500 11 225278

523 B22 22 9 2500 20 131594

633 B62 62 17 2500 9 300846

739 B11 11 9 2500 16 185942

987 B62 62 9 2500 12 248342

235

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 57032

168 A88 88 17 1200 17 87103

176 A198 198 9 800 23 48309

262 A88 88 9 1200 23 71915

335 B22 22 17 2500 17 173688

474 B11 11 1as7 2500 11 245755

523 B22 22 9 2500 20 143555

633 B62 62 17 2500 9 328192

739 B11 11 9 2500 16 202844

987 B62 62 9 2500 12 270916

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 76042

168 A88 88 17 1200 17 116137

176 A198 198 9 800 23 64412

262 A88 88 9 1200 23 95887

335 B22 22 17 2500 17 231583

474 B11 11 17 2500 11 327673

523 B22 22 9 2500 20 191407

633 B62 62 17 2500 9 437589

739 B11 11 9 2500 16 270459

987 B62 62 9 2500 12 361221

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 80796

168 A88 88 17 1200 17 122339

176 A198 198 9 800 23 68439

262 A88 88 9 1200 23 101881

335 B22 22 17 2500 17 24606

474 B11 11 17 2500 11 348157

523 B22 22 9 2500 20 203372

633 B62 62 17 2500 9 464943

236

739 B11 11 9 2500 16 287365

987 B62 62 9 2500 12 383802

Frequency

(kHz)

Capacitor

Array

TypeVal

ue

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 95054

168 A88 88 17 1200 17 145173

176 A198 198 9 800 23 80516

262 A88 88 9 1200 23 119859

335 B22 22 17 2500 17 289482

474 B11 11 17 2500 11 409595

523 B22 22 9 2500 20 239261

633 B62 62 17 2500 9 546992

739 B11 11 9 2500 16 338077

987 B62 62 9 2500 12 451531

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 109312

168 A88 88 17 1200 17 166949

176 A198 198 9 800 23 92594

262 A88 88 9 1200 23 137838

335 B22 22 17 2500 17 332905

474 B11 11 17 2500 11 471035

523 B22 22 9 2500 20 275151

633 B62 62 17 2500 9 629041

739 B11 11 9 2500 16 388788

987 B62 62 9 2500 12 519261

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 118817

168 A88 88 17 1200 17 181467

176 A198 198 9 800 23 100645

262 A88 88 9 1200 23 149824

335 B22 22 17 2500 17 361853

474 B11 11 17 2500 11 511995

237

523 B22 22 9 2500 20 299077

633 B62 62 17 2500 9 68374

739 B11 11 9 2500 16 422596

987 B62 62 9 2500 16 564414

- Spring 4-15-2019

ii

IRON-CONTAINING

IN CYSTIC FIBROSIS

by

LEISHA MARIE MARTIN

DISSERTATION

May 2019

iii

DEDICATION

사랑해

iv

ACKNOWLEDGEMENTS

v

vi

vii

IN CYSTIC FIBROSIS

by

Leisha Marie Armijo

BS Biology

ABSTRACT

viii

extensive treatment

ix

Table of Contents

x

xi

xii

xiii

xiv

xv

List of Figures

xvi

xvii

xviii

xix

List of Tables

xx

AI auto-inducer

CF cystic fibrosis

DI deionized

hydrochloride

I intermediate

LB Luria-Bertani

xxi

NP nanoparticle

OD optical density

QS quorum sensing

R resistant

r radius

S sensitive

xxii

1

Chapter 1

death

2

3

Table 11

Austria

13500 [Southern 2007]

Belgium

12850 [Lucotte 1995] [Chung 2002]

Bulgaria

12500 [Chung 2002]

Czech Republic

12833 [Romeo 1989] [Lucoette 1995]

Faroe Islands

11775 [Kaplan 1968]

Finland

125000 [Denning 1968]

125000 [Kere 1994] [Klaassen 1998]

France

14700 [Southern 2007]

Germany

4

Italy

14238 [Siegel 1960]

14238 [Bossi 2004]

Italy (Milan)

13170 [Chernick 1959]

Netherlands

14750 [Spock 1967]

14750 [Slieker 2005]

11857 [Noblett 1969]

Norway

16574 [Johnson 1984]

Poland

15000 [Southern 2007]

Republic of Ireland

11353 [Farrell 2007]

Romania

12056 [Popa 1997]

Scotland

11984 [Hide 1969]

Slovakia

11800 [Kadasi 1997]

Spain

13750 [Lucotte 1995] [Chung 2002]

Sweden

12200-4500 [Rosan 1962]

United Kingdom

12415 [Gracey 1969]

12381 [Dodge 2007]

North America

United States

[Bowman 1969]

[Palomaki 2004]

Canada

12500 [Mearns 1974] 13608 [Dupuis 2005]

(Quebec)

1895 [Weaver 1994]

Amish OH USA

1569 [Stutman 2002]

5

Zuni Tribe NM USA

Middle East

14000-18000 [Crozier 1974]

Bahrain

15800 [Corey 1988]

Jordan

12560 [Nielsen 1982]

Oceana

New Zealand

Australia

[Jensen 1987]

Other

Japan

South Africa

12000 (Caucasian) [Allan 1973] 1784-13924

6

Foundation 2015]

in the Population

7

Armijo 2016

8

future

9

10

11

cell results

12

13

of the lungs

Treatments

medicine was built

14

15

16

CF

17

Table 12

Class

Effect on CFTR

Types of Mutation

Therapy Potential

Therapy

(nonsense or frame

shift)

Aminoglycosides

Gene transfer read-

through compounds

Amino acid deletion

mutation)

Correctors

Butyrates

Gene transfer

impaired gating

Missense mutation

(ie G551D)

Potentiators

Genistein

Gene transfer

Missense mutation

(ie R117H or

R347P)

Milrinone

Potentiators

Gene transfer

(ie A445E or 5T)

Aminoglycosides

Antisense

oligonucleotides

Correctors Gene

transfer

transcription

Aminoglycosides

Stabilizers

Gene transfer

Bypass therapies

18

19

20

absence of mRNA

21

Fibrosis Patients

22

disease and illness

23

water

24

25

26

27

28

biofilm

29

30

31

32

33

34

35

36

37

38

Chapter 2

OXIDE NANOPARTICLES

39

40

211 Materials

41

42

43

44

45

46

217 Cost Reduction

47

48

49

50

bar is 5 nm

51

52

53

12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

0

1000

2000

3000

4000

5000

Inte

nsity (

cps)

2-Theta (degree)

[111]

[110]

[311]

[220][422]

[011]

54

55

Chapter 3

NANOPARTICLES AND

56

2013]

57

31 Theory

mp = Msυ (31)

58

2001]

τN = τ0exp(σ) (33)

59

32 Experimental

AB Sweden)

60

0 50 100 150 200 250 300 350

00004

00006

00008

00010

00012

00014

00016

Field-Cooled

Zero Field-Cooled

Mag

ne

tizatio

n (

em

u)

Temperature (K)

2012a]

61

observed

62

63

thermometer

cooling water

64

65

Wire (black)

0 5 10 15 20 25 30 35 40 4515

18

21

24

27

30

33

36

39

42

45

48

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

0 5 10 15 20 25 30 35

24

26

28

30

32

34

36

38

40

42

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

1111 kHz

6292 kHz

Spherical (blue)

Poloymorphus (red)

Figure 37

22 nm spheres

(b) 6292 kHz 9 mT

66

4 degC

67

experiments

68

pronounced

69

0 10 20 30 40 5020

30

40

50

Tem

per

ature

[d

egre

es C

elsi

us]

Time [min]

Fe3O

4 NPs in water

Fe3O

70

71

72

73

Chapter 4

in Chapter 6

74

75

Oe [Hattori 2001]

42 Theory

76

many researchers

2003]

gas

77

431 Materials

in Appendix I

78

solid

79

80

gas for 30 minutes

81

82

chloroform and heat

83

84

magnetometer

hysteresis loops of

iron oxide

-50E4 00 50E4-1

0

1

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

-50E4 00 50E4-80

-60

-40

-20

0

20

40

60

80

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

85

00 20E4 40E40

20

40

60

80

100

(

Am

2k

g)

H (mT)

temperature

86

87

Chapter 5

88

[Prencipe 2009]

51 Experimental

tobramycin molecule

89

511 Materials

90

119901119867 = 119901119870119886 + 11989711990011989210([119860 minus]

[119867119860] (1)

119901119870119886 = minus11989711990011989210([1198673119874+][119860minus]

[119867119860] (2)

91

119870119886 = [119900119897119890119886119905119890 119894119900119899][1198673119874+]

[119900119897119890119894119888 119886119888119894119889] = 1 times 10-5 pKa = 5

92

MarvinSketch

93

513 Citrate Capping

94

95

original synthesis

group

96

(PEG) 5000 powder

97

200 300 400 500 600 700

00

02

04

06

08

10

12

14

Abs

orba

nce

[OD

cm

]

Wavelength [nm]

glycol (PEG) 5000

98

99

Appendix II

MarvinSketch

100

101

102

103

to be ~2

104

105

Chapter 6

SENSITIVITY TESTING

106

positive control

107

108

Figure 61 Pyocyanin

Image showing the

produced by P

aeruginosa cultures

grown on agar for

Image by L Armijo

2014

109

61

611 Materials

110

111

112

strains

113

114

micropipette

115

recorded

growth period

116

117

118

119

62 Results

120

121

time

122

123

a 1 mg biomass

Table 61

Disk diffusion

[mm]

ge 15 13-14 le 12

124

Table 62

Disk

with PEG-OH 17 mm S

with PEG-OH 0 R

tobramycin 17 mm S

125

capped NPs

126

day 3

ZOI (mm)

day 60

5 μg 175S 16S

25 μg 11R 10R

5 μg 0R 0R

25 μg 0R 0R

5 μg 16S 15S

25 μg 10R 8R

5 μg 11R 15I

25 μg 7R 5R

5 μg 21S 22S

25 μg 20S 20S

100 μg 25R 15R

1000 μg 35R 32R

127

inactivate microbes

128

129

130

Table 64

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

4 Iron Oxide NP-

Tobramycin

25S 19S 14I

Table 65

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

9 Iron Oxide NP-

Tobramycin

0R 0R 0R

131

Table 66

Alginate and

Mucin

(No magnet)

Alginate

(magnet)

Mucin

(magnet)

Alginate and

Mucin

(magnet)

DI Water R R R R

Tobramycin R S R S

NP-alginate-

drug

R I S S

NP-citrate-

drug

R I I I

132

motility test for P

133

134

toxic dose ranges

135

136

137

138

Chapter 7

139

140

141

142

143

nm

144

716 Viability Assay

145

717 Apoptosis Assay

146

147

significant

148

Perc

en

t In

ten

sit

y R

ed

ucti

on

co

ntr

ol

1x

10

-3

1x

10

-2

1x

10

-1 1

10

0

20

40

60

reduction 2 M dye

149

measured value

72 Results

150

151

152

153

154

Viability

Time Point

Flu

ore

scen

ce (

RF

U)

6 hou

rs

12 h

ours

24 h

ours

0

20000

40000

60000

Digitonin

Staurosporine

Ionomycin

Untreated Cells

viability

155

156

157

Time Point

Lu

min

escen

ce (

RL

U)

6 hou

r

12 h

our

24 h

our

0

2100 5

4100 5

6100 5

Staurosporine

NPs (1 mgmL)

NPs (05 mgmL)

Untreated Cells

Ionomycin

observed

158

73 Discussion

159

160

161

162

163

CHapter 8

164

165

166

167

168

169

83 Conclusions

170

84 Future Work

171

bactericidal effect

172

173

REFERENCES

2762-2768

Wiley amp Sons 2007

(2003) 451-462

174

(2014) 688ndash99

pp)

(12 pp) doi101117122043340

175

pp)

208-213

Chest 59 no 1 (1965) 9-8

286ndash92

176

196ndash200

638-644

25 no 23 (2004) 5405-5413

3-14

no 3 (2004) 455-467

no 1 (2009)1ndash12

177

(2004) 55ndash60

Engineering 53 no 10 (2006) 2075-2083

Medica 36 no 1-4 (1989) 13-15

178

(2007) 366-370

(2006) 1675-1682

1453-1464

7551-7553

179

Research 45 no 5 (2011) 1995-2001

(1959) 739-745

(2005) 956ndash60

(2013) 4476-4489

(2007) 411-437

180

Thorax 67 no 1 (2012) 12-18

(2009) 2563ndash8

Bacteriology 185 no 3 (2003) 1027-1036

181

Research 67 no 1 (2006) 55-60

(2007) 522-526

no 1 (2012) 9 (9 pp)

Pediatrics 147 no 3 (2005) 312-315

8

182

(2014) 1ndash5

187-191

183

85 no 7 (2011) 743ndash50

277 no 2 (1998) 347-362

(2015) 8856ndash74

1986

184

13593-13599

1048-1059

60 no 3 (1996) 539-574

Childhood 44 no 235 (1969) 404-405

802-808

185

4021

186

(1908) 173ndash9

19095-19098

Press 2013

Press 2003

187

pp)

no 22 (2009) 3645-3651

(2007) 51-84

3 (2010) 1035-1042

200-215

188

(2016) e0154445 (13 pp)

838

11 (1997) 51-56

217

189

Medicine 279 no 2 (1968) 65-69

no 5 (1968) 810-818

Research 6 no 21 (2012) 4620-4622

190

Chemistry 27 no 9 (2008) 1825-1851

Screening 5 no 1 (1998) 16-19

109 no 6 (2004) 799-809

3 Pt 2 (1967) 388-392

1750-1754

191

Science 29 no 5 (1998) 511-535

(2011) 789-808

619

9 (2002) 681-685

(12 pp)

no 6 (2008) 2064-2110

(2012) 045007 (10 pp)

192

1529-1540

(2011) 136-146

Pharmacology 411 no 3 (2001) 255-260

193

Valley 1994

1442

(2014) 227 (9 pp)

(2012) 181-205

875

194

(2005) 106102

(1997) 1122-1127

oncology 103 no 2 (2011) 317-324

Sciences 32 no 3-4 (2007) 203-215

195

(2008) 1343ndash8

A 100 no 6 (2012) 1637-1646

(1974) 497-506

196

Materials 21 no 23 (2011) 4573-4581

no 2 (2017) 105

Health 213 no 3 (2010) 190-197

(2004) 2161-2175

16 no 10 (2005) 2346ndash53

197

175

Biology 12 no 7 (2005) 789-796

Science 75 no 1 (2018) 13-18

198

(1988) 836-841

190-197

1712-1720

199

no 33 (2006) 16248-16253

Materials 3 no 12 (2004) 891-895

167 (14 pp)

28 no 16 (2007) 2572-2581

200

Pediatrics 156 no 3 (1997) 212-213

131 no 13 (2009) 4783-4787

201

(1974)454ndash63

(2008)1079ndash81

4922 (1989) 1066-1073

202

no 3 (1976) 287ndash95

(2012) 22ndash6

(2000) 1905-1911

2906

pp)

203

Pharmacology 153 no 6 (2008) 1311-1323

Surgery 3 no 5 (1986) 732-740

204

(2017) 291ndash7

no 42 (2007) 5712-5720

506

205

(2017) 15464

(1974) 153-169

(1992) 222-228

Chemistry C 111 no 49 (2007) 18078-18086

689-699

440

206

(2014) 40 (9 pp)

Immunology 74 no 3 (2006) 1673-1682

no 4 (2002) 1132-1139

1275

207

267-281

(2007) 57-65

188ndash94

173-177

731

Pediatrics 140 no 3 (2002) 299-305

208

Letters 61 no 26 (2007) 4769-4772

Materials 55 no 1 (2009) 22-45

behind-genius

467-474

209

142 no 3 (1990) 523-528

1427-1433

210

warning (2008)

59 no 10 (1993) 3280-3286

1008

Reviews 62 no 3 (2010) 284-304

1 (2003) 30-39

211

167ndash73

212

(2009) 397-415

no 41 (2013) 9859-9866

11 (2010) 3016-3022

23 (2005) 9263-9269

213

no 2 (2000) 117-133

214

Journal Papers

Impact factor 7877

215

3105

2011 p 56

2011 p 62

82320M (11 pp)

216

13-18 May 2012 Paper Th-73

Paper Th-74

8548 Paper 85480E (12 pp)

pp)

217

2013

July 21-27 2013

218

2014 p 53

Patent Applications

filed on 6 Feb 2015

219

APPENDIX I

1 CAS No 67-64-1

Density 0791 gmL

Vapor density 2

Hazard codes F Xi T

2 CAS No 9005-32-7

Synonyms Alginate

Hazard codes Xi

3 CAS No 7664-41-7

Synonyms Ammonia

Flash point 1111

Density 1023 gmL

Vapor density 06

220

4 CAS No 67-66-3

Melting point -63

Boiling point 61

Flash point 1492

Density 075 gmL

Vapor density 41

Hazard codes Xi

5 CAS No 13754-17-1

Synonyms Citrate

hydroxy- ion(3-) (13754-17-1)

Hazard codes None

6 CAS No 64-17-5

Melting point -114

Boiling point 78

Flash point 12

221

Density 079

Hazard codes Xi

CAS No 112-40-3

Synonyms Dodecane

Flash point 83 degC

Density 075 gmL

Vapor density 596

Hazard codes Xn

CAS No 629-97-0

Synonyms Docosane

Flash point 95 degC

Density 0778 gmL

Vapor density 108

Hazard codes Xi

CAS No 112-95-8

222

Density 07886 gmL

Vapor density 98

Hazard codes Xi

7 CAS No 106627-54-7

salt

8 CAS No 1333-74-0

Synonyms Hydrogen

Density 00899

Vapor density 007

Hazard codes F+

9 CAS No 73513-42-5

223

Density 0672 gmL

Vapor density 3

Hazard codes F Xn N

10 CAS No 1317-61-9

oxide

Density 48-51 gmL

Hazard codes Xi

11 CAS No 7439-89-6

Chemical name Iron

Synonyms Iron

Density 105 gmL

Hazard codes F Xi

12 CAS No None

Synonyms PBS

Hazard codes Xi

224

13 CAS No 25322-68-3

Density 127 gmL

Vapor density gt1

Hazard codes Xi T

14 CAS No 23335-74-2

Hazard codes none

15 CAS No 10025-77-1

chloride

Density 182 gmL

Hazard codes Xn C

16 CAS No 110-86-1

225

Synonyms Azabenzine

Flash point 20 degC

Vapor density 272

17 CAS No 1332-37-2

iron(III) oxide

Hazard codes Xi

18 CAS No 85721-33-1

Synonyms Cipro CPFX

Hazard codes Xi

19 CAS No 77-86-1

Synonyms TRIS

226

Density 1353 gmL

Hazard codes Xi

20 CAS No 143-19-1

Hazard codes None

21 CAS No 108-30-5

Density 1572 gmL

Vapor density 358

Hazard codes Xi Xn

22 CAS No 32986-56-4

Melting point 178

Hazard codes Xi

227

23 CAS No 67-56-1

Density 0791 gmL

Vapor density 111

Hazard codes Xn T F

24 CAS No 7647-01-0

Synonyms HCl

Density 12 gmL

Vapor density 13

25 CAS No 1310-73-2

Synonyms NaOH

228

Density 1515 gMl

Vapor density lt1

2)

Hazard codes C Xi

2014

harmonizedhtml

229

APPENDIX II

SULFO-NHS

Source

35 mM Tris-OH

230

tube

for 2 min

231

APPENDIX III

119885 =120596119871lowast

1

120596119862

120596119871+1

120596119862

=120596119871

1205962119871119862+1=

1

120596119862+1

120596119871

=1

0= infin

119889

119889120596[120596119862 +

1

120596119871= 119862 minus

1

1205962119871= 0 and

2016

232

119881119871 = minus119881119862 and 119868119871119883119871 = minus119868119862119883119862

When

119883119871 = 119883119862

120596119871 =1

120596119862

parallel or series

119897119868

by L Armijo2016

233

Φ=BA

of the coil

Φ= 1205830119873119868119860

119897= 120583119899119868119860

119871 =119873120567

119868

119871 = 1205830

1198732119860

119897

Rearranging we get

1205830119873

119897=

119871

119873119860

and 119861 =119871119868

119873119860

119861 = (119871

119873119860) lowast (

119881

120596119871) =

119881

119873119860120596

234

119913 =119933

119925119912120654

Frequency

(kHz)

Capacitor

Part No

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 42774

168 A88 88 17 1200 17 65328

176 A198 198 9 800 23 36232

262 A88 88 9 1200 23 53937

335 B22 22 17 2500 17 130267

474 B11 11 17 2500 11 184318

523 B22 22 9 2500 20 107668

633 B62 62 17 2500 9 246146

739 B11 11 9 2500 16 152135

987 B62 62 9 2500 12 203189

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 5228

168 A88 88 17 1200 17 79845

176 A198 198 9 800 23 42284

262 A88 88 9 1200 23 65923

335 B22 22 17 2500 17 159215

474 B11 11 17 2500 11 225278

523 B22 22 9 2500 20 131594

633 B62 62 17 2500 9 300846

739 B11 11 9 2500 16 185942

987 B62 62 9 2500 12 248342

235

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 57032

168 A88 88 17 1200 17 87103

176 A198 198 9 800 23 48309

262 A88 88 9 1200 23 71915

335 B22 22 17 2500 17 173688

474 B11 11 1as7 2500 11 245755

523 B22 22 9 2500 20 143555

633 B62 62 17 2500 9 328192

739 B11 11 9 2500 16 202844

987 B62 62 9 2500 12 270916

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 76042

168 A88 88 17 1200 17 116137

176 A198 198 9 800 23 64412

262 A88 88 9 1200 23 95887

335 B22 22 17 2500 17 231583

474 B11 11 17 2500 11 327673

523 B22 22 9 2500 20 191407

633 B62 62 17 2500 9 437589

739 B11 11 9 2500 16 270459

987 B62 62 9 2500 12 361221

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 80796

168 A88 88 17 1200 17 122339

176 A198 198 9 800 23 68439

262 A88 88 9 1200 23 101881

335 B22 22 17 2500 17 24606

474 B11 11 17 2500 11 348157

523 B22 22 9 2500 20 203372

633 B62 62 17 2500 9 464943

236

739 B11 11 9 2500 16 287365

987 B62 62 9 2500 12 383802

Frequency

(kHz)

Capacitor

Array

TypeVal

ue

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 95054

168 A88 88 17 1200 17 145173

176 A198 198 9 800 23 80516

262 A88 88 9 1200 23 119859

335 B22 22 17 2500 17 289482

474 B11 11 17 2500 11 409595

523 B22 22 9 2500 20 239261

633 B62 62 17 2500 9 546992

739 B11 11 9 2500 16 338077

987 B62 62 9 2500 12 451531

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 109312

168 A88 88 17 1200 17 166949

176 A198 198 9 800 23 92594

262 A88 88 9 1200 23 137838

335 B22 22 17 2500 17 332905

474 B11 11 17 2500 11 471035

523 B22 22 9 2500 20 275151

633 B62 62 17 2500 9 629041

739 B11 11 9 2500 16 388788

987 B62 62 9 2500 12 519261

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 118817

168 A88 88 17 1200 17 181467

176 A198 198 9 800 23 100645

262 A88 88 9 1200 23 149824

335 B22 22 17 2500 17 361853

474 B11 11 17 2500 11 511995

237

523 B22 22 9 2500 20 299077

633 B62 62 17 2500 9 68374

739 B11 11 9 2500 16 422596

987 B62 62 9 2500 16 564414

- Spring 4-15-2019

Page 4: Iron-containing Nanoparticles for the Treatment of ...

iii

DEDICATION

사랑해

iv

ACKNOWLEDGEMENTS

v

vi

vii

IN CYSTIC FIBROSIS

by

Leisha Marie Armijo

BS Biology

ABSTRACT

viii

extensive treatment

ix

Table of Contents

x

xi

xii

xiii

xiv

xv

List of Figures

xvi

xvii

xviii

xix

List of Tables

xx

AI auto-inducer

CF cystic fibrosis

DI deionized

hydrochloride

I intermediate

LB Luria-Bertani

xxi

NP nanoparticle

OD optical density

QS quorum sensing

R resistant

r radius

S sensitive

xxii

1

Chapter 1

death

2

3

Table 11

Austria

13500 [Southern 2007]

Belgium

12850 [Lucotte 1995] [Chung 2002]

Bulgaria

12500 [Chung 2002]

Czech Republic

12833 [Romeo 1989] [Lucoette 1995]

Faroe Islands

11775 [Kaplan 1968]

Finland

125000 [Denning 1968]

125000 [Kere 1994] [Klaassen 1998]

France

14700 [Southern 2007]

Germany

4

Italy

14238 [Siegel 1960]

14238 [Bossi 2004]

Italy (Milan)

13170 [Chernick 1959]

Netherlands

14750 [Spock 1967]

14750 [Slieker 2005]

11857 [Noblett 1969]

Norway

16574 [Johnson 1984]

Poland

15000 [Southern 2007]

Republic of Ireland

11353 [Farrell 2007]

Romania

12056 [Popa 1997]

Scotland

11984 [Hide 1969]

Slovakia

11800 [Kadasi 1997]

Spain

13750 [Lucotte 1995] [Chung 2002]

Sweden

12200-4500 [Rosan 1962]

United Kingdom

12415 [Gracey 1969]

12381 [Dodge 2007]

North America

United States

[Bowman 1969]

[Palomaki 2004]

Canada

12500 [Mearns 1974] 13608 [Dupuis 2005]

(Quebec)

1895 [Weaver 1994]

Amish OH USA

1569 [Stutman 2002]

5

Zuni Tribe NM USA

Middle East

14000-18000 [Crozier 1974]

Bahrain

15800 [Corey 1988]

Jordan

12560 [Nielsen 1982]

Oceana

New Zealand

Australia

[Jensen 1987]

Other

Japan

South Africa

12000 (Caucasian) [Allan 1973] 1784-13924

6

Foundation 2015]

in the Population

7

Armijo 2016

8

future

9

10

11

cell results

12

13

of the lungs

Treatments

medicine was built

14

15

16

CF

17

Table 12

Class

Effect on CFTR

Types of Mutation

Therapy Potential

Therapy

(nonsense or frame

shift)

Aminoglycosides

Gene transfer read-

through compounds

Amino acid deletion

mutation)

Correctors

Butyrates

Gene transfer

impaired gating

Missense mutation

(ie G551D)

Potentiators

Genistein

Gene transfer

Missense mutation

(ie R117H or

R347P)

Milrinone

Potentiators

Gene transfer

(ie A445E or 5T)

Aminoglycosides

Antisense

oligonucleotides

Correctors Gene

transfer

transcription

Aminoglycosides

Stabilizers

Gene transfer

Bypass therapies

18

19

20

absence of mRNA

21

Fibrosis Patients

22

disease and illness

23

water

24

25

26

27

28

biofilm

29

30

31

32

33

34

35

36

37

38

Chapter 2

OXIDE NANOPARTICLES

39

40

211 Materials

41

42

43

44

45

46

217 Cost Reduction

47

48

49

50

bar is 5 nm

51

52

53

12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

0

1000

2000

3000

4000

5000

Inte

nsity (

cps)

2-Theta (degree)

[111]

[110]

[311]

[220][422]

[011]

54

55

Chapter 3

NANOPARTICLES AND

56

2013]

57

31 Theory

mp = Msυ (31)

58

2001]

τN = τ0exp(σ) (33)

59

32 Experimental

AB Sweden)

60

0 50 100 150 200 250 300 350

00004

00006

00008

00010

00012

00014

00016

Field-Cooled

Zero Field-Cooled

Mag

ne

tizatio

n (

em

u)

Temperature (K)

2012a]

61

observed

62

63

thermometer

cooling water

64

65

Wire (black)

0 5 10 15 20 25 30 35 40 4515

18

21

24

27

30

33

36

39

42

45

48

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

0 5 10 15 20 25 30 35

24

26

28

30

32

34

36

38

40

42

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

1111 kHz

6292 kHz

Spherical (blue)

Poloymorphus (red)

Figure 37

22 nm spheres

(b) 6292 kHz 9 mT

66

4 degC

67

experiments

68

pronounced

69

0 10 20 30 40 5020

30

40

50

Tem

per

ature

[d

egre

es C

elsi

us]

Time [min]

Fe3O

4 NPs in water

Fe3O

70

71

72

73

Chapter 4

in Chapter 6

74

75

Oe [Hattori 2001]

42 Theory

76

many researchers

2003]

gas

77

431 Materials

in Appendix I

78

solid

79

80

gas for 30 minutes

81

82

chloroform and heat

83

84

magnetometer

hysteresis loops of

iron oxide

-50E4 00 50E4-1

0

1

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

-50E4 00 50E4-80

-60

-40

-20

0

20

40

60

80

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

85

00 20E4 40E40

20

40

60

80

100

(

Am

2k

g)

H (mT)

temperature

86

87

Chapter 5

88

[Prencipe 2009]

51 Experimental

tobramycin molecule

89

511 Materials

90

119901119867 = 119901119870119886 + 11989711990011989210([119860 minus]

[119867119860] (1)

119901119870119886 = minus11989711990011989210([1198673119874+][119860minus]

[119867119860] (2)

91

119870119886 = [119900119897119890119886119905119890 119894119900119899][1198673119874+]

[119900119897119890119894119888 119886119888119894119889] = 1 times 10-5 pKa = 5

92

MarvinSketch

93

513 Citrate Capping

94

95

original synthesis

group

96

(PEG) 5000 powder

97

200 300 400 500 600 700

00

02

04

06

08

10

12

14

Abs

orba

nce

[OD

cm

]

Wavelength [nm]

glycol (PEG) 5000

98

99

Appendix II

MarvinSketch

100

101

102

103

to be ~2

104

105

Chapter 6

SENSITIVITY TESTING

106

positive control

107

108

Figure 61 Pyocyanin

Image showing the

produced by P

aeruginosa cultures

grown on agar for

Image by L Armijo

2014

109

61

611 Materials

110

111

112

strains

113

114

micropipette

115

recorded

growth period

116

117

118

119

62 Results

120

121

time

122

123

a 1 mg biomass

Table 61

Disk diffusion

[mm]

ge 15 13-14 le 12

124

Table 62

Disk

with PEG-OH 17 mm S

with PEG-OH 0 R

tobramycin 17 mm S

125

capped NPs

126

day 3

ZOI (mm)

day 60

5 μg 175S 16S

25 μg 11R 10R

5 μg 0R 0R

25 μg 0R 0R

5 μg 16S 15S

25 μg 10R 8R

5 μg 11R 15I

25 μg 7R 5R

5 μg 21S 22S

25 μg 20S 20S

100 μg 25R 15R

1000 μg 35R 32R

127

inactivate microbes

128

129

130

Table 64

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

4 Iron Oxide NP-

Tobramycin

25S 19S 14I

Table 65

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

9 Iron Oxide NP-

Tobramycin

0R 0R 0R

131

Table 66

Alginate and

Mucin

(No magnet)

Alginate

(magnet)

Mucin

(magnet)

Alginate and

Mucin

(magnet)

DI Water R R R R

Tobramycin R S R S

NP-alginate-

drug

R I S S

NP-citrate-

drug

R I I I

132

motility test for P

133

134

toxic dose ranges

135

136

137

138

Chapter 7

139

140

141

142

143

nm

144

716 Viability Assay

145

717 Apoptosis Assay

146

147

significant

148

Perc

en

t In

ten

sit

y R

ed

ucti

on

co

ntr

ol

1x

10

-3

1x

10

-2

1x

10

-1 1

10

0

20

40

60

reduction 2 M dye

149

measured value

72 Results

150

151

152

153

154

Viability

Time Point

Flu

ore

scen

ce (

RF

U)

6 hou

rs

12 h

ours

24 h

ours

0

20000

40000

60000

Digitonin

Staurosporine

Ionomycin

Untreated Cells

viability

155

156

157

Time Point

Lu

min

escen

ce (

RL

U)

6 hou

r

12 h

our

24 h

our

0

2100 5

4100 5

6100 5

Staurosporine

NPs (1 mgmL)

NPs (05 mgmL)

Untreated Cells

Ionomycin

observed

158

73 Discussion

159

160

161

162

163

CHapter 8

164

165

166

167

168

169

83 Conclusions

170

84 Future Work

171

bactericidal effect

172

173

REFERENCES

2762-2768

Wiley amp Sons 2007

(2003) 451-462

174

(2014) 688ndash99

pp)

(12 pp) doi101117122043340

175

pp)

208-213

Chest 59 no 1 (1965) 9-8

286ndash92

176

196ndash200

638-644

25 no 23 (2004) 5405-5413

3-14

no 3 (2004) 455-467

no 1 (2009)1ndash12

177

(2004) 55ndash60

Engineering 53 no 10 (2006) 2075-2083

Medica 36 no 1-4 (1989) 13-15

178

(2007) 366-370

(2006) 1675-1682

1453-1464

7551-7553

179

Research 45 no 5 (2011) 1995-2001

(1959) 739-745

(2005) 956ndash60

(2013) 4476-4489

(2007) 411-437

180

Thorax 67 no 1 (2012) 12-18

(2009) 2563ndash8

Bacteriology 185 no 3 (2003) 1027-1036

181

Research 67 no 1 (2006) 55-60

(2007) 522-526

no 1 (2012) 9 (9 pp)

Pediatrics 147 no 3 (2005) 312-315

8

182

(2014) 1ndash5

187-191

183

85 no 7 (2011) 743ndash50

277 no 2 (1998) 347-362

(2015) 8856ndash74

1986

184

13593-13599

1048-1059

60 no 3 (1996) 539-574

Childhood 44 no 235 (1969) 404-405

802-808

185

4021

186

(1908) 173ndash9

19095-19098

Press 2013

Press 2003

187

pp)

no 22 (2009) 3645-3651

(2007) 51-84

3 (2010) 1035-1042

200-215

188

(2016) e0154445 (13 pp)

838

11 (1997) 51-56

217

189

Medicine 279 no 2 (1968) 65-69

no 5 (1968) 810-818

Research 6 no 21 (2012) 4620-4622

190

Chemistry 27 no 9 (2008) 1825-1851

Screening 5 no 1 (1998) 16-19

109 no 6 (2004) 799-809

3 Pt 2 (1967) 388-392

1750-1754

191

Science 29 no 5 (1998) 511-535

(2011) 789-808

619

9 (2002) 681-685

(12 pp)

no 6 (2008) 2064-2110

(2012) 045007 (10 pp)

192

1529-1540

(2011) 136-146

Pharmacology 411 no 3 (2001) 255-260

193

Valley 1994

1442

(2014) 227 (9 pp)

(2012) 181-205

875

194

(2005) 106102

(1997) 1122-1127

oncology 103 no 2 (2011) 317-324

Sciences 32 no 3-4 (2007) 203-215

195

(2008) 1343ndash8

A 100 no 6 (2012) 1637-1646

(1974) 497-506

196

Materials 21 no 23 (2011) 4573-4581

no 2 (2017) 105

Health 213 no 3 (2010) 190-197

(2004) 2161-2175

16 no 10 (2005) 2346ndash53

197

175

Biology 12 no 7 (2005) 789-796

Science 75 no 1 (2018) 13-18

198

(1988) 836-841

190-197

1712-1720

199

no 33 (2006) 16248-16253

Materials 3 no 12 (2004) 891-895

167 (14 pp)

28 no 16 (2007) 2572-2581

200

Pediatrics 156 no 3 (1997) 212-213

131 no 13 (2009) 4783-4787

201

(1974)454ndash63

(2008)1079ndash81

4922 (1989) 1066-1073

202

no 3 (1976) 287ndash95

(2012) 22ndash6

(2000) 1905-1911

2906

pp)

203

Pharmacology 153 no 6 (2008) 1311-1323

Surgery 3 no 5 (1986) 732-740

204

(2017) 291ndash7

no 42 (2007) 5712-5720

506

205

(2017) 15464

(1974) 153-169

(1992) 222-228

Chemistry C 111 no 49 (2007) 18078-18086

689-699

440

206

(2014) 40 (9 pp)

Immunology 74 no 3 (2006) 1673-1682

no 4 (2002) 1132-1139

1275

207

267-281

(2007) 57-65

188ndash94

173-177

731

Pediatrics 140 no 3 (2002) 299-305

208

Letters 61 no 26 (2007) 4769-4772

Materials 55 no 1 (2009) 22-45

behind-genius

467-474

209

142 no 3 (1990) 523-528

1427-1433

210

warning (2008)

59 no 10 (1993) 3280-3286

1008

Reviews 62 no 3 (2010) 284-304

1 (2003) 30-39

211

167ndash73

212

(2009) 397-415

no 41 (2013) 9859-9866

11 (2010) 3016-3022

23 (2005) 9263-9269

213

no 2 (2000) 117-133

214

Journal Papers

Impact factor 7877

215

3105

2011 p 56

2011 p 62

82320M (11 pp)

216

13-18 May 2012 Paper Th-73

Paper Th-74

8548 Paper 85480E (12 pp)

pp)

217

2013

July 21-27 2013

218

2014 p 53

Patent Applications

filed on 6 Feb 2015

219

APPENDIX I

1 CAS No 67-64-1

Density 0791 gmL

Vapor density 2

Hazard codes F Xi T

2 CAS No 9005-32-7

Synonyms Alginate

Hazard codes Xi

3 CAS No 7664-41-7

Synonyms Ammonia

Flash point 1111

Density 1023 gmL

Vapor density 06

220

4 CAS No 67-66-3

Melting point -63

Boiling point 61

Flash point 1492

Density 075 gmL

Vapor density 41

Hazard codes Xi

5 CAS No 13754-17-1

Synonyms Citrate

hydroxy- ion(3-) (13754-17-1)

Hazard codes None

6 CAS No 64-17-5

Melting point -114

Boiling point 78

Flash point 12

221

Density 079

Hazard codes Xi

CAS No 112-40-3

Synonyms Dodecane

Flash point 83 degC

Density 075 gmL

Vapor density 596

Hazard codes Xn

CAS No 629-97-0

Synonyms Docosane

Flash point 95 degC

Density 0778 gmL

Vapor density 108

Hazard codes Xi

CAS No 112-95-8

222

Density 07886 gmL

Vapor density 98

Hazard codes Xi

7 CAS No 106627-54-7

salt

8 CAS No 1333-74-0

Synonyms Hydrogen

Density 00899

Vapor density 007

Hazard codes F+

9 CAS No 73513-42-5

223

Density 0672 gmL

Vapor density 3

Hazard codes F Xn N

10 CAS No 1317-61-9

oxide

Density 48-51 gmL

Hazard codes Xi

11 CAS No 7439-89-6

Chemical name Iron

Synonyms Iron

Density 105 gmL

Hazard codes F Xi

12 CAS No None

Synonyms PBS

Hazard codes Xi

224

13 CAS No 25322-68-3

Density 127 gmL

Vapor density gt1

Hazard codes Xi T

14 CAS No 23335-74-2

Hazard codes none

15 CAS No 10025-77-1

chloride

Density 182 gmL

Hazard codes Xn C

16 CAS No 110-86-1

225

Synonyms Azabenzine

Flash point 20 degC

Vapor density 272

17 CAS No 1332-37-2

iron(III) oxide

Hazard codes Xi

18 CAS No 85721-33-1

Synonyms Cipro CPFX

Hazard codes Xi

19 CAS No 77-86-1

Synonyms TRIS

226

Density 1353 gmL

Hazard codes Xi

20 CAS No 143-19-1

Hazard codes None

21 CAS No 108-30-5

Density 1572 gmL

Vapor density 358

Hazard codes Xi Xn

22 CAS No 32986-56-4

Melting point 178

Hazard codes Xi

227

23 CAS No 67-56-1

Density 0791 gmL

Vapor density 111

Hazard codes Xn T F

24 CAS No 7647-01-0

Synonyms HCl

Density 12 gmL

Vapor density 13

25 CAS No 1310-73-2

Synonyms NaOH

228

Density 1515 gMl

Vapor density lt1

2)

Hazard codes C Xi

2014

harmonizedhtml

229

APPENDIX II

SULFO-NHS

Source

35 mM Tris-OH

230

tube

for 2 min

231

APPENDIX III

119885 =120596119871lowast

1

120596119862

120596119871+1

120596119862

=120596119871

1205962119871119862+1=

1

120596119862+1

120596119871

=1

0= infin

119889

119889120596[120596119862 +

1

120596119871= 119862 minus

1

1205962119871= 0 and

2016

232

119881119871 = minus119881119862 and 119868119871119883119871 = minus119868119862119883119862

When

119883119871 = 119883119862

120596119871 =1

120596119862

parallel or series

119897119868

by L Armijo2016

233

Φ=BA

of the coil

Φ= 1205830119873119868119860

119897= 120583119899119868119860

119871 =119873120567

119868

119871 = 1205830

1198732119860

119897

Rearranging we get

1205830119873

119897=

119871

119873119860

and 119861 =119871119868

119873119860

119861 = (119871

119873119860) lowast (

119881

120596119871) =

119881

119873119860120596

234

119913 =119933

119925119912120654

Frequency

(kHz)

Capacitor

Part No

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 42774

168 A88 88 17 1200 17 65328

176 A198 198 9 800 23 36232

262 A88 88 9 1200 23 53937

335 B22 22 17 2500 17 130267

474 B11 11 17 2500 11 184318

523 B22 22 9 2500 20 107668

633 B62 62 17 2500 9 246146

739 B11 11 9 2500 16 152135

987 B62 62 9 2500 12 203189

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 5228

168 A88 88 17 1200 17 79845

176 A198 198 9 800 23 42284

262 A88 88 9 1200 23 65923

335 B22 22 17 2500 17 159215

474 B11 11 17 2500 11 225278

523 B22 22 9 2500 20 131594

633 B62 62 17 2500 9 300846

739 B11 11 9 2500 16 185942

987 B62 62 9 2500 12 248342

235

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 57032

168 A88 88 17 1200 17 87103

176 A198 198 9 800 23 48309

262 A88 88 9 1200 23 71915

335 B22 22 17 2500 17 173688

474 B11 11 1as7 2500 11 245755

523 B22 22 9 2500 20 143555

633 B62 62 17 2500 9 328192

739 B11 11 9 2500 16 202844

987 B62 62 9 2500 12 270916

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 76042

168 A88 88 17 1200 17 116137

176 A198 198 9 800 23 64412

262 A88 88 9 1200 23 95887

335 B22 22 17 2500 17 231583

474 B11 11 17 2500 11 327673

523 B22 22 9 2500 20 191407

633 B62 62 17 2500 9 437589

739 B11 11 9 2500 16 270459

987 B62 62 9 2500 12 361221

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 80796

168 A88 88 17 1200 17 122339

176 A198 198 9 800 23 68439

262 A88 88 9 1200 23 101881

335 B22 22 17 2500 17 24606

474 B11 11 17 2500 11 348157

523 B22 22 9 2500 20 203372

633 B62 62 17 2500 9 464943

236

739 B11 11 9 2500 16 287365

987 B62 62 9 2500 12 383802

Frequency

(kHz)

Capacitor

Array

TypeVal

ue

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 95054

168 A88 88 17 1200 17 145173

176 A198 198 9 800 23 80516

262 A88 88 9 1200 23 119859

335 B22 22 17 2500 17 289482

474 B11 11 17 2500 11 409595

523 B22 22 9 2500 20 239261

633 B62 62 17 2500 9 546992

739 B11 11 9 2500 16 338077

987 B62 62 9 2500 12 451531

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 109312

168 A88 88 17 1200 17 166949

176 A198 198 9 800 23 92594

262 A88 88 9 1200 23 137838

335 B22 22 17 2500 17 332905

474 B11 11 17 2500 11 471035

523 B22 22 9 2500 20 275151

633 B62 62 17 2500 9 629041

739 B11 11 9 2500 16 388788

987 B62 62 9 2500 12 519261

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 118817

168 A88 88 17 1200 17 181467

176 A198 198 9 800 23 100645

262 A88 88 9 1200 23 149824

335 B22 22 17 2500 17 361853

474 B11 11 17 2500 11 511995

237

523 B22 22 9 2500 20 299077

633 B62 62 17 2500 9 68374

739 B11 11 9 2500 16 422596

987 B62 62 9 2500 16 564414

- Spring 4-15-2019

Page 5: Iron-containing Nanoparticles for the Treatment of ...

iv

ACKNOWLEDGEMENTS

v

vi

vii

IN CYSTIC FIBROSIS

by

Leisha Marie Armijo

BS Biology

ABSTRACT

viii

extensive treatment

ix

Table of Contents

x

xi

xii

xiii

xiv

xv

List of Figures

xvi

xvii

xviii

xix

List of Tables

xx

AI auto-inducer

CF cystic fibrosis

DI deionized

hydrochloride

I intermediate

LB Luria-Bertani

xxi

NP nanoparticle

OD optical density

QS quorum sensing

R resistant

r radius

S sensitive

xxii

1

Chapter 1

death

2

3

Table 11

Austria

13500 [Southern 2007]

Belgium

12850 [Lucotte 1995] [Chung 2002]

Bulgaria

12500 [Chung 2002]

Czech Republic

12833 [Romeo 1989] [Lucoette 1995]

Faroe Islands

11775 [Kaplan 1968]

Finland

125000 [Denning 1968]

125000 [Kere 1994] [Klaassen 1998]

France

14700 [Southern 2007]

Germany

4

Italy

14238 [Siegel 1960]

14238 [Bossi 2004]

Italy (Milan)

13170 [Chernick 1959]

Netherlands

14750 [Spock 1967]

14750 [Slieker 2005]

11857 [Noblett 1969]

Norway

16574 [Johnson 1984]

Poland

15000 [Southern 2007]

Republic of Ireland

11353 [Farrell 2007]

Romania

12056 [Popa 1997]

Scotland

11984 [Hide 1969]

Slovakia

11800 [Kadasi 1997]

Spain

13750 [Lucotte 1995] [Chung 2002]

Sweden

12200-4500 [Rosan 1962]

United Kingdom

12415 [Gracey 1969]

12381 [Dodge 2007]

North America

United States

[Bowman 1969]

[Palomaki 2004]

Canada

12500 [Mearns 1974] 13608 [Dupuis 2005]

(Quebec)

1895 [Weaver 1994]

Amish OH USA

1569 [Stutman 2002]

5

Zuni Tribe NM USA

Middle East

14000-18000 [Crozier 1974]

Bahrain

15800 [Corey 1988]

Jordan

12560 [Nielsen 1982]

Oceana

New Zealand

Australia

[Jensen 1987]

Other

Japan

South Africa

12000 (Caucasian) [Allan 1973] 1784-13924

6

Foundation 2015]

in the Population

7

Armijo 2016

8

future

9

10

11

cell results

12

13

of the lungs

Treatments

medicine was built

14

15

16

CF

17

Table 12

Class

Effect on CFTR

Types of Mutation

Therapy Potential

Therapy

(nonsense or frame

shift)

Aminoglycosides

Gene transfer read-

through compounds

Amino acid deletion

mutation)

Correctors

Butyrates

Gene transfer

impaired gating

Missense mutation

(ie G551D)

Potentiators

Genistein

Gene transfer

Missense mutation

(ie R117H or

R347P)

Milrinone

Potentiators

Gene transfer

(ie A445E or 5T)

Aminoglycosides

Antisense

oligonucleotides

Correctors Gene

transfer

transcription

Aminoglycosides

Stabilizers

Gene transfer

Bypass therapies

18

19

20

absence of mRNA

21

Fibrosis Patients

22

disease and illness

23

water

24

25

26

27

28

biofilm

29

30

31

32

33

34

35

36

37

38

Chapter 2

OXIDE NANOPARTICLES

39

40

211 Materials

41

42

43

44

45

46

217 Cost Reduction

47

48

49

50

bar is 5 nm

51

52

53

12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

0

1000

2000

3000

4000

5000

Inte

nsity (

cps)

2-Theta (degree)

[111]

[110]

[311]

[220][422]

[011]

54

55

Chapter 3

NANOPARTICLES AND

56

2013]

57

31 Theory

mp = Msυ (31)

58

2001]

τN = τ0exp(σ) (33)

59

32 Experimental

AB Sweden)

60

0 50 100 150 200 250 300 350

00004

00006

00008

00010

00012

00014

00016

Field-Cooled

Zero Field-Cooled

Mag

ne

tizatio

n (

em

u)

Temperature (K)

2012a]

61

observed

62

63

thermometer

cooling water

64

65

Wire (black)

0 5 10 15 20 25 30 35 40 4515

18

21

24

27

30

33

36

39

42

45

48

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

0 5 10 15 20 25 30 35

24

26

28

30

32

34

36

38

40

42

Tem

pera

ture

(degC

)

Time (minutes)

Wires

Polymorphous

Spheres

1111 kHz

6292 kHz

Spherical (blue)

Poloymorphus (red)

Figure 37

22 nm spheres

(b) 6292 kHz 9 mT

66

4 degC

67

experiments

68

pronounced

69

0 10 20 30 40 5020

30

40

50

Tem

per

ature

[d

egre

es C

elsi

us]

Time [min]

Fe3O

4 NPs in water

Fe3O

70

71

72

73

Chapter 4

in Chapter 6

74

75

Oe [Hattori 2001]

42 Theory

76

many researchers

2003]

gas

77

431 Materials

in Appendix I

78

solid

79

80

gas for 30 minutes

81

82

chloroform and heat

83

84

magnetometer

hysteresis loops of

iron oxide

-50E4 00 50E4-1

0

1

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

-50E4 00 50E4-80

-60

-40

-20

0

20

40

60

80

(A

m2k

g)

H (mT)

Fe3O

4

Fe16

N2

85

00 20E4 40E40

20

40

60

80

100

(

Am

2k

g)

H (mT)

temperature

86

87

Chapter 5

88

[Prencipe 2009]

51 Experimental

tobramycin molecule

89

511 Materials

90

119901119867 = 119901119870119886 + 11989711990011989210([119860 minus]

[119867119860] (1)

119901119870119886 = minus11989711990011989210([1198673119874+][119860minus]

[119867119860] (2)

91

119870119886 = [119900119897119890119886119905119890 119894119900119899][1198673119874+]

[119900119897119890119894119888 119886119888119894119889] = 1 times 10-5 pKa = 5

92

MarvinSketch

93

513 Citrate Capping

94

95

original synthesis

group

96

(PEG) 5000 powder

97

200 300 400 500 600 700

00

02

04

06

08

10

12

14

Abs

orba

nce

[OD

cm

]

Wavelength [nm]

glycol (PEG) 5000

98

99

Appendix II

MarvinSketch

100

101

102

103

to be ~2

104

105

Chapter 6

SENSITIVITY TESTING

106

positive control

107

108

Figure 61 Pyocyanin

Image showing the

produced by P

aeruginosa cultures

grown on agar for

Image by L Armijo

2014

109

61

611 Materials

110

111

112

strains

113

114

micropipette

115

recorded

growth period

116

117

118

119

62 Results

120

121

time

122

123

a 1 mg biomass

Table 61

Disk diffusion

[mm]

ge 15 13-14 le 12

124

Table 62

Disk

with PEG-OH 17 mm S

with PEG-OH 0 R

tobramycin 17 mm S

125

capped NPs

126

day 3

ZOI (mm)

day 60

5 μg 175S 16S

25 μg 11R 10R

5 μg 0R 0R

25 μg 0R 0R

5 μg 16S 15S

25 μg 10R 8R

5 μg 11R 15I

25 μg 7R 5R

5 μg 21S 22S

25 μg 20S 20S

100 μg 25R 15R

1000 μg 35R 32R

127

inactivate microbes

128

129

130

Table 64

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

4 Iron Oxide NP-

Tobramycin

25S 19S 14I

Table 65

Barrier

Alginate

Barrier

Mucin +

Alginate

Barriers

9 Iron Oxide NP-

Tobramycin

0R 0R 0R

131

Table 66

Alginate and

Mucin

(No magnet)

Alginate

(magnet)

Mucin

(magnet)

Alginate and

Mucin

(magnet)

DI Water R R R R

Tobramycin R S R S

NP-alginate-

drug

R I S S

NP-citrate-

drug

R I I I

132

motility test for P

133

134

toxic dose ranges

135

136

137

138

Chapter 7

139

140

141

142

143

nm

144

716 Viability Assay

145

717 Apoptosis Assay

146

147

significant

148

Perc

en

t In

ten

sit

y R

ed

ucti

on

co

ntr

ol

1x

10

-3

1x

10

-2

1x

10

-1 1

10

0

20

40

60

reduction 2 M dye

149

measured value

72 Results

150

151

152

153

154

Viability

Time Point

Flu

ore

scen

ce (

RF

U)

6 hou

rs

12 h

ours

24 h

ours

0

20000

40000

60000

Digitonin

Staurosporine

Ionomycin

Untreated Cells

viability

155

156

157

Time Point

Lu

min

escen

ce (

RL

U)

6 hou

r

12 h

our

24 h

our

0

2100 5

4100 5

6100 5

Staurosporine

NPs (1 mgmL)

NPs (05 mgmL)

Untreated Cells

Ionomycin

observed

158

73 Discussion

159

160

161

162

163

CHapter 8

164

165

166

167

168

169

83 Conclusions

170

84 Future Work

171

bactericidal effect

172

173

REFERENCES

2762-2768

Wiley amp Sons 2007

(2003) 451-462

174

(2014) 688ndash99

pp)

(12 pp) doi101117122043340

175

pp)

208-213

Chest 59 no 1 (1965) 9-8

286ndash92

176

196ndash200

638-644

25 no 23 (2004) 5405-5413

3-14

no 3 (2004) 455-467

no 1 (2009)1ndash12

177

(2004) 55ndash60

Engineering 53 no 10 (2006) 2075-2083

Medica 36 no 1-4 (1989) 13-15

178

(2007) 366-370

(2006) 1675-1682

1453-1464

7551-7553

179

Research 45 no 5 (2011) 1995-2001

(1959) 739-745

(2005) 956ndash60

(2013) 4476-4489

(2007) 411-437

180

Thorax 67 no 1 (2012) 12-18

(2009) 2563ndash8

Bacteriology 185 no 3 (2003) 1027-1036

181

Research 67 no 1 (2006) 55-60

(2007) 522-526

no 1 (2012) 9 (9 pp)

Pediatrics 147 no 3 (2005) 312-315

8

182

(2014) 1ndash5

187-191

183

85 no 7 (2011) 743ndash50

277 no 2 (1998) 347-362

(2015) 8856ndash74

1986

184

13593-13599

1048-1059

60 no 3 (1996) 539-574

Childhood 44 no 235 (1969) 404-405

802-808

185

4021

186

(1908) 173ndash9

19095-19098

Press 2013

Press 2003

187

pp)

no 22 (2009) 3645-3651

(2007) 51-84

3 (2010) 1035-1042

200-215

188

(2016) e0154445 (13 pp)

838

11 (1997) 51-56

217

189

Medicine 279 no 2 (1968) 65-69

no 5 (1968) 810-818

Research 6 no 21 (2012) 4620-4622

190

Chemistry 27 no 9 (2008) 1825-1851

Screening 5 no 1 (1998) 16-19

109 no 6 (2004) 799-809

3 Pt 2 (1967) 388-392

1750-1754

191

Science 29 no 5 (1998) 511-535

(2011) 789-808

619

9 (2002) 681-685

(12 pp)

no 6 (2008) 2064-2110

(2012) 045007 (10 pp)

192

1529-1540

(2011) 136-146

Pharmacology 411 no 3 (2001) 255-260

193

Valley 1994

1442

(2014) 227 (9 pp)

(2012) 181-205

875

194

(2005) 106102

(1997) 1122-1127

oncology 103 no 2 (2011) 317-324

Sciences 32 no 3-4 (2007) 203-215

195

(2008) 1343ndash8

A 100 no 6 (2012) 1637-1646

(1974) 497-506

196

Materials 21 no 23 (2011) 4573-4581

no 2 (2017) 105

Health 213 no 3 (2010) 190-197

(2004) 2161-2175

16 no 10 (2005) 2346ndash53

197

175

Biology 12 no 7 (2005) 789-796

Science 75 no 1 (2018) 13-18

198

(1988) 836-841

190-197

1712-1720

199

no 33 (2006) 16248-16253

Materials 3 no 12 (2004) 891-895

167 (14 pp)

28 no 16 (2007) 2572-2581

200

Pediatrics 156 no 3 (1997) 212-213

131 no 13 (2009) 4783-4787

201

(1974)454ndash63

(2008)1079ndash81

4922 (1989) 1066-1073

202

no 3 (1976) 287ndash95

(2012) 22ndash6

(2000) 1905-1911

2906

pp)

203

Pharmacology 153 no 6 (2008) 1311-1323

Surgery 3 no 5 (1986) 732-740

204

(2017) 291ndash7

no 42 (2007) 5712-5720

506

205

(2017) 15464

(1974) 153-169

(1992) 222-228

Chemistry C 111 no 49 (2007) 18078-18086

689-699

440

206

(2014) 40 (9 pp)

Immunology 74 no 3 (2006) 1673-1682

no 4 (2002) 1132-1139

1275

207

267-281

(2007) 57-65

188ndash94

173-177

731

Pediatrics 140 no 3 (2002) 299-305

208

Letters 61 no 26 (2007) 4769-4772

Materials 55 no 1 (2009) 22-45

behind-genius

467-474

209

142 no 3 (1990) 523-528

1427-1433

210

warning (2008)

59 no 10 (1993) 3280-3286

1008

Reviews 62 no 3 (2010) 284-304

1 (2003) 30-39

211

167ndash73

212

(2009) 397-415

no 41 (2013) 9859-9866

11 (2010) 3016-3022

23 (2005) 9263-9269

213

no 2 (2000) 117-133

214

Journal Papers

Impact factor 7877

215

3105

2011 p 56

2011 p 62

82320M (11 pp)

216

13-18 May 2012 Paper Th-73

Paper Th-74

8548 Paper 85480E (12 pp)

pp)

217

2013

July 21-27 2013

218

2014 p 53

Patent Applications

filed on 6 Feb 2015

219

APPENDIX I

1 CAS No 67-64-1

Density 0791 gmL

Vapor density 2

Hazard codes F Xi T

2 CAS No 9005-32-7

Synonyms Alginate

Hazard codes Xi

3 CAS No 7664-41-7

Synonyms Ammonia

Flash point 1111

Density 1023 gmL

Vapor density 06

220

4 CAS No 67-66-3

Melting point -63

Boiling point 61

Flash point 1492

Density 075 gmL

Vapor density 41

Hazard codes Xi

5 CAS No 13754-17-1

Synonyms Citrate

hydroxy- ion(3-) (13754-17-1)

Hazard codes None

6 CAS No 64-17-5

Melting point -114

Boiling point 78

Flash point 12

221

Density 079

Hazard codes Xi

CAS No 112-40-3

Synonyms Dodecane

Flash point 83 degC

Density 075 gmL

Vapor density 596

Hazard codes Xn

CAS No 629-97-0

Synonyms Docosane

Flash point 95 degC

Density 0778 gmL

Vapor density 108

Hazard codes Xi

CAS No 112-95-8

222

Density 07886 gmL

Vapor density 98

Hazard codes Xi

7 CAS No 106627-54-7

salt

8 CAS No 1333-74-0

Synonyms Hydrogen

Density 00899

Vapor density 007

Hazard codes F+

9 CAS No 73513-42-5

223

Density 0672 gmL

Vapor density 3

Hazard codes F Xn N

10 CAS No 1317-61-9

oxide

Density 48-51 gmL

Hazard codes Xi

11 CAS No 7439-89-6

Chemical name Iron

Synonyms Iron

Density 105 gmL

Hazard codes F Xi

12 CAS No None

Synonyms PBS

Hazard codes Xi

224

13 CAS No 25322-68-3

Density 127 gmL

Vapor density gt1

Hazard codes Xi T

14 CAS No 23335-74-2

Hazard codes none

15 CAS No 10025-77-1

chloride

Density 182 gmL

Hazard codes Xn C

16 CAS No 110-86-1

225

Synonyms Azabenzine

Flash point 20 degC

Vapor density 272

17 CAS No 1332-37-2

iron(III) oxide

Hazard codes Xi

18 CAS No 85721-33-1

Synonyms Cipro CPFX

Hazard codes Xi

19 CAS No 77-86-1

Synonyms TRIS

226

Density 1353 gmL

Hazard codes Xi

20 CAS No 143-19-1

Hazard codes None

21 CAS No 108-30-5

Density 1572 gmL

Vapor density 358

Hazard codes Xi Xn

22 CAS No 32986-56-4

Melting point 178

Hazard codes Xi

227

23 CAS No 67-56-1

Density 0791 gmL

Vapor density 111

Hazard codes Xn T F

24 CAS No 7647-01-0

Synonyms HCl

Density 12 gmL

Vapor density 13

25 CAS No 1310-73-2

Synonyms NaOH

228

Density 1515 gMl

Vapor density lt1

2)

Hazard codes C Xi

2014

harmonizedhtml

229

APPENDIX II

SULFO-NHS

Source

35 mM Tris-OH

230

tube

for 2 min

231

APPENDIX III

119885 =120596119871lowast

1

120596119862

120596119871+1

120596119862

=120596119871

1205962119871119862+1=

1

120596119862+1

120596119871

=1

0= infin

119889

119889120596[120596119862 +

1

120596119871= 119862 minus

1

1205962119871= 0 and

2016

232

119881119871 = minus119881119862 and 119868119871119883119871 = minus119868119862119883119862

When

119883119871 = 119883119862

120596119871 =1

120596119862

parallel or series

119897119868

by L Armijo2016

233

Φ=BA

of the coil

Φ= 1205830119873119868119860

119897= 120583119899119868119860

119871 =119873120567

119868

119871 = 1205830

1198732119860

119897

Rearranging we get

1205830119873

119897=

119871

119873119860

and 119861 =119871119868

119873119860

119861 = (119871

119873119860) lowast (

119881

120596119871) =

119881

119873119860120596

234

119913 =119933

119925119912120654

Frequency

(kHz)

Capacitor

Part No

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 42774

168 A88 88 17 1200 17 65328

176 A198 198 9 800 23 36232

262 A88 88 9 1200 23 53937

335 B22 22 17 2500 17 130267

474 B11 11 17 2500 11 184318

523 B22 22 9 2500 20 107668

633 B62 62 17 2500 9 246146

739 B11 11 9 2500 16 152135

987 B62 62 9 2500 12 203189

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 5228

168 A88 88 17 1200 17 79845

176 A198 198 9 800 23 42284

262 A88 88 9 1200 23 65923

335 B22 22 17 2500 17 159215

474 B11 11 17 2500 11 225278

523 B22 22 9 2500 20 131594

633 B62 62 17 2500 9 300846

739 B11 11 9 2500 16 185942

987 B62 62 9 2500 12 248342

235

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 57032

168 A88 88 17 1200 17 87103

176 A198 198 9 800 23 48309

262 A88 88 9 1200 23 71915

335 B22 22 17 2500 17 173688

474 B11 11 1as7 2500 11 245755

523 B22 22 9 2500 20 143555

633 B62 62 17 2500 9 328192

739 B11 11 9 2500 16 202844

987 B62 62 9 2500 12 270916

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 76042

168 A88 88 17 1200 17 116137

176 A198 198 9 800 23 64412

262 A88 88 9 1200 23 95887

335 B22 22 17 2500 17 231583

474 B11 11 17 2500 11 327673

523 B22 22 9 2500 20 191407

633 B62 62 17 2500 9 437589

739 B11 11 9 2500 16 270459

987 B62 62 9 2500 12 361221

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 80796

168 A88 88 17 1200 17 122339

176 A198 198 9 800 23 68439

262 A88 88 9 1200 23 101881

335 B22 22 17 2500 17 24606

474 B11 11 17 2500 11 348157

523 B22 22 9 2500 20 203372

633 B62 62 17 2500 9 464943

236

739 B11 11 9 2500 16 287365

987 B62 62 9 2500 12 383802

Frequency

(kHz)

Capacitor

Array

TypeVal

ue

Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 95054

168 A88 88 17 1200 17 145173

176 A198 198 9 800 23 80516

262 A88 88 9 1200 23 119859

335 B22 22 17 2500 17 289482

474 B11 11 17 2500 11 409595

523 B22 22 9 2500 20 239261

633 B62 62 17 2500 9 546992

739 B11 11 9 2500 16 338077

987 B62 62 9 2500 12 451531

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 109312

168 A88 88 17 1200 17 166949

176 A198 198 9 800 23 92594

262 A88 88 9 1200 23 137838

335 B22 22 17 2500 17 332905

474 B11 11 17 2500 11 471035

523 B22 22 9 2500 20 275151

633 B62 62 17 2500 9 629041

739 B11 11 9 2500 16 388788

987 B62 62 9 2500 12 519261

Frequency

(kHz)

Capacitor Capacitor

Value C

(nF)

Inductor

Coil

Turns (N)

Maximum

(Vp-p)

Nominal

Coil Field

at Max

Vp-p (mT)

Applied

Vp-p

110 A198 198 17 1200 25 118817

168 A88 88 17 1200 17 181467

176 A198 198 9 800 23 100645

262 A88 88 9 1200 23 149824

335 B22 22 17 2500 17 361853

474 B11 11 17 2500 11 511995

237

523 B22 22 9 2500 20 299077

633 B62 62 17 2500 9 68374

739 B11 11 9 2500 16 422596

987 B62 62 9 2500 16 564414

- Spring 4-15-2019

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