Applications of multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles in enzyme stabilization and drug delivery Tristan DeVere Clemons, BSc (Hons)
This thesis is presented for the degree of Doctor of Philosophy of the University of Western Australia
School of Chemistry and Biochemistry
School of Animal Biology
2013
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Abstract Nanotechnology, although a science in its infancy, has the potential to revolutionize the
medical profession by improving on traditional drug delivery methods and transforming
how disease and injury are currently diagnosed, monitored and treated. The effective
delivery of small molecule drugs, peptides and proteins to a diseased or injury site has
faced considerable barriers in the past including premature clearance from the body, off
site toxicity and poor bioavailability or pharmacokinetics. Nanoparticles can be used to
help improve these characteristics by aiding delivery of therapeutics which otherwise
show little efficacy without assisted delivery. In this work, poly(glycidyl methacrylate)
(PGMA) nanoparticles have been synthesized as delivery vehicles incorporating a range
of surface functionalities and imaging probes to allow successful tracking of these
nanoparticles throughout testing. These delivery vehicles have been used in a range of
applications suggesting the broad applicability and suitability of functionalized PGMA
nanoparticles in medicine. The nanoparticles were shown to aid in the delivery of a
therapeutic peptide to modulate activity of the L-type calcium channel of cardiac tissue
as well as thermally stabilize industrially relevant enzymes through nanosurface
interactions. Finally, the potential of the nanoparticle’s as DNA delivery vectors for
gene silencing in cancer models was investigated. Further to these three delivery
applications of the functionalized PGMA nanoparticles, work will be presented herein
on the development of a novel spectrophotometric assay suitable for the detection of the
activity of the therapeutic enzyme chondroitinase ABC (chABC). This newly presented
assay was superior when compared to the traditional methods used for detecting the
activity of chABC. This assay was used to investigate a range of formulations including
functionalized PGMA nanoparticles, in an attempt to stabilize the therapeutically
relevant chABC at 37 °C, to prolong its activity and in turn improve its effectiveness as
a therapeutic in the treatment of central nervous system injuries.
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Table of Contents
Abstract ................................................................................................................................... iii Table of Contents ................................................................................................................. iv Acknowledgements ............................................................................................................. ix Abbreviations ....................................................................................................................... xi Published Works ................................................................................................................. xv Conference presentations .............................................................................................. xvii Statement of candidate contribution .......................................................................... xix Thesis Prelude .................................................................................................................... xxi Chapter 1 ................................................................................................................................. 1 Introduction and literature review ................................................................................ 1
1.1 Nanoparticles in modern medicine ................................................................... 1 1.1.1 Nanoparticles for drug delivery ............................................................................................ 2 1.1.2 Micelles, liposomes and dendrimers for drug delivery applications .................... 5
1.2 Polymeric nanoparticles and nanocapsules as drug delivery vehicles .......... 8 1.2.1 Methods for the preparation of polymeric nanoparticles .......................................... 9
1.3 Nanoparticle and cell interactions .................................................................. 13 1.3.1 Nanoparticle endocytosis ....................................................................................................... 16 1.3.2 Strategies to enhance cellular internalization ............................................................... 18
1.4 Multifunctional nanoparticles ........................................................................ 19 1.4.1 Targeted nanoparticles and ‘stealth’ coatings ............................................................... 21 1.4.2 Imaging agents and multifunctional nanoparticles .................................................... 24 1.4.3 Magnetic resonance imaging ................................................................................................ 29 1.4.4 Magnetic resonance contrast agents ................................................................................. 31 1.4.5 Fluorescent probes for biological imaging ..................................................................... 33 1.4.6 Theranostic nanoparticles and the combination of imaging and treatment together ......................................................................................................................................................... 34
1.5 Assessing nanoparticle toxicity ....................................................................... 35 1.6 Summary of the literature and thesis rationale ............................................. 37 1.7 Introduction to series of chapters ................................................................... 39
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Chapter 2 ............................................................................................................................... 43 Poly(Glycidyl Methacrylate) (PGMA) nanoparticle synthesis and characterization ................................................................................................................. 43
2.1 An introduction to polymeric nanoparticles for drug delivery .................... 43 2.2 Multimodal PGMA nanoparticles with a PEI functionalized surface ........ 45 2.3 Multimodal PGMA nanoparticles with a PEGylated surface ...................... 51 2.4 In vitro toxicity and cellular internalization studies ...................................... 54 2.5 Conclusion ......................................................................................................... 55 2.6 Detailed methods of nanoparticle synthesis and characterization .............. 55 2.6.1 Nanoparticle synthesis ............................................................................................................ 55 2.6.2 Nanoparticle characterization ............................................................................................. 57 2.6.3 In vitro testing of nanoparticles ........................................................................................... 57
Chapter 3 ............................................................................................................................... 59 Multifunctional polymeric nanoparticles for the delivery of the therapeutic AID peptide in cardiac ischemia-‐reperfusion injuries .......................................... 59
3.1 Current treatment of cardiac ischemia-reperfusion injuries ....................... 59 3.2 Loading of the therapeutic AID peptide to the nanoparticles ...................... 62 3.3 A comparison of cellular uptake and biodistribution ................................... 64 3.4 Nanoparticle and TAT-mediated delivery of the AID peptide reduces
damage following ischemia-reperfusion injury .............................................. 68 3.5 Conclusions and future work .......................................................................... 72 3.6 Detailed methods .............................................................................................. 73 3.6.1 Nanoparticle synthesis and characterization ................................................................ 73 3.6.2 Synthesis of the AID peptide ................................................................................................. 73 3.6.3 AID peptide attachment to PGMA nanoparticles ......................................................... 73 3.6.4 Isolation of guinea-‐pig ventricular myocytes ................................................................ 73 3.6.5 Uptake studies with cardiac myocytes ............................................................................. 74 3.6.6 in vitro fluorescence assays ................................................................................................... 74 3.6.7 Ischemia-‐reperfusion model ................................................................................................. 75 3.6.8 CK and LDH assays .................................................................................................................... 75 3.6.9 Cardiac biodistribution studies ........................................................................................... 76 3.6.10 Statistical analysis ................................................................................................................... 77
Chapter 4 ............................................................................................................................... 79 Polymeric nanoparticles as enzyme stabilization agents .................................... 79
4.1 The importance of enzyme stabilization ........................................................ 79
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4.2 Nanomaterials are ideal enzyme stabilization agents ................................... 81 4.3 Multifunctional polymeric nanoparticles impart thermal stability to
enzymes .............................................................................................................. 84 4.4 Conclusions and potential future directions .................................................. 91 4.5 Materials and detailed methods ...................................................................... 92 4.5.1 Nanoparticle synthesis and characterization ................................................................ 92 4.5.2 Thermal stabilization studies ............................................................................................... 92 4.5.3 Persistence of enzyme activity studies ............................................................................. 93 4.5.4 Enzyme activity determination ........................................................................................... 93 4.5.5 SDS-‐PAGE analysis .................................................................................................................... 93 4.5.6 Assessment of enzyme kinetics ........................................................................................... 94
Chapter 5 ............................................................................................................................... 95 The development of a spectrophotometric assay suitable for quantitative and kinetic analysis of chondroitinase ABC (chABC) activity ...................................... 95
5.1 chABC as a potential therapeutic intervention for central nervous system
injuries ................................................................................................................ 95 5.2 Current assay methods for chABC activity can be improved ..................... 97 5.3 Comparison of the novel WST-1 assay to traditional assays for chABC
activity ................................................................................................................ 98 5.4 Conclusion and potential applications of the WST-1 assay ....................... 105 5.5 Materials and detailed methods .................................................................... 106 5.5.1 Materials ..................................................................................................................................... 106 5.5.2 WST-‐1 chABC assay ............................................................................................................... 106 5.5.3 DMMB chABC assay ............................................................................................................... 106 5.5.4 Absorbance at 232 nm chABC assay ............................................................................... 106 5.5.5 Substrate inhibition experiments .................................................................................... 107 5.5.6 Kinetic analysis with the WST-‐1 assay .......................................................................... 107 5.5.7 Activity of chABC versus trehalose stabilised chABC .............................................. 107
Chapter 6 ............................................................................................................................ 109 Attempts at stabilizing the therapeutically relevant enzyme chondroitinase ABC (chABC) ...................................................................................................................... 109
6.1 chABC for the treatment of central nervous system injury ....................... 109 6.2 PGMA nanoparticles do not impart thermal stability to chABC .............. 110 6.3 Investigation of PEG as a thermal stabilizing agent and cryoprotectant . 112 6.4 Assessment of chABC activity at 37 °C ........................................................ 115
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6.5 Recent advancements for the removal of chondroitin sulfate proteoglycans
and the use of chABC as a therapeutic intervention .................................... 117 6.6 Materials and detailed methods .................................................................... 120 6.6.1 Materials ..................................................................................................................................... 120 6.6.2 WST-‐1 chABC assay ............................................................................................................... 120 6.6.3 Time course experiment comparing PGMA nanoparticles and trehalose ..... 121 6.6.4 Evaluating chABC activity in the presence of free PEI ............................................ 121 6.6.5 Evaluating chABC activity in basic conditions ........................................................... 121 6.6.6 Investigation of PEG as a thermal stabilization agent ............................................ 121 6.6.7 Investigation of PEG as a cryoprotectant ..................................................................... 122 6.6.8 Time course analysis of chABC activity ......................................................................... 122
Chapter 7 ............................................................................................................................. 125 Multifunctional polymeric nanoparticles for gene delivery and RNAi in breast and colon cancer models. .............................................................................................. 125
7.1 c-Myc an appropriate target gene ................................................................ 126 7.2 RNAi technology and the problem of delivery ............................................ 126 7.3 Design of the nanoparticle non-viral vectors ............................................... 129 7.4 Summary of key findings ............................................................................... 132
Chapter 8 ............................................................................................................................. 133 Conclusions and future work ....................................................................................... 133
8.1 PGMA multifunctional nanoparticles .......................................................... 134 8.2 PGMA nanoparticles and peptide delivery to cardiac ischemia-reperfusion
injury ................................................................................................................ 136 8.3 PGMA nanoparticles for the stabilization of chABC ................................. 138 8.4 PGMA nanoparticles for RNAi technology ................................................. 139 8.5 Final remarks ................................................................................................. 140
Appendices ......................................................................................................................... 143
Appendix A – Elemental analysis calculation of PEI attachment to nanoparticle surface by mass ........................................................................................................ 143 Appendix B – 1H-NMR spectra of carboxylic end functionalized poly(ethylene glycol) and poly(ethylene glycol) bound to poly(glycidyl methacrylate). ........... 144
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Appendix C - Standard curve used for the calculation of WST-1 molar absorption coefficient. ............................................................................................. 145 Appendix D – Chapter 7 – in vitro and in vivo analysis ....................................... 146 Appendix E – Published works not included directly in the thesis .................... 167
References ......................................................................................................................... 187
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Acknowledgements I would like to firstly and whole-heartedly thank my coordinating supervisor Dr.
Swaminathan Iyer for his continued support, guidance and for igniting a passion within
me for research science. I have also been very fortunate to have four supporting
supervisors for this PhD project all whom have added their own individual twists on this
body of work and on my own development as an academic. Dr. Keith Stubbs for your
interest in my project and continued support in the laboratory as well as on the hockey
field; Dr. Lindy Fitzgerald for your keen eye and attention to detail in all my work
throughout my PhD; W/Prof. Sarah Dunlop for being able to make me smile and see the
positives in my work despite the setbacks and W/Prof. Alan Harvey for constantly
challenging my knowledge and understanding, always pushing me to improve. A
special thankyou must be made to Dr. Cameron Evans for his mentorship, help and
guidance throughout my time as a PhD student.
I thank my collaborators and their research teams whom have all helped to contribute to
this body of work, with special mention to Prof. Alan Clarke from Cardiff University,
Wales, Prof. James Fawcett from Cambridge University, United Kingdom, Dr. Lekha
Dinesh Kumar from the Centre of Cellular and Molecular Biology, India, Assoc./Prof.
Mike House from the School of Physics and Assoc./Prof. Livia Hool from the School of
Anatomy, Physiology and Human Biology both at the University of Western Australia,
Perth whom all allowed me to work and visit within their laboratories.
I would like to thank the support from the School of Chemistry and Biochemistry staff
both academic and administrative for hosting me throughout my PhD candidature and
supporting me through this journey as well as the imaging and training support I have
received from the Centre for Microscopy, Characterization and Analysis at the
University of Western Australia. I would like to thank Dr. Nicole Smith for her final
proof reading of this thesis before submission along with the keen eye of all my
supervisors.
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I would like to thank the funding bodies that have supported me financially throughout
this journey including the Australian Research Council, National Health and Medical
Research Council, The University of Western Australia Research Collaboration Award
and the Australian Nanotechnology Network.
Finally, I would like to thank my girlfriend, family and friends whom have put up with
me, supported me and allowed me to pursue wholeheartedly and at times selfishly my
passion for science and also hockey over the past 4 years. For this I thank you all.
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Abbreviations AID Alpha interacting domain
AID-NP Alpha interacting domain peptide-nanoparticle complex
AID(S)-NP Alpha interacting domain peptide scrambled-nanoparticle complex
AID(S)-TAT Alpha interacting domain peptide scrambled-TAT peptide
AID-TAT Alpha interacting domain peptide-TAT peptide
ANOVA Analysis of variance
BSA Bovine serum albumin
chABC Chondroitinase ABC
chPF Chondroitin polymerizing factor
CK Creatine kinase
CNS Central nervous system
CPP Cell penetrating peptide
CSPG Chondroitin sulfate proteoglycan
CT Computed tomography
DHE Dihydroethidium
DMMB 1,9-dimethylmethylene blue
ECM Extracellular matrix
EPR effect Enhanced permeability and retention effect
FACE Fluorophore assisted carbohydrate electrophoresis
FDA United States food and drug administration
GAG Glycosaminoglycan
GMNPs Gadolinium labeled magnetite nanoparticles
HIV Human immunodeficiency virus
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HPLC High pressure liquid chromatography
HSV Human simplex virus
KHB Krebs-Henseleit buffer
LbL Layer-by-layer
LDH Lactate dehydrogenase
MMP Matrix metalloproteinase
MPS Mononuclear phagocyte system
MRI Magnetic resonance imaging
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
nav Neutravidin
NHMRC National Health and Medical Research Council
Nisol Nisoldipine
NMR Nuclear magnetic resonance
NP Nanoparticle
O/W Oil in water emulsion
PAMAM Poly(amidoamine)
PBS Phosphate buffered saline
PEG Poly(ethylene glycol)
PEI Poly(ethyleneimine)
PET Positron emission tomography
PGMA Poly(glycidyl methacrylate)
PLA Poly(lactic acid)
PLG Poly(D-L-glycolide)
PLGA Poly(lactic-co-glycolic acid)
PLL Poly-L-lysine
PNNs Perineuronal nets
PpIX Protoporphyrin IX
QD Quantum dot
rf Radio frequency
RhB Rhodamine B
RNAi RNA interference
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SCF Super critical fluid
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SE Standard error
siRNA Short interfering RNA
SPECT Single photon emission computed tomography
SQUID Superconducting quantum interference device
SWCNT Single walled carbon nanotubes
TAMRA Carboxytetramethyl rhodamine
TAT Trans activator of transcription
US Ultrasound
VIP Vasoactive intestinal peptide
W/O Water in oil emulsion
W/O/W Water in oil in water emulsion
WST-1 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate
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Published Works
1. Clemons, T.; Fitzgerald, M.; Dunlop, S.; Harvey, A.; Swaminathan, I.; Stubbs,
K., An Improved Assay for the Spectrophotometric Determination of
Chondroitinase ABC Activity. New J Chem, 2013, Published online
DOI:10.1039/c3nj00168g. (Impact Factor 2.61, 0 citations, Journal Ranking 44th
out of 154).
2. Clemons, T.; Viola, H.; House, M.; Swaminathan, I.; Hool, L., Examining
Efficacy of “TAT-less” Delivery of a Peptide against the L-Type Calcium
Channel in Cardiac Ischemia-Reperfusion Injury. ACS Nano 2012, Published
online DOI:10.1021/nn305211f. (Impact Factor 11.42, 0 citations, Journal
Ranking 5th out of 66).
3. Evans, C. W.; Latter, M. J.; Ho, D.; Peerzade, S. A. M. A.; Clemons, T. D.;
Fitzgerald, M.; Dunlop, S. A.; Iyer, K. S., Multimodal and Multifunctional
Stealth Polymer Nanospheres for Sustained Drug Delivery. New J Chem 2012,
36, 1457. (Impact Factor 2.61, 1 citation, Journal Ranking 44th out of 154).
4. Harrison, J.; Bartlett, C.; Cowin, G.; Nicholls, P.; Evans, C.; Clemons, T.;
Zdyrko, B.; Luzinov, I.; Harvey, A.; Swaminathan, I.; Dunlop, S.; Fitzgerald, M.,
in-vivo imaging and biodistribution of multimodal polymeric nanoparticles
delivered to the central nervous system. Small, 2012, 8, 1579. (Impact Factor
8.35, 1 citation, journal ranking 7th out of 66).
5. Clemons, T. D.; Evans, C. W.; Zdyrko, B.; Luzinov, I.; Fitzgerald, M.; Dunlop,
S. A.; Harvey, A. R.; Iyer, K. S.; Stubbs, K. A., Multifunctional nanoadditives
for the thermodynamic and kinetic stabilization of enzymes. Nanoscale 2011, 3,
4085. (Impact Factor 5.91, 0 citations, 11th out of 66).
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6. Evans, C. W.; Fitzgerald, M.; Clemons, T. D.; House, M. J.; Padman, B. S.;
Shaw, J. A.; Saunders, M.; Harvey, A. R.; Zdyrko, B.; Luzinov, I.; Silva, G. A.;
Dunlop, S. A.; Iyer, K. S., Multimodal Analysis of PEI-Mediated Endocytosis of
Nanoparticles in Neural Cells. ACS Nano 2011, 5, 8640. (Impact Factor 11.421,
4 citations, journal ranking 5th out of 66).
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Conference presentations
1. T. Clemons, N. K. Tangudu, V. K. Verma, S. S. Beevi, G. Mahidhara, T. Hay, M.
Raja, R. A. Nair, L. E. Alexander, A. B. Patel, J. Jose, N. M. Smith, B. Zdyrko,
A. Bourdoncle, I. Luzinov, A. R. Clarke, L. D. Kumar & K. S. Iyer. Breast and
colon cancer tumour regression through the delivery of c-Myc shRNA
conjugated to multifunctional polymeric nanoparticles. 4th International
Nanomedicine Conference, 1st July 2013. Sydney, Australia.
2. (Invited) T. Clemons. How tiny science can have a big impact on students. 2013
Conference of the Science Teachers Association of Western Australia, 18th May
2013. Perth, Australia.
3. T. Clemons, H. Viola, I. Swaminathan, L. Hool. Polymeric nanoparticles for the
treatment of ischemia-reperfusion injury. Australian Nanotechnology Network
Early Career Research Symposium 2012, 15th December 2012. Melbourne,
Australia.
4. (Invited) T. Clemons. How tiny science can have a big impact on students –
Nanotechnology outreach. Australian Nanotechnology Network Early Career
Research Symposium 2012, 16th December 2012. Melbourne, Australia.
5. T. Clemons, H. Viola, I. Swaminathan, L. Hool. Nanoparticles as a delivery
vehicle for the alleviation of cardiac ischemia-reperfusion injury. University of
Western Australia, School of Chemistry and Biochemistry 2012 research forum,
2nd November 2012. Perth, Australia.
6. T. Clemons, M. Fitzgerald, S. Dunlop, A. Harvey, B. Zydyrko, I. Luzinov, S.
Iyer, K. Stubbs. Nanoparticles for enzyme stabilization. XI International
conference on Nanostructured Materials (Nano2012), 28th August 2012. Rhodes,
Greece.
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7. T. Clemons, H. Viola, I. Swaminathan, L. Hool. Polymeric nanoparticles for
delivery to cardiac myocytes. Molecular Imaging Symposium, 1st May 2012.
Sydney, Australia.
8. T. Clemons, M. Fitzgerald, S. Dunlop, A. Harvey, B. Zydyrko, I. Luzinov, S.
Iyer, K. Stubbs. Thermal stabilization of industrial and medically relevant
enzymes in the presence of nanoadditives. International Conference on
Nanoscience and Nanotechnology, 6th February 2012. Perth, Australia.
9. (Invited) T. Clemons, C. Evans, D. Ho. Because the small things make a big
difference. Science Teachers Association of Western Australia - Future Science
Conference, 2nd December 2011. Perth, Australia.
10. T. Clemons, C. Evans, B. Zydyrko, I. Luzinov, M. Fitzgerald, S. Dunlop, A.
Harvey, S. Iyer, K. Stubbs. Nanoparticles for multimodal enzymatic therapy in
the central nervous system. Combined Biological Sciences Meeting, 26th August
2011. Perth, Australia.
11. T. Clemons, C. Evans, B. Zydyrko, I. Luzinov, M. Fitzgerald, S. Dunlop, A.
Harvey, S. Iyer, K. Stubbs. Stabilization of enzymes against thermal inactivation
with multifunctional polymeric nanoparticles. Fifth International Conference on
Advanced Materials and Nanotechnology, 10th February 2011. Wellington, New
Zealand.
12. T. Clemons, C. Evans, K. Stubbs, M. Fitzgerald, S. Dunlop, A. Harvey, I.
Luzinov, B. Zydyrko, I. Swaminathan. Multifunctional polymeric nanoparticles
for the stabilization of enzymes against thermal inactivation. OzBio 2010 The
molecules of Life – From Discovery to Biotechnology, 29th September 2010.
Melbourne, Australia.
13. T. Clemons, C. Evans, K. Stubbs, M. Fitzgerald, S. Dunlop, A. Harvey, I.
Luzinov, B. Zydyrko, I. Swaminathan. Multifunctional polymeric nanoparticles
for the stabilization of enzymes against thermal inactivation. Australian
Research Network for Advanced Materials/Australian Research Council
Nanotechnology Network joint conference, July 22nd 2010. Adelaide, Australia.
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Statement of candidate contribution This thesis contains the results of work carried out by the author within the School of
Chemistry and Biochemistry and the School of Animal Biology at the University of
Western Australia during the period of January 2010 to June 2013.
The work presented herein contains no materials which the author has submitted or
accepted for the award of another degree or diploma at any university and, to the best of
the author’s knowledge and belief, contains no material previously published or written
by another person, except where due reference is made in the text.
Tristan DeVere Clemons
2013
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Thesis Prelude Nanotechnology and its integration into biology and medicine has been a rapidly
developing field with significant breakthroughs constantly being made. The work
conducted during this PhD was aimed at furthering this relationship between
nanotechnology and biology by improving the delivery of therapeutics to treat a range
of diseases and injuries. Specifically, the aims of the research were to:
1. Synthesize and characterize a novel multimodal PGMA nanoparticle platform
with the potential for use as a drug delivery vehicle.
2. Assess the efficacy of this delivery vehicle for the delivery of biologically
relevant payloads such as peptides and plasmid DNA in in vitro, ex vivo and in
vivo models of cardiac ischemia-reperfusion injury and breast and colon cancer
respectively.
3. Assess the ability of the synthesized polymeric nanoparticles to impart thermal
stability to enzymes for the end goal of producing a delivery vehicle for the
enzyme chondroitinase ABC to central nervous system injuries.
For the aforementioned aims to be achieved, a comprehensive understanding and
review of the literature regarding nanoparticle design, synthesis and characterization
techniques, nanoparticle structures and the addition of imaging agents and surface
ligands was required. The review of the literature, presented in Chapter One, covers
a range of fields as is expected from a multidisciplinary project. The use of
nanoparticles in drug delivery was explored, providing a review of the different
types of nanoparticles currently being used for medical applications and their
inherent merits and pitfalls. The synthesis, use and advantages of polymeric based
nanoparticle systems were discussed in order to provide a clear rationale for the
choice of using polymeric nanoparticles in this work.
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Secondly, as the nanoparticles that have been developed are intended for medical
applications, an understanding of the interactions of these nanoparticles at a cellular
level as well as the clearance mechanisms by which nanoparticles are removed from
the body are also discussed. The incorporation of imaging modalities into
nanoparticles to allow them to act in both a therapeutic as well as a diagnostic role is
important for nanoparticle tracking in vivo, and hence is also explored in the review.
Finally, the review covers a range of clinically relevant imaging modalities
appropriate for integration into the polymeric nanoparticle system. The review in
Chapter One provides a broad background of nanoparticles in medicine while also
assessing the considerations to be made in synthesizing new nanoparticles to be
suitable as drug delivery vehicles. Each subsequent chapter in the thesis will begin
with a short introduction to the literature specific to that chapter to provide the
relevant information required to understand the research problem, injury or disease
and the role in which nanoparticles can play in addressing these issues directly.
Chapter 1
Introduction and literature review
1.1 Nanoparticles in modern medicine
Nanotechnology is characterized by the creation and use of engineered materials or
devices that have at least one dimension in the range of 1-100 nm in size.1
Nanotechnology exploits the physical and chemical properties of nanoparticles, which
as a result of their size, are remarkably different from both atomic species and bulk
materials.2 Since the properties depend on the dimensions of the nanostructure, reliable
and continual changes can be achieved by changing the size of single particles. The best
example of this is quantum dots where altering the size of the quantum dot particle can
change the optical emission properties of the material (Figure 1.1).
Not only is nanotechnology interesting from a synthetic approach but this scale also
mirrors that of many biological targets and systems. Many proteins, viruses and
important biological molecules are in the size range of 1-10 nm and as a result,
structures that can be accurately designed on the nanometer scale have the ability to
interact on the cellular, sub-cellular and molecular levels with unique specificity.1, 3 This
specificity can result in explicit interactions within cells and tissues without causing
undesirable side effects.3 A major field of nanotechnology research is the synthesis of
nanoparticles for medical applications including disease diagnosis, imaging and most
importantly treatment through the delivery of therapeutics. It is envisaged that the
global market for nanotechnology related applications in the medical field could
increase to between $70-160 billion US by the year 2015.4, 5
2
Figure 1.1. A) Quantum dots possess unique photo-physical properties making them
ideal for applications in biological imaging due to the ability to tune the emission colour
by altering the quantum dot size (particle size increasing from left to right). B) Narrow
emission spectra along with efficient light absorption throughout a wide spectrum of
wavelengths make quantum dots suitable for a range of applications, especially in
biological imaging. Figure and caption modified from Zrazhevskiy et al. 2009.6
Nanotechnology and nanoparticle drug delivery vehicles provide an exciting prospect
for the delivery of therapeutics in the treatment of a range of diseases and injuries in
comparison to current clinical methods.7, 8 Nanoparticles in particular possess a range of
advantages as drug delivery vehicles including drug protection from clearance and
degradation, high levels of drug loading, the potential for multiple therapeutics to be
delivered from the same entity, preferential drug release at target tissues, modifiable
drug release kinetics and finally ease of nanoparticle modification for the incorporation
of imaging probes, targeting moieties and surface structure functionalities.9 This review
provides insight into some significant breakthroughs and also highlights some of the
challenges still facing this field as a prelude to the work conducted in this thesis.
1.1.1 Nanoparticles for drug delivery
In drug discovery it is easy to find a long list of drug candidates that, although
possessing high potency, are unsuitable for clinical application due to poor solubility or
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poor circulation within the body. Often these candidates have been overlooked in
preference for drugs possessing lower potency but better solubility and half-lives.4
Nanotechnology has the potential to change this by rewriting the rules of drug discovery
and improving drug characteristics, which were previously seen as limiting or
significant enough to warrant a drug’s rejection.4 Nanoparticle based drug delivery
systems have been developed to ultimately improve the efficiency of delivery and to
reduce systemic toxicity of a wide range of therapeutics. The application of
nanoparticles and nanocapsules for drug encapsulation has looked to build on this
concept down to the nanoscale. The first generation of nanoparticles developed for drug
delivery often only provided one function: drug coating and protection to either enhance
drug solubility or circulation time. These nanoparticles are now currently being tested in
clinical trials with some gaining recent approval for clinical applications (Table 1.1).10
A wide variety of nanoparticle formulations have been used for drug delivery
applications including liposomes, dendrimers, microemulsions, micelles, solid lipid and
polymer nanoparticles, and soluble polymers that have a therapeutic attached via
biodegradable linkages (Figure 1.2). Particles already approved for clinical use include
those based on liposomes, biodegradable polymeric nanoparticles and polyethylene
glycol (PEG) or protein based nanoparticle drug conjugates.10, 11
4
Table 1.1. Nontargeted nanoparticles that have been approved for clinical use or undergoing clinical trials.10 PLA, poly(l-lactide); pAsp, poly(l-aspartic acid); PEG, poly(ethylene glycol) Pglu, polyglutamate; PAA, poly(l-aspartate); HPMA, N-(2-hydroxypropyl)-methacrylamide-copolymer
Brand Name Composition Indication Status
Liposome-based nanoparticle
Doxil/Caelyx PEGylated liposomal doxorubicin Ovarian cancer, Kaposi's sarcoma Approved DaunoXome (Galen) Liposomal daunorubicin Kaposi's sarcoma Approved Myocet (Sopherion) Non-PEGylated liposomal doxorubicin Breast cancer Approved
Micelle-based nanoparticle
Genexol-PM Paclitaxel-loaded PEG-PLA micelle Breast cancer, lung cancer Approved NK911 Doxorubicin-loaded PEG-pAsp micelle Various cancers Phase 2 NK012 SN-38-loaded PEG-Pglu (SN-38) micelle Breast cancer Phase 2
NC-6004 Cisplatin-loaded PEG-Pglu micelle Various cancers Phase 1 SP1049C Doxorubicin-loaded pluronic micelle Gastric cancer Phase 3 NK105 Paclitaxel-loaded PEG-PAA micelle Breast cancer Phase 3
Polymer-drug conjugates-based nanoparticle
OPAXIO (Cell Therapeutics)
Paclitaxel combined with a polyglutamate polymer Ovarian cancer Phase 3
IT-101 Camptothecin conjugated to cyclodextrin-based polymer Various cancers Phase 1/2 HPMA-DOX (PK1) Doxurubicin bound to HPMA Lung cancer, breast cancer Phase 2
HPMA-DOX-galactosamine (PK2)
Doxorubicin linked to HPMA bearing galactosamine Hepatocellular carcinoma Phase 1/2
CT-2106 Camptothecin poly-l-glutamate conjugate Various cancers Phase 1/2
Albumin-based nanoparticle
Abraxane Albumin-bound paclitaxel nanoparticles Metastatic breast cancer Approved
5
Figure 1.2. Schematic structure of a range of nanoparticle formulations currently being
prepared for drug delivery applications. Nanospheres and nanocapsules are basically
small vesicles used to transport materials. Nanocapsules are a shell with an inner space
loaded with the drug of interest. Both systems are useful for controlling the release of a
drug and/or protecting it from the surrounding environment. A micelle is a spherical
conglomeration of amphiphilic molecules, such as cholesterol. In aqueous environments,
the molecules form a tight ball with the hydrophobic groups on the inside and the
hydrophilic groups on the outside. The reverse occurs in a non-aqueous environment.
Micelles are useful for encapsulating non-water soluble drugs to be administered
intravenously. Dendrimers are highly branched polymers with a controlled three-
dimensional structure around a central core. Dendrimers are easily functionalized and
can accommodate more than 100 terminal groups. Liposomes are spherical vesicles that
comprise one or more lipid bilayer structures enclosing an aqueous core. Liposomes can
also be functionalized to improve cell targeting and solubility. Figure from Sanna et al.
2013 and caption has been modified from Orive et al. 2009.12, 13
1.1.2 Micelles, liposomes and dendrimers for drug delivery applications
Micellar nanoparticles consist of a hydrophobic core which is surrounded by
amphiphilic block copolymers that have assembled around this hydrophobic core to
produce a core/shell architecture in aqueous media.13, 14 The hydrophobic core region of
6
the micelle acts as a reservoir for hydrophobic drugs; the hydrophilic exterior of the
micelle allows for nanoparticle stability in aqueous media.14 Micelles have the ability to
encapsulate a range of therapeutic cargoes including hydrophobic drugs,
oligonucleotides, proteins and imaging agents with considerably high loading levels (up
to 30% w/w).13, 15 Micelle nanoparticles have shown great promise as delivery systems
with a number currently in phase 3 clinical trials (Table 1.1). Micelles can also be
produced from stimuli responsive block copolymers to allow disassembly in the
presence of triggers such as pH, temperature, light or ultrasound.16 This allows for
targeted release of the therapeutic payload held within the micelle structure.
A recent study by Lee et al. encapsulated the photosensitive Protoporphyrin IX (PpIX)
within a pH responsive micelle based on the block copolymer of PEG-poly(β-amino
ester) (Figure 1.3).16 The pH of the microenvironment surrounding tumour tissue is
lower (pH 6.4-6.8) than that of normal tissue (pH 7.4).17, 18 This reduction in pH allows
for protonation of the tertiary amines present in the amino ester, resulting in an increase
in the hydrophilicity of the polymer.17 This change results in rapid demicellization in
the regions surrounding the tumour tissue and leads to the release of the encapsulated
photosensitizer PpIX. PpIX produces a strong fluorescent signal allowing the location
of PpIX surrounding the tumour microenvironment to be identified (Figure 1.3B).
Furthermore, when irradiated with the appropriate wavelength of light, PpIX produces
cytotoxic singlet oxygen (photodynamic therapy), which in turn destroys nearby tumour
cells (Figure 1.3D).
7
Figure 1.3. Polymeric micelles for optical imaging and photodynamic therapy. A)
Schematic illustration of PpIX-encapsulated pH-responsive polymeric micelles for
tumor diagnosis and photodynamic therapy. B) Fluorescence images after injection of
PpIX-encapsulated pH-responsive polymeric micelles. C) Ex vivo images of organs and
tumors. D) Tumour growth after injection and laser irradiation. Figure and caption from
Lee et al. 2012.16
Similar to micelles, liposomes are closed colloidal structures consisting of an aqueous
core surrounded by a phospholipid bilayer with their main application in the delivery of
aqueous biomolecules and hydrophilic drugs.13 Liposomes have the potential to entrap
relatively large amounts of hydrophilic drugs within their aqueous core or between the
lipid bilayer shell structure if the therapeutic is lipophilic.15 A major advantage of
liposomes is that they form spontaneously in solution and they essentially possess no
inherent toxicity due to the presence of the components of liposomes throughout the
body in all cell membranes.13 Liposomes have had great success in the delivery of
anthracycline based chemotherapeutics including doxorubicin, and daunorubicin for the
treatment of metastatic breast cancer,19, 20 ovarian cancer,19 and for the treatment of
AIDs related Kaposi’s sarcoma.21 An interesting application of liposomes for drug
8
delivery is the utilization of liposomes for the encapsulation and aerosol delivery of
vasoactive intestinal peptide (VIP) for the treatment of various lung diseases such as
asthma and pulmonary hypertension. A recent study by Hajos et al. found that
encapsulation of VIP within liposomes was successful in allowing VIP to avoid
enzymatic degradation once inhaled and deposited within the bronchi.22 This study
found that loading of the VIP within the liposomes for inhalation therapy improved the
pharmacological and biological activity of the VIP treatment in comparison to the
delivery of free VIP.22
Dendrimers are not nanoparticles per se but more strictly defined as a polymeric
macromolecule of nanometer dimensions composed of highly branched monomers that
emerge radially from a central core.14 Dendrimers can be biodegradable or non-
biodegradable structures. Natural polymers such as glycogen, and some proteoglycans
consist of a dendrimer like structure. However, for drug delivery, the synthetic polymer
poly(amidoamine) (PAMAM) is the most extensively studied.14, 23 PAMAM has been
shown to be effective for the binding and subsequent delivery of cisplatin both in vitro
and in vivo where it shows improved efficacy in comparison to cisplatin delivered
without the dendrimer.24 Properties that make dendrimers attractive for drug delivery
applications include monodispersed size distributions, modifiable surface chemistry,
multivalency, water solubility and an internal cavity available for drug loading.23 Due to
the ease with which dendrimer surface chemistry can be modified, the addition of
contrast agents, imaging probes and targeting ligands can be coupled with a therapeutic
for delivery, resulting in the production of dendrimer based multifunctional drug
delivery systems.23 Dendrimers can be produced with low cytotoxicity and surface
decoration of the dendritic structure with PEG can prolong its circulation half-life.
Although there is significant interest in dendrimers as drug delivery vehicles, few have
translated into clinical trials with Vivagel® the most promising candidate, currently in
phase 2 clinical trials.25 Vivagel® is a L-lysine dendrimer that contains a polyanionic
outer surface which exhibits antiviral activity against the sexually transmitted herpes
simplex virus (HSV) and the human immunodeficiency virus (HIV).25
1.2 Polymeric nanoparticles and nanocapsules as drug delivery vehicles
Polymeric nanoparticles and nanocapsules are solid formulations ranging in size from
10-1000 nm in diameter and can be synthesized from natural or artificial polymers.
9
Generally speaking, the major advantage of polymeric nanosystems over other nano-
delivery systems is their inherent stability and structural rigidity.13 These polymeric
nanoparticles often incorporate their therapeutic for delivery via drugs that are adsorbed,
dissolved, entrapped, encapsulated or covalently linked to the nanoparticle.26, 27 The
most commonly used synthetic materials for the synthesis of biodegradable polymeric
nanoparticles are poly(lactic acid) (PLA), poly(D-L-glycolide (PLG) or the copolymer
of these synthetic polymers being poly(lactic-co-glyoclic acid) (PLGA). due to their low
toxicity, biodegradability, FDA approval and tissue compatibility.27, 28 Biodegradable
nanoparticles based on these aforementioned polymers have been used for the delivery
of a range of therapeutics in vivo for the treatment of cancers,29 neurodegenerative
disorders,30 and for the controlled release of contraceptive steroids and fertility control
systems.31, 32
Nanoparticles synthesized from naturally occurring polymers such as chitosan, albumin
and heparin have been popular choices for the delivery of oligonucleotides, proteins and
small molecule drugs. Despite significant research in the use of polymers for
nanoparticle drug delivery systems only one, Abraxane, has been approved for clinical
applications to date.33 Abraxane is an albumin based nanoparticle system developed for
the delivery of paclitaxel, a proven chemotherapeutic agent, to metastatic breast
cancers.33 Furthermore, Abraxane is currently undergoing clinical trials for delivery to a
variety of other cancers including non-small-cell lung cancer (phase 2 trial)34 and
advanced nonhematologic malignancies (phase 1 and pharmacokinetics trials).35 Since
the use of polymeric nanoparticles forms the crux of this PhD thesis, the following
section will cover in detail the common methods used for their preparation.
1.2.1 Methods for the preparation of polymeric nanoparticles
A number of approaches have been developed for the synthesis of polymeric
nanoparticles most of which involve the use of block copolymers consisting of polymer
chains of differing solubilities. The more common techniques for polymeric
nanoparticle formulations include layer-by-layer (LbL) approaches, nanoprecipitation
(sometimes referred to as the solvent displacement method), emulsification, solvent
evaporation methods, and the salting out method. Further to these traditional methods,
techniques that make use of microfluidics, super critical technology and the premix
membrane emulsification method are increasingly favoured due to their potential for
10
producing highly monodispersed nanoparticles in high yields.28 Usually, the choice of
nanoparticle formulation method is dictated by the physicochemical properties of the
drug, the polymer intended for encapsulation and particle size requirements.9 The
common techniques for the preparation of polymeric nanoparticles from pre-formed
polymer are discussed in the following paragraphs paying special attention to the
associated merits and pitfalls of each method.
The LbL approach to producing polymeric nanoparticles is a highly versatile and
interesting nanoparticle engineering method. Typically, LbL particles are formed
through the consecutive deposition of polymers which interact with one another (e.g.
through electrostatic interactions or hydrogen bonding) onto a core particle template.36
This results in the formation of core-shell particles consisting of an ultrathin, highly
tunable multilayered polymer coating on particles of varying size (from 10 nm up to a
few microns), shape and composition.37, 38 Furthermore, if a sacrificial core is used in
this method the subsequent removal of this template allows the production of hollow
polymeric capsules.36 LbL particles have been successful in their efficient encapsulation
of cargoes, triggered release or degradation and targeted delivery through the
incorporation of antibodies.39, 40 An interesting recent study by Poon et al. developed
LbL nanoparticles with a pH responsive shell in order to improve cellular uptake of the
nanoparticles in an acidic tumour microenvironment.41 Carboxyl terminated quantum
dots were sequentially coated with iminobiotin-functionalized poly(L-lysine) (PLL),
neutravidin, and biotin-functionalized PEG. The nanoparticles produced consisted of a
fluorescent quantum dot core and alternate layers of PLL and PEG held together by the
strong physical interaction between biotin and avidin.41 The incorporation of PEG on
the nanoparticle surface increased circulation time until this layer was selectively
eroded by the acidity in the hypoxic tumour microenvironment.36, 41 This erosion of the
outer surface exposed the positively charged layers beneath the PEG coating resulting in
rapid cellular internalization and, in turn, tumour retention of the LbL nanoparticles.41
Despite the considerable promise surrounding LbL nanoparticles, these systems are still
in their infancy and thus the pharmacokinetics in disease models, the biocompatibility
and toxicity in vivo following delivery, are yet to be determined.36 This method can
also be time consuming with wash steps between layer depositions, and often the
requirement of a highly precise pairing of polymers to ensure a strong interaction
between layers and thus structural integrity once the sacrificial core is removed from the
polymeric shell.
11
The nanoprecipitation method involves an organic solvent that is miscible with an
aqueous phase and can also dissolve both the polymer and drug intended for
encapsulation.9 The organic phase (solvent), containing polymer and drug, is added
drop wise to the stirring aqueous phase (non-solvent) where, upon contact with the
water, the hydrophobic polymers and drug precipitate and spontaneously self assemble
into core shell like spherical structures in an attempt to reduce the system’s free energy.9
The nanoprecipitation method is simple and can be easily scaled up to industrial levels
as it only requires gentle stirring and no high stress shear. This method however is
limited to hydrophobic drugs, which are highly soluble in non-polar solvents but only
slightly soluble in water. The method also has challenges in determining an appropriate
polymer/drug/solvent/non-solvent system, which allows for nanoparticle formation with
high drug loading efficiency.28 Using a diblock copolymer of PLGA and PEG,
docetaxel loaded polymeric nanoparticles consisting of a PLGA core and a PEGylated
outer shell have been successfully produced with the nanoprecipitation method for in
vivo chemotherapeutic treatments.42 When targeted to and tested in an in vivo model of
prostate cancer, these targeted nanoparticles resulted in 100% viability (all animals
reached 109 days survival) and tumour reduction compared to docetaxel alone where
only 14% of mice reached the 109 day target.42
Emulsion techniques involving either a water in oil (W/O), an oil in water (O/W) or a
double emulsion (W/O/W) require the formation of an emulsion followed by an input of
high powered sonication or homogenization to produce nanoparticles from the emulsion.
The single O/W emulsion technique is the most common and is used for the preparation
of hydrophobic polymeric nanoparticles containing hydrophobic drugs. The organic
components (drug and polymer) are dissolved in a water immiscible organic solvent (e.g.
dichloromethane), which is then emulsified under intense shear stress in an aqueous
phase containing an appropriate surfactant to aid in particle stabilization.9 The volatile
organic solvents are allowed to evaporate resulting in the self-assembly of nanoparticles
containing the encapsulated drug once again as a result of the system aiming to reduce
free energy.9 This method produces nanoparticles with high drug entrapment efficiency
although it is limited to drugs that are soluble in the same solvent as that used for the
polymer. Furthermore, particle monodispersity is difficult to achieve with emulsion
techniques.
12
Questions remain surrounding the scale up potential of this technology due to the high
energy requirements in homogenization and the use of toxic chlorinated solvents.28 The
single O/W emulsion technique is limited in its ability to only encapsulate hydrophobic
drugs and hence a double emulsion of W/O/W can be used for the incorporation of
hydrophilic drugs. The W/O/W method is still an emulsion technique, where a W/O
emulsion is first produced before emulsifying the mixture again to produce a final
nanoparticle in aqueous media.9 The salting out method is an extension of the emulsion
techniques described above where the mixing of the organic and the aqueous phases is
prevented by saturating the aqueous phase with electrolytes such as magnesium acetate,
magnesium chloride or calcium chloride.28 This method is advantageous in that it does
not require elevated temperatures and also avoids the use of toxic chlorinated solvents.
However, this method does introduce extensive nanoparticle washing steps to remove
the excess salts and also raises concerns with regards to waste and recyclability of the
large amounts of solvent and salts required.28
New approaches for polymeric nanoparticle preparations have looked to address some
of the limiting factors of the aforementioned technologies. Methods based on
microfluidic technology to produce rapid mixing techniques in microchannels such as
hydrodynamic flow focusing have been shown to produce polymeric nanoparticles
exhibiting narrow size distributions when compared to similar nanoparticles produced
by bulk synthesis techniques.43, 44 An interesting finding from a study by Karnik et al.
found that the polymeric nanoparticles produced from microfluidic methods were able
to achieve higher drug loading then those produced by bulk methods, a highly desirable
characteristic of nanoparticles intended for drug delivery.43 Super critical fluid (SCF)
methods based on the rapid expansion of a super critical solution containing the
polymer and drug to produce the nanoparticles have had great success recently. One
success in this area is that of the UK based spin off company, Critical Pharamceuticals,
which has commercialized a range of products through the use of supercritical methods
for advanced drug delivery of growth hormones. As solubility in SCFs can be up to a
million times higher than that under ideal gas conditions, the rapid expansion from
supercritical pressure to ambient pressures produces extremely high super saturated
solutions.28 These solutions when released from super critical conditions, rapidly result
in very homogenous nucleation conditions for the solute (i.e. polymer) producing
nanoparticles of narrow and reproducible size distributions.45 The premix emulsification
13
method combines the emulsification technique to produce a coarse ‘premix’ emulsion as
per techniques mentioned previously. This premix is then extruded through a Shirasu
porous glass membrane with high pressure to produce uniform nanodroplets.28 Wei et al.
prepared PLA nanoparticles by this method and found that several factors play a key
role in influencing the uniformity in nanoparticles produced including organic solvent
selection, the volume ratio of organic to the external aqueous phase, the pore size of the
microporous membrane and finally the transmembrane pressure used during
collection.46 This method has the advantage of high productivity, simplicity in operation
and the potential for industrial scale up by increasing the surface area of the membrane
or by connecting membranes in parallel.46
While polymeric nanoparticles have the potential to revolutionize drug delivery and
how diseases are treated, important issues still remain. Current synthetic methods,
although very successful at producing a large range of functionalized polymeric
nanoparticles have only been achieved on a small scale. Issues surrounding efficiency of
loading and recycling of by products must be addressed before these technologies can
be scaled up to industrial production. It is considered that this is a major limiting factor
preventing the successful integration of polymeric nanoparticles into the clinic and
market.28
1.3 Nanoparticle and cell interactions
In addition to accurate synthesis and drug loading, another integral consideration for
nanoparticles developed for drug delivery is how they interact with biological systems
once introduced into the body. For drugs with intracellular targets, often the cell
membrane can loom as a formidable barrier. The concept of nanoparticles, which can be
tailored to carry these drugs across the cell membrane and to relevant sub cellular
compartments, provides an attractive means to achieve improved drug trafficking. Proof
of concept studies in the 1970s have shown that sub-micron sized liposomes,47 as well
as synthetic polymer nanoparticles,48 were able to deliver and concentrate in cells,
therapeutics which previously were unable to do so on their own. The plasma
membrane is the barrier which protects the cell against unwanted intruders such as
pathogens, macromolecules and even nanoparticles from entering the cell from the extra
cellular space.49 It consists of a self-assembled bi-layer of lipids where the hydrophobic
interior of this layer is responsible for restricting the passage of water-soluble
14
substances into the cell. Although the passage of small molecules, amino acids and ions
occurs through specialized membrane protein pumps and selected ion channels on the
cell surface, the majority of nanoparticles must undergo some form of membrane
interaction before the process of endocytosis can occur.50 Endocytosis can occur
through a range of mechanisms (Figure 1.4) which can be broadly categorized into
either phagocytosis (cell ‘eating’ for solid particles) or non-phagocytic pathways (cell
‘drinking’ processes).5, 50 With reference to nanoparticles however, these classical
references of cell eating and drinking are not as relevant due to the ability of solid
nanoparticles to still be internalized through non-phagocytic pathways.51 It is important
to have an understanding of the relevant pathways of cell entry which could act on or
affect nanoparticle uptake as this will have direct effects on the drug physicochemical
characteristics as well as the intracellular fate of the nanoparticle carrier and in turn its
therapeutic cargo.51
Phagocytosis for the internalization of macromolecules and indeed most nanoparticles
occurs primarily in specialized cells known as phagocytes, which include macrophages,
monocytes, neutrophils, astrocytes and dendritic cells.52 Phagocytosis can be described
as a general three-step process. An important first step is recognition of the nanoparticle
by opsonin proteins in the bloodstream to tag the nanoparticle for phagocytosis.
Secondly, this signaling triggers the plasma membrane to form an invagination
preparing for the nanoparticle to be internalized and, finally the plasma membrane will
‘pinch off’ from the surrounding plasma membrane to engulf the nanoparticle producing
a discrete package bound by plasma membrane proteins within the cell (Figure 1.4).5, 53,
54 The internalized vesicle, known as a phagosome, is trafficked within the cytoplasm
until it becomes accessible to early endosomes. The phagosome then begins to acidify
and matures, fusing with late endosomes and finally lysosomes to form a
phagolysosome.52 The speed with which this process occurs is highly dependent on the
particle and its surface characteristics but typically the process can take from minutes to
hours.52
15
Figure 1.4. Pathways of entry into cells. Large particles can be internalized by
phagocytosis, whereas fluid uptake occurs by macropinocytosis. Both processes appear
to be triggered by and are dependent on actin-mediated remodeling of the plasma
membrane at a large scale. Compared with the other endocytotic pathways, the size of
the intracellular vesicles formed by phagocytosis and macropinocytosis are much larger.
Numerous cargoes can be endocytosed by mechanisms that are independent of the coat
protein clathrin and the fission GTPase, dynamin. Most internalized cargoes are
delivered to the early endosome via vesicular (clathrin- or caveolin-coated vesicles) or
tubular intermediates (known as clathrin- and dynamin- independent carriers (CLIC))
that are derived from the plasma membrane. Some pathways may first traffic to
intermediate compartments, such as the caveosome or glycosyl phosphatidylinositol-
anchored protein enriched early endosomal compartments (GEEC), en route to the early
endosome. Figure and caption from Mayor et al. 2007.54
Phagolysosomes become acidified due to the proton pump ATPase located in the
membrane of the phagolysosome; the recruitment of an enzyme cocktail to aid in the
degradation of the foreign body also occurs at this time.55 Although a minimum size of
0.5 µm is often considered the limit for phagocytosis, previous studies have shown
nanoparticles ranging from 250 nm to 3 µm in diameter can undergo in vitro
phagocytosis.51 Careful control of the nanoparticle surface coating and nanoparticle size
can play important roles in producing nanoparticles that can avoid phagocytic uptake.56
It is generally accepted however that the in vivo fate of nanoparticles is opsonization
followed by phagocytosis with little discrimination for nanoparticle composition, unless
16
the particles are very small in size (less then 100 nm), or more importantly possess a
specific hydrophilic coating (such as PEG) to aid in the avoidance of opsonin
recognition.51
Non-phagocytic pathways, normally referred to as pinocytosis, are not restricted to
specialized cells and contain processes that are used by almost all cells for the
internalization of fluids and solutes alike. Non-phagocytic uptake into cells can occur
through four main mechanisms: clathrin-mediated endocytosis, caveolae-mediated
endocytosis, macropinocytosis and other clathrin and caveolae independent processes
(Figure 1.4).51, 54 Clathrin-mediated endocytosis, the most common mechanism for
uptake, results in trafficking of cargoes into the lysosomal pathway for
biodegradation.53 Conversely, caveolae-mediated uptake has been shown to produce
caveolar vesicles which do not contain a degradative enzymatic cocktail and hence
caveolae-dependent uptake is seen as a mechanism which if targeted could avoid
trafficking of nanoparticles to the degradative lysosomal pathway.56 A third process
known as macropinocytosis is where actin derived protrusions from the cell membrane
can engulf cargoes, upon which the protrusion collapses to again fuse with the cell
membrane. The fate of cargoes which are internalized by macropinosomes can vary
however often they will fuse with lysosomes, which in turn acidify for the degradation
of the cargo.51 By having a better understanding of the variety of internalization
pathways by which nanoparticles can be internalized, a clearer understanding will be
gained as to what kind of environment nanoparticles may be exposed to once they are
internalized. This information is important, for example, when developing new
nanoparticles with site-specific drug release capabilities or biodegradation qualities, or
if the nanoparticle vehicle is engineered with specific escape mechanisms to avoid
degradation in endosomes.57, 58
1.3.1 Nanoparticle endocytosis
Nanoparticle size, shape and relative hardness can dictate which endocytosis pathway is
activated and utilized for nanoparticle uptake. A study by Rejman et al. investigated the
internalization of uniform spherical polystyrene nanoparticles of differing sizes in
murine melanoma cells (B16-F10).56 This study demonstrated that polystyrene spherical
nanoparticles with diameters of 50 and 100 nm were rapidly internalized in less than 30
minutes by a clathrin-mediated pathway.56 In comparison, larger nanoparticles (200 and
17
500 nm in diameter), also made from polystyrene, were internalized much more slowly
(2-3 h) and exhibit an 8-10 fold decrease in internalization when compared to the
smaller particles.56
The shape of nanoparticles has also been recently investigated to see the role it plays on
nanoparticle internalization. Gratton et al. investigated the internalization of a series of
nanoparticles in HeLa cells where the nanoparticles were fabricated to have differing
aspect ratios.59 High aspect ratio rod shaped nanoparticles were internalized in HeLa
cells at a greater rate than spherical nanoparticles of similar internal volume, a
phenomenon similar to that of the appreciable increase seen in the uptake of rod-shaped
bacteria in non-phagocytic cell lines.59 Even nanoparticle hardness can influence the
interactions of nanoparticles with the cell membrane and in turn can have a direct
influence over cell internalization. A recent study by Banquy et al. investigated the
internalization of similar particles of differing hardness, i.e. Young’s modulus.60 This
study found that 150 nm hydrogel nanoparticles with intermediate Young’s modulus
(35 and 136 kPa) were internalized by a range of different mechanisms in macrophages
whereas softer nanoparticles (150 nm diameter hydrogel nanoparticles, 18 kPa) were
preferentially internalized by macropinocytosis and stiffer nanoparticles (150 nm
diameter hydrogel nanoparticles, 211 kPa) via clathrin-mediated endocytosis.60 Further
to this, these nanoparticles with intermediate hardness experienced approximately 67%
higher internalization then softer nanoparticles and approximately 25% higher
internalization in comparison to the harder nanoparticles.60
It is evident that size, shape and hardness can affect nanoparticle endocytosis but
another important characteristic is that of nanoparticle surface charge. The surface
charge of nanoparticles plays an integral role in determining which endocytosis pathway
nanoparticles are internalized through.5 Positively charged nanoparticles are the most
efficient at plasma membrane interactions and in turn internalization as they interact
favourably with the negatively charged residues present on the cell surface.5
Nonetheless, uptake of nanoparticles with negative surface charges has also been
observed despite the unfavourable electrostatic interactions which occur between the
nanoparticles and the negatively charged cell membrane.5, 61 For example, a study by
Harush-Frenkel et al. investigating the internalization of cationic and anionic
nanoparticles in epithelial Madin-Darby Canine Kidney cells found that cationic
nanoparticles experienced rapid uptake, while the anionic nanoparticles, although at a
18
slower rate, still experienced effective cellular internalization.61 The majority of both
nanoparticle formulations was targeted mainly to the clathrin-dependent endocytosis
pathways with a small proportion of both formulations experiencing macropinocytosis-
dependent uptake.61 Further studies from the same group investigated a similar effect in
HeLa cells where it was determined that the cationic nanoparticles once again
experienced rapid clathrin-dependent uptake compared to the anionic nanoparticles,
being internalized more slowly by a different endocytosis pathway.62
1.3.2 Strategies to enhance cellular internalization
Poly(ethyleneimine) (PEI) is a synthetic polycation well known for its long history as a
non-viral transfection agent. Studies have used PEI for intracellular delivery of a range
of cargoes including nanoparticles, proteins and small molecule drugs.63-66 PEI is also
suitable for the delivery of siRNA and DNA due to the ability of this positively charged
polymer to condense around oligonucleotides thus enabling transfection of the anionic
cell membrane.67-70 PEI can promote and facilitate endosomal escape due to its strong
buffering characteristics in what is referred to as the “proton sponge” effect.63, 64 After
endocytosis, the natural acidification within the endosome protonates PEI, inducing
chloride ion influx, osmotic swelling and destabilization of the vesicle, resulting in the
nanoparticles being released into the cytoplasm.71,72 The major downfall associated with
PEI as a non-viral vector is its inherent toxicity which has been shown to scale with its
molecular weight and transfection efficiency.64
Another method other than cationic polymers that has had success in transfecting
therapeutic cargoes and nanoparticles across cellular membranes is the incorporation of
cell penetrating peptides (CPPs). CPPs are short cationic peptide sequences, which were
first discovered by investigating the ability of the HIV trans-activator of transcription
protein to penetrate cells and subsequently effectively deliver the HIV-1 specific
genes.73 There is a broad spectrum of cell penetrating peptides available, with most
consisting of fewer then 20 amino acids, the most common of these being the Trans-
Activator of Transcription (TAT) or as it is more commonly known, the TAT peptide.49
TAT is an 11 residue long peptide taken from the protein transduction domain of the
HIV-1 TAT protein, this domain is responsible for viral transfection.74 The TAT peptide
is rich in arginine and lysine residues, making it a highly positively charged, basic and
hydrophilic peptide suitable for attachment to the anionic cell membrane and in turn
19
subsequent internalization.74 The TAT peptide sequence has also been widely used to
improve the cellular delivery of a plethora of molecular cargoes from small molecule
drugs through to large proteins and nanoparticles.75
The exact mechanism for TAT transfection is still an area of debate and contradicting
theories. However, most studies agree on the importance of direct contact between the
TAT peptide and the negative residues on the cell surface as a preliminary requirement
for successful transfection to occur.74-77 However despite this, TAT mediated
therapeutic delivery still has some major drawbacks yet to be fully addressed. These
include the non-specificity of the current TAT sequence, as well as the associated social
stigma surrounding its use due to the origins of this sequence in the debilitating and
currently incurable HIV virus.42 Finally, and most importantly, is the possible
immunogenicity of the TAT delivery system. It has been speculated that TAT,
especially through repeated dosing would produce a significant immunogenic response
thus limiting its clinical applications, an issue yet to be examined further.73
1.4 Multifunctional nanoparticles
A major downside to the nanoparticles that are currently being investigated for clinical
trials (Table 1.1) is their ability to perform only one primary role as delivery agents.
More recent work has resulted in the production of multifunctional nanoparticles for
drug delivery which aim to achieve combinations including imaging probes, high drug
loading, modifiable drug release kinetics, drug release triggers, targeting ligands (such
as antibodies, proteins and peptides) and nanoparticle coatings to improve circulation
times. Figure 1.5 provides a schematic of such a nanoparticle conveying broadly the
different aspects that scientists may look to incorporate into a multifunctional
nanoparticle system.78 Research in this field has resulted in a plethora of nanoparticle
formulations and combinations of functions being presented within the literature.
A recent study by Zhou et. al. describes an octa-functional nanoparticle suitable for the
delivery of short interfering RNA (siRNA) to tumours for RNA interference (RNAi).79
The octa-functional nanoparticle included: 1) a biodegradable PLGA polymer matrix
for controlled release; the core of the particle contained 2) siRNA for gene knockdown;
3) an agent to facilitate endosomal escape; 4) an agent to enhance siRNA potency; with
the nanoparticle surface containing a range of functionalities including; 5) the
20
attachment of a cell penetrating peptide; 6) a peptide to aid in endosomal escape; 7) a
tumour homing peptide and finally 8) PEGylation of the surface to improve circulation
time.79 It is important to realize however that, with the addition of extra functions, the
cost to produce, time associated with production and purification as well as the
complexity of the nanoparticle system all increase. As a result, there is an ongoing
battle in evaluating the benefits of added functionalities versus the extra cost of adding
that function in these multicomponent nanoparticle systems. The following sections will
address some of the key considerations and functionalities currently being investigated
in the application of multifunctional nanoparticles for drug delivery applications, paying
special attention to some pivotal examples making use of these additions.
Figure 1.5. Multifunctional nanoparticles for drug delivery. Multifunctional
nanocarriers can combine a specific targeting agent (usually an antibody or peptide)
with nanoparticles for imaging (such as quantum dots or magnetic nanoparticles), a cell-
penetrating agent (e.g. the polyArg peptide, TAT), a stimulus-sensitive element for drug
release (i.e. pH or photo sensitive material), a stabilizing polymer to ensure
biocompatibility (PEG most frequently used) and the therapeutic compound for delivery.
Development of novel strategies for controlled released of drugs as well as timed
release will provide nanoparticles with the capability to deliver two or more therapeutic
agents on differing time scales. Figure and caption from Sanvicens et al. 2008.78
21
1.4.1 Targeted nanoparticles and ‘stealth’ coatings
Probably the most significant efforts following on from first generation nanoparticles
for drug delivery is that of nanoparticle targeting. Targeting can be achieved by two
main avenues, the first by passive targeting (nanoparticle targeting resulting from
disease pathophysiology) and the second by active targeting where targeting ligands and
moieties are added to the nanoparticles to produce preferential nanoparticle binding, and
at times cellular uptake in target tissue.80 Nanoparticles produced for the treatment of
malignant tumours and cancers can be considered to be targeting tumour tissue through
a passive process known as the enhanced permeability and retention (EPR) effect
(Figure 1.6).81 This is an effect directly due to the leakiness of tumour vasculature
combined with poor lymphatic drainage and the high fluid flow often seen with many
solid tumours.11 Tumour vasculature enables nanoparticles to accumulate within tumour
tissue without the addition of specific targeting moieties to the nanoparticle surface for
tumour recognition.82 Animal studies have shown that a 50-fold increase in nanoparticle
accumulation can be achieved through this passive process when compared with healthy
tissue.83 Hence, to optimize uptake due to the passive process of the EPR effect, an
important characteristic of nanoparticle systems is to ensure long blood circulation
times to avoid nanoparticle removal before accumulation can occur. Both nanoparticles
and liposomes are known to be rapidly recognized and cleared from the blood by cells
of the mononuclear phagocytic system (MPS), particularly macrophages present in the
liver and spleen.84-87 As alluded to earlier, nanoparticle removal by macrophages is
initiated by interactions with the hydrophobic nanoparticle surface and plasma opsonin
proteins including immunoglobulins, albumin and fibronectin in a process known as
opsonization.52, 80 These plasma proteins are recognized by specific receptors on
macrophages, which then phagocytose the nanoparticles (as discussed in detail in
section 1.3).86, 87
22
Figure 1.6. Schematic representation of nanoparticle active and passive targeting via
the EPR effect. The schematic demonstrates the increased ‘leaky’ vasculature consistent
with a tumour’s rich vascular network in comparison to that of normal healthy tissue.
Figure and caption from McNeil et al. 2010.88
Studies have shown that through alteration of the nanoparticle surface with hydrophilic,
flexible, and non-ionic polymer chains, avoidance of macrophage removal or ‘stealth
like’ nanoparticles can be produced.89 The coating of nanoparticle surfaces with PEG, a
process sometimes referred to as PEGylation, has been extensively used in providing
nanoparticles with a ‘stealth’ like coating.90 The addition of neutrally charged
hydrophilic polymer chains to a nanoparticle’s surface can provide a means for avoiding
phagocytosis and recognition by macrophages, and in turn experiencing longer
23
circulation times.90, 91 Improved circulation times enhance the EPR effect as generally
the longer the nanoparticle circulation time the greater the EPR induced nanoparticle
accumulation experienced within tumour tissue.92 Due to the large variability seen
between tumours, it is difficult to assess the optimum nanoparticle surface charge and
size of nanoparticles to best exploit the EPR effect. However, generally nanoparticles
on the scale of 10-100 nm typically demonstrate the most effective tumour uptake,93
although reports also show nanoparticles of 400 nm penetrating tumours as a result of
the EPR effect.81 Passive targeting for nanoparticles via the EPR effect still face some
challenges. The longer circulation times of drug loaded nanoparticles can lead to
adverse effects, as has been seen with DOXIL, which can cause severe hand-foot
syndrome (Hand-foot syndrome causes redness, swelling, and pain on the palms of the
hands and/or the soles of the feet similar to severe sunburn. Sometimes blisters may
also appear).94 Further to this, recent studies have shown that repeated doses of PEG
coated particles (including liposomes, nanoparticles and micelles) results in activation
of the accelerated blood clearance (ABC) phenomenon where an increased immune
response is generated towards the PEG-conjugated systems resulting in accelerated
clearance and reduced efficacy of PEG-conjugated systems in repeated dosing.95, 96 This
is an area requiring further investigation especially with the increasing number of PEG
conjugated drug delivery systems obtaining FDA approval and in the latter stages of
clinical trials. In addition to this, the tumour vasculature is highly dependent on tumour
type and age of the tumour and hence the EPR effect is not suitable for all tumours or
all tumour stages of development.97 The heterogeneous nature of tumours emphasizes
the need to identify and develop alternate targeting strategies to enhance the
effectiveness of particle based delivery systems and therapies.11, 97
Active targeting of nanoparticles has the ability to further complement the EPR effect.
Active targeting can be achieved through the addition of proteins, ligands or antibodies
to the nanoparticle surface to exploit receptors and other signaling molecules associated
with diseased states. Targeted nanoparticles possess major advantages in drug delivery
such as the potential for lower dosing, reduced systemic toxicity and the potential for
the safe delivery of more potent therapeutics. These moieties with corresponding
specific receptors in target tissue can greatly enhance specific cell uptake of a
nanoparticle vehicle and its subsequent drug payload.10 One example is a study
conducted by Poon et al. where the authors found folate targeted paclitaxel-loaded
micelles resulted in a significant increase in tumour accumulation and retention when
24
compared to non-targeted micelles.98 There was a four-fold increase in the efficiency of
paclitaxel when delivered in the targeted nanoparticle system while also significantly
reducing in vivo toxicity of the chemotherapeutic treatment.98 Folate is an attractive
targeting moiety for cancer with folate receptors, responsible for delivery of folic acid
into cells, showing a 100 to 300-fold overexpression in a wide spectrum of cancer
cells.99 Furthermore, the application of active targeted nanoparticles for the treatment of
cancer has an added advantage with regards to the treatment of small metastases (<100
mm3), as these sites are poorly vascularized and do not evoke a significant EPR effect
suitable for passive nanoparticle targeting.88
However, despite the significant upside to nanoparticle targeting, both passive and
active, there are still major problems to be overcome. This is further supported by the
fact that currently only a handful of targeted formulations have made it into clinical
trials with none so far being clinically approved despite targeted nanoparticles being
around for some decades now.10 The added difficulty and time during synthesis required
for the addition of ligands, the potential for off target effects if the ligand-receptor pair
is not highly specific and also increased monetary expense are all valid reasons to
question the balance of cost to benefits when discussing targeted nanoparticles.
Furthermore, the attachment of targeting moieties can compromise the stealth
capabilities of the nanoparticles and in turn accelerate their clearance by the host
through recognition by opsonin proteins.10, 100 It has been shown that non-targeted
liposomes can achieve comparable tumour accumulation to that of folic acid conjugated
liposomes as they benefit from higher circulation times and in turn a longer period of
accumulation resulting from the EPR effect.100 Hence, the addition of targeting moieties
has the potential for producing very selective drug delivery vehicles however substantial
consideration must be given to the choice of targeting ligand and in turn the estimated
costs and benefits of its use in a given application.
1.4.2 Imaging agents and multifunctional nanoparticles
Recent trends in multifunctional nanoparticles have endeavoured to not only improve
pharmacokinetic properties of a therapeutic or increased blood circulation time and
targeting but to also look to include imaging agents within the nanoconstruct suitable
for imaging in a clinical setting. The introduction of magnetic resonance imaging (MRI)
25
contrast agents and fluorescent/optical-imaging probes are two of the most common
modifications that still have strong prevalence and applicability in disease diagnosis and
detection. Further to these two, positron emission tomography (PET), computed
tomography (CT), ultrasound (US) or single photon emission computed tomography
(SPECT) are all finding applications in nanoparticles developed for drug delivery.
Figure 1.7 shows the characteristics of each of these imaging modalities currently used
in the clinical field, displaying each mode’s advantages along with their intrinsic
limitations.16 The ability to use imaging tools to follow a nanoparticle in vivo has great
applications in drug delivery due to the ability to provide key information to help
physicians make better informed decisions regarding drug dosage, timing of drug
delivery, and drug choice resulting in a powerful addition to personalized treatment
strategies.10
Figure 1.7. Characteristics of imaging modalities currently used for biomedical
applications. Figure and caption from Lee et al. 2012.16
26
Furthermore, the integration of two or more imaging modalities, when chosen carefully,
can allow for the advantages of one imaging mode to overlap with the disadvantages of
another to produce an entity which is detectable and suitable for imaging across a range
of length scales, instrumentations, time points and resolutions. With this, nanoparticles
have the potential to overcome a common conundrum of modality selection in clinical
diagnostic imaging where the modalities with the highest sensitivities have relatively
poor resolutions, while those which can achieve high resolution often have relatively
poor sensitivity.101 Another important consideration further to the type of imaging
modality is the relative amount or dose of imaging or contrast agent. For example, PET
or fluorescent probes often only require low concentrations when compared to the doses
required for MR or CT imaging.16
PET imaging is a technique, which can potentially provide functional information about
a disease with high sensitivity. This complements a technique such as MRI or CT which
both offer high-resolution images for anatomical information. A recent study by de
Blower et al. combining PET with MRI demonstrated these benefits. Blower and
colleagues designed a novel bifunctional chelator to allow for radiolabelling of iron
oxide nanoparticles with 64Cu. In their study they demonstrated the ability for the
bifunctional chelator to bind 64Cu to Endorem®, a commercially available iron oxide
MRI contrast agent, and demonstrated the PET/MRI capabilities of this nanoparticle
conjugate in the detection of lymph nodes in vivo (Figure 1.8).102
27
Figure 1.8. Iron oxide nanoparticles for MR chelated with 64Cu for PET imaging. A)
Schematic representations of the conjugation between the bisphosphate (BP)-based PET
tracer [64Cu-(DTCBP)2] and the dextran-coated iron oxide for a dual-modality PET-
MRI agent. B) In vivo PET-MR images showing uptake of [64Cu-(DTCBP)2]-Endorem
in the popliteal (solid arrows) and iliac lymph (hollow arrows) nodes. T2 weighted MR
images before (left) and after (right) footpad injection of [64Cu-(DTCBP)2]-Endorem. C)
PET images from coronal (top), short-axis (bottom). D) Whole body PET-CT image.
Image and captions modified from Blower et al. 2011.102
CT imaging is a clinically relevant technique due to its affordable price, high spatial
resolution and unlimited depth. Accurate anatomical information can be determined
with CT generated reconstructed three dimensional images.16 In CT imaging, X-rays are
emitted from a focused source, which rotates around a subject placed in the centre of the
CT scanner. As X-rays pass through the subject, they are absorbed in inverse proportion
28
to the density of the subject’s tissue before being detected by detectors on the other side
of the subject.103 This information can then be used to produce high resolution
tomographic anatomical images by reconstruction through a series of back
calculations.104 One of the major downfalls of this technique however is its lack of soft
tissue contrast resulting in large amounts (gram quantities) of CT compatible contrast
agents (e.g. iodine often required).103 However, iodine has also been problematic as a
CT contrast agent due to its rapid renal clearance, poor sensitivity and issues
surrounding its toxicity. The use of gold nanoparticles has overcome some of these
problems due to their X-ray absorbing and fluorescent quenching characteristics. A
recent study looking to combine CT and fluorescence imaging made use of gold
nanoparticles conjugated with a Cy5.5 metalloproteinase (MMP) sensitive peptide,
which upon degradation produced a near infrared fluorescent signal (Figure 1.9A).105 In
vivo accumulation of the nanoparticles in the tumour resulted in dual imaging of the
tumours through the X-ray absorption of the gold nanoparticles providing CT contrast
and the evolution of Cy5.5 fluorescence upon the MMP degradation of the peptide for
optical imaging (Figure 1.9B-D).105
29
Figure 1.9. Gold nanoparticles for optical and CT imaging. A) Schematic diagram of
MMP peptide probe conjugated gold nanoparticles with glycol chitosan coating for
biocompatibility (MMP-GC-AuNPs). B) Cross-sectional CT images of tumour before
and after injection of GC-AuNPs. C) Near infrared fluorescent (NIRF) images of HT-29
tumor-bearing mice after injection of the MMP-GC-AuNPs with and without an
inhibitor of matrix metalloproteinase. D) CT/optical dual imaging of the HT-29 tumor-
bearing mouse model. Figure and caption modified from Sun et al. 2011.105
1.4.3 Magnetic resonance imaging
Magnetic resonance imaging (MRI) is primarily a diagnostic tool that allows non-
invasive visualization of organs and other structures within the body.106 In 1946 two
scientists (Felix Bloch and Edward Mills Purcell) through independent experiments
observed that when substances such as water or paraffin were placed in a strong
magnetic field and then barraged with magnetic oscillations at radio frequencies, they
would absorb and release energy.106 This was the beginning of Nuclear Magnetic
Resonance (NMR), which in turn resulted in the development of MRI for the imaging
of bodily tissues, a feat that resulted in both Bloch and Purcell receiving the Nobel Prize
30
in Physics in 1952. The NMR phenomenon, which is the foundation of MRI image
generation, is based on the interaction between an external magnetic field and nuclei,
such as 1H, which has a non-zero magnetic moment.107 When such nuclei are placed in a
large external magnetic field, they will either align parallel or antiparallel to the applied
magnetic field and will precess at the Larmor frequency as they align.107, 108 The Larmor
or precession frequency is the rate at which these nuclei wobble when placed in the
magnetic field and is directly proportional to the applied magnetic field which generates
the nuclear spin alignment.108 Although nuclear spins can align both parallel and
antiparallel to the applied magnetic field, parallel alignment with the magnetic field is
energetically favourable and hence a slightly larger fraction of spins align with the
field.108 The application of a radio frequency (rf) pulse perpendicular to the applied
magnetic field will result in further precessional motion, this time the nuclei will
precess about the axis of the rf pulse. Resonance between the frequency of the rf pulse
and the Larmor frequency can be achieved if these frequencies are equal. This
resonance results in a spiral motion of the nuclear spins into the plane, which is
perpendicular to the applied field.109 For example, if the applied field is orientated in the
z-direction, then a 90° rf pulse will force the nuclear spins into the xy plane. This pulse
will force all of the spins to be in phase with one another and also for the system to now
be in an excited state due to the addition of energy from the rf pulse.109
The key to MRI imaging is how this additional energy is released as the spins relax and
return to align with the initial applied magnetic field.107, 108 There are two mechanisms
for which this spin relaxation can occur, the first T1 relaxation is a result of spin-lattice
interactions (longitudinal) and T2 or T2* relaxations resulting from spin-spin
(transverse) relaxation.108, 110 These relaxation times vary greatly depending on the
immediate chemical environment surrounding the nuclei which is the reason that
differences in tissue provide slightly different contrast.110 However, often differences in
T1 or T2 of tissue alone are not great enough to notice certain anatomical differences,
tissue differences or changes in pathology. To improve the visibility of abnormal
pathology in MRI, contrast agents are applied to shorten relaxation times and in turn
improve contrast between tissues.111
31
1.4.4 Magnetic resonance contrast agents
The use of contrast enhancing agents has become an integral part of MR imaging and its
application in a clinical setting.111 Under most conditions, differences in longitudinal
and transverse relaxation times are usually high enough to provide sufficient contrast in
MR images. However, some pathological conditions do not display sufficient
differences in tissue to clearly discriminate from surrounding healthy tissue. All MR
contrast agents work by shortening the T1 or the T2 relaxation times of the target tissue
and as a result they are often classified as ‘T1 agents’ or ‘T2 agents’ depending on the
signal which is predominantly influenced.111 The ability for these agents to reduce T1
and T2 are described by the r1 and r2 relaxivity values of the agent respectively. A higher
relaxivity means the greater the effect of the contrast agent on nearby nuclear spins and
thus the faster the relaxation time observed.108, 111 MRI contrast agents can be based
broadly into two main categories, those based on paramagnetic materials which mainly
induce a shortening of the T1 relaxation signal and superparamagnetic materials which
produce a more pronounced affect on the T2 relaxation times.
Metal ions with one or more unpaired electrons are considered to be paramagnetic and
as a result have a permanent magnetic moment.111 Gd3+ contains seven unpaired
electrons and due to this is the most popular choice as a T1 contrast agent with
significant research also being conducted on Mn2+.108 Although these ions are able to
produce high r1 relaxivities, their use must be carefully considered due to the high
toxicity these elements produce in their ionic form and hence they must be chelated or
chemically bound in a nanoformulation for safe use as contrast agents.111 Despite this,
there are a range of contrast agents based on gadolinium currently on the market
including Magnevist®, Dotarem® and Gadovist® which are approved for clinical use.
Agents that shorten the T2 relaxation times usually consist of iron oxide nanoparticles
with magnetite (Fe3O4) and maghemite (γ-Fe2O3) both seen as the most popular
candidates for this application. Nanoparticles of iron oxide can consist of several
thousand magnetic ions and as a result of this are said to have superparamagnetic
properties if these magnetic ions within the particle are aligned.111, 112 If the magnetic
moments of the iron ions within the nanoparticle are mutually aligned this will result in
a permanent net magnetic moment for the nanoparticle which when exposed to a
magnetic field is very large.111 Iron oxides are considered to be advantageous due to
32
their relatively low toxicity with the majority of these particles being endocytosed by
Kupffer cells (specialized macrophages of the liver) where they are degraded within
lysosomes within approximately 7 days.111 Sinorem®, Feridex®, Resovist® and
Endorem® are all examples of commercially available MR contrast agents based on
iron oxide nanoparticles.
Recent studies have endeavored to combine both T1 and T2 contrast agents in a single
construct to produce nanoparticles suitable for the enhancement of both imaging signals.
A study by Bae et al. synthesized gadolinium labeled magnetite nanoparticles (GMNPs)
through a bio-inspired method to be used as dual T1 and T2 weighted contrast agents in
MRI.113 The success of these dual contrast agents was demonstrated in vivo in mice
when their contrast was compared to commercially available contrast agents for T1 and
T2 weighted MRI respectively (Figure 1.10).113
Figure 1.10. A) T1-weighted and B) T
2-weighted magnetic resonance images of a
mouse injected with Feridex® and Magnevist® (the orange arrows indicate the
injection sites of Feridex®, and the green arrows indicate the injection sites of
Magnevist®). C) T1-weighted and D) T
2-weighted magnetic resonance images of a
mouse injected with GMNPs and the hydrogel solution as a control (the white arrow
indicates the injection site of the hydrogel solution, and the blue arrows indicate the
injection sites of GMNPs). Figure and caption modified from Bae et al. 2010.113
33
1.4.5 Fluorescent probes for biological imaging
Fluorescence microscopy still stands as one of the most common and powerful
technique for both in vitro and in vivo imaging due to the ability to image intracellular
events and differing tissues with high specificity. The synthesis of traditional organic
dyes and fluorescent quantum dot nanoparticles has seen these two imaging probes be
developed to have emission spectra which cover the entire visible spectrum as well as
into the near infrared region.114 Organic fluorophores, the most commonly used imaging
probes in biology, suffer from fast photo bleaching and broad sometimes overlapping
emission and excitation spectra.115 This limits the application of these probes for long-
term imaging and/or when used as one of multiple probes due to the spectral overlap.
Colloidal semiconductor nanoparticles, more commonly known as quantum dots, are
robust bright fluorescence emitters with size dependent emission wavelengths. The
extreme brightness of these nanoparticles and resistance to photo bleaching make them
ideal candidates for long term imaging requirements such as the acquisition of z-stacks
or 3D reconstruction imaging.115 The size dependent, narrow and tunable emission
wavelength is also advantageous in the application of these imaging probes for
multispectral imaging as they can often be tuned to avoid overlap unlike that of the
traditional organic dyes.115 However, despite the considerable upside to the use of
quantum dots for biological imaging concerns have been raised with regards to their
toxicity with the majority of quantum dot cores usually containing highly toxic elements
such as cadmium and selenium.115 Care must be taken to ensure proper capping of these
cores is achieved with biocompatible coatings such as ZnS (a common capping agent)
to ensure these toxic elements do not leach.115
Another interesting fluorescence imaging tool is that of fluorescent proteins. Naturally
fluorescent proteins have become an incredibly useful tool for biologists and
biochemists alike especially in the fields of cancer research, neuroscience and drug
delivery. These proteins have allowed researchers to visualize important aspects of
cancer in living animals, including tumour cell mobility, metastasis and angiogenesis all
in real time.116 Fluorescent proteins of many different colours have now been
characterized which in turn can be used to label cells of specific genotypes or
phenotypes.116 This allows for single cell resolution and the ability to easily
differentiate between a range of different biological processes. One area where this is
finding great promise is that of neuroscience, for the mapping of synaptic connections.
34
Mapping neural circuits to learn how these account for mental activities and behaviours
and more importantly how alterations to this circuitry can ultimately result in
neurological or psychiatric disorders.117 Recently, Livet and colleagues have developed
a technique that could allow neurologists to draw a detailed wiring plan of the
mammalian brain through the insertion of genes coding for a range of fluorescent
proteins in mice.118 This technique nicknamed ‘brainbow’ can reveal individual neurons
within the nervous system in high resolution and up to 160 distinctly different colours
(Figure 1.11).117, 118 This differential expression allowed researchers to map glial
territories and follow glial cells and neurons independently over time in vivo.118
Figure 1.11. A) A motor nerve innervating ear muscle. B) An axon tract in the
brainstem. C) The hippocampal dentate gyrus. In the Brainbow mice from which these
images were taken, up to ~160 colours were observed as a result of the co-integration of
several tandem copies of the transgene into the mouse genome and the independent
recombination of each by Cre recombinase. Figure and caption modified from Lichtman
et. al. 2008.117
1.4.6 Theranostic nanoparticles and the combination of imaging and treatment together
Recent trends in multifunctional nanoparticles look to combine both the therapeutic
delivery capabilities of nanoparticles with that of imaging modalities for diagnostic
purposes. Further to this, research has focused on ‘theranostic agents’ where the aim is
for the nanoparticle itself to be used for both the treatment (therapy) and imaging
(diagnosis) of disease.16, 119 Theranostic particles also have the potential to provide
information surrounding the localization of drugs, direct visualization of
pharmacokinetics and clearance, which can in turn provide direct insight into the
differences evident between patients and diseases. One example of a theranostic
nanoparticle would be the use of iron oxide nanoparticles not only for MRI contrast but
35
also for thermal therapy.120 Similarly the use of gold nanoparticles as contrast agents in
CT imaging and radiation therapy or gold nanoparticles for the enhancement of X-ray
imaging combined with thermal ablation therapy have both been achieved with success
in vivo.121 Questions have been raised with regards to the level of contrast achievable
with theranostic nanoparticles when compared to that of state of the art contrast agents
currently in clinical use as often the dosage for appropriate therapy will differ from that
required for imaging enhancement. However, despite this, theranostic nanoparticles
provide an exciting area of research going forward.
1.5 Assessing nanoparticle toxicity
The assessment of nanoparticle toxicity is of paramount importance for all
nanoengineered materials not only for those prepared for intended biomedical
applications. Due to the infancy of this science, the effects of nanoparticles on human
health in general, as well as on the environment and ecosystems which these
nanoparticles can potentially inadvertently reach, is integral for the future of the
technology. The evolution of nanoparticle synthesis and technology has historically
developed faster than testing and protocol development for the assessment of
nanoparticle toxicity. This however is changing with the ever-increasing interest in the
field of nanotoxicology, which deals with the assessment of nanoparticles for their
toxicity and environmental effects. An evaluation of this rapid growth is provided in
Figure 1.12 where it is clear that the rate of peer reviewed publications mentioning the
term ‘nanotoxicology’ has undergone exponential growth over the past decade.
36
Figure 1.12. Number of citations recorded per year (2002-2012) for the search term
‘nanotoxicology’ as assessed in Google scholar.
A recent report published by the US National Science Foundation in conjunction with
the US Environmental Protection Agency identified five critical areas of risk associated
with manufactured nanoparticles.122 These five critical areas include:
1. Exposure measurement and assessment of manufactured nanoparticles.
2. Toxicology of manufactured nanoparticles.
3. Ability to extrapolate manufactured nanoparticle toxicity using existing particle
and fiber toxicological databases.
4. Environmental and biological fate, transport, persistence, and transformation of
manufactured nanoparticles.
5. Recyclability and overall sustainability of manufactured nanomaterials.
One of the main reasons for nanotoxicology lagging behind the production of
nanoparticles is due to the difficulty of accurately predicting toxicity of specific
nanomaterials. Toxicity is highly dependent on the dose, exposure and pathway of
cellular entry of the nanoparticles not to mention the array of computations possible for
nanoparticles with regards to composition, size, shape, structure and morphology all
adding the potential of variation.123
37
The most common approach to assess the toxicity of nanoparticles and nanomaterials
alike is through in vitro toxicity tests for cell viability or by indirectly measuring cell
numbers through the use of cell metabolism assays such as the MTT or tetrazolium-
based assays.123 However, these in vitro assays only provide preliminary data and to
better understand the toxicology including distribution, fate and clearance of
nanoparticles from a biological system, in vivo comparisons are required.
Two recent studies investigating comparative toxicological assessments of single walled
carbon nanotubes (SWCNT) in rodent models found that the severity of the induced
pulmonary granulomas followed a dose dependent response.124, 125 Current material
safety data sheets classify SWCNT as “a new form of graphite”. However, results from
these studies would suggest that simply extrapolating exposure limits from those set out
for graphite would not be sufficient for protection against SWCNT exposure.122 With
the above in mind, it is clearly evident that before practical applications of nanoparticles
for diagnosis or therapeutic purposes in humans, comprehensive assessments for
potential toxicity of the nanoparticles must be carefully considered.16
1.6 Summary of the literature and thesis rationale
It is evident that a plethora of nanoparticle constructs have been developed for the
biomedical industry including areas such as drug delivery, therapy, diagnosis and
biological imaging. Advantages and disadvantages of a particular construct depend on
the choice of material used to formulate the nanoparticle and the intended final use.
Questions remain with regards to bionanotechnology and the safety of engineered
nanomaterials and their incorporation into clinical settings. Hence, it is important that
any undertaking involving the generation of engineered nanoparticles should
incorporate toxicology assessments of these constructs. Furthermore, nanoparticles
developed for drug delivery applications must incorporate some form of probe suitable
for imaging in a clinical setting. This is of paramount importance especially with the
newfound ability to deliver potentially more potent drugs within nanoparticles, which
were once unsuitable due to solubility issues, to be certain of the site of delivery. The
concept of nanoparticle targeting is an interesting one, which requires further
exploration. The attachment of targeting moieties and ligands to actively target
nanoparticles has been very successful in achieving site specific nanoparticle delivery.
38
Similarly, long circulating nanoparticles without targeting ligands, which make use of
passive uptake mechanisms such as the EPR effect, have also shown promise. The
potential for the delivery and rapid transfection of the nanoparticles and cargo at the
desired treatment site may also be an effective delivery strategy. Finally, a nanoparticle
construct developed for drug delivery must have the potential for encapsulation,
protection and delivery of a range of therapeutics while achieving high levels of
therapeutic loading. It is with these criteria in mind that the nanoparticles and work in
this thesis was developed.
Multifunctional polymeric nanoparticles for drug delivery are an exciting area of
research. The use of polymers to form nanoparticles provides structural rigidity and
integrity to the nanoparticle allowing for therapeutic protection and integrity when
compared to other nanoparticle delivery methods. Further to this, the ability to easily
modify the nanoparticle chemistry for the attachment of ligands and imaging probes
make polymeric particles an ideal core structure to build from. The choice of polymer
core is important to allow for fast and easy surface modification to occur. Poly(glycidyl
methacrylate) (PGMA) is an ideal choice, the epoxide functionality of this polymer
being well suited for epoxide ring opening reactions, thus making the addition of
functionalized ligands and imaging probes straightforward.
PEI is a polyplex, which has shown to be able to enhance cellular uptake of a range of
therapeutics both in vitro and in vivo, however issues remain surrounding free PEI and
its toxicity. Covalently binding this polymer to a polymeric surface of a nanoparticle
has allowed for the excellent transfection capabilities to remain without the inherent
toxicity associated with free PEI. Furthermore, polymeric nanoparticles provide an ideal
platform for the incorporation of multiple imaging probes, a limiting factor of other
nanoparticle technologies where functionality is an issue.
The emulsion technique used to synthesize polymeric nanoparticles is well suited to the
incorporation of hydrophobic drugs and therapeutics as well as the incorporation of PEI
to the nanoparticle surface. PEI can enhance the electrostatic attachment of a range of
biologically relevant payloads including plasmid DNA, peptides and proteins to the
nanoparticle surface. From the reviewed literature it is clear that nanoparticles
synthesized for drug delivery should aim to contain a means for cellular transfection,
high drug loading, therapeutic release from nanoparticles and also means for imaging
39
and nanoparticle tracking. In this thesis such a nanoparticle has been synthesized and
characterized; and data describing their use in a range of applications are described.
1.7 Introduction to series of chapters
The multifunctional PGMA nanoparticles developed and used throughout this thesis
were developed for the delivery of a range of therapeutics including small molecule
drugs, peptides, DNA and proteins to a range of different injury models. Throughout
this project the nanoparticles were tested in a variety of disease models including
delivery of a therapeutic peptide for the alleviation of cardiac ischemia-reperfusion
injury and for gene delivery in models of both breast and colon cancer. Chapter Two,
the first to contain experimental work, explores the synthesis of two similar multimodal
nanoparticle systems, and the subsequent characterization of these nanoparticles. Herein,
a cationic PGMA nanoparticle coated in PEI and a neutrally charged PEGylated PGMA
nanoparticle will be presented along with the initial toxicity data and cellular uptake
data showing key differences between both systems.
Chapter Three describes the application of the nanoparticles for therapeutic delivery.
The PEI coated multimodal PGMA nanoparticles were used for the delivery of a
therapeutic peptide designed to act on the L-type Ca2+ channel to help alleviate cardiac
ischemia-reperfusion injury by regulating channel function. Data presented in this
chapter include therapeutic peptide loading onto the nanoparticles as well as
comparative assessment of the delivery of peptide loaded on the nanoparticles with that
of the therapeutic peptide bound to the TAT peptide which has well known cell
penetrating capabilities.126 Comparisons made include cellular uptake efficiencies in
cardiac myocytes, biodistribution in cardiac tissue, effects on intracellular calcium
levels assessed in vitro and finally efficacy of preventing heart tissue damage in an ex
vivo model of cardiac ischemia-reperfusion injury.
Chapters Four, Five and Six delve into the use of the PEI coated multimodal PGMA
nanoparticles for their application as enzyme stabilization agents with the aim of using
the nanoparticles for the delivery of the therapeutically relevant chondroitinase ABC
(chABC). This enzyme has been shown in vivo127, 128 to have success in degrading
chondroitin sulfate proteoglycans (CSPGs), an important class of molecules that are
upregulated at the time of a central nervous system (CNS) injury as a result of the
40
formation of dense glial scar tissue around the primary injury site, which is rich in
CSPGs.129 The delivery of chABC to CNS injury has been shown to degrade the
inhibitory CSPGs, breaking down the glial scar and in turn producing an environment at
the site of injury more permissive to axon regeneration and plasticity, two factors seen
as integral to improving function post CNS injury.130 However, this enzyme has been
shown to have poor thermal stability at 37 °C,131 thus providing the rationale for use of
nanoparticles as both a stabilization agent and delivery vehicle for this enzyme in CNS
injuries. As a proof of concept, the use of nanoparticles as enzyme stabilization agents
was first investigated with industrially relevant enzymes with strong results observed
for the stabilization of all three test enzymes chosen, a β-glucosidase, β-galactosidase
and acid phosphatase. This proof of concept work is presented in Chapter Four.
For stabilization studies with the therapeutically relevant enzyme chABC, it was first
important to develop a suitable spectrophotometric activity assay for this enzyme as
previous methods used were indirect and only semi-quantitative in nature. Chapter
Five presents data on the assay development for an accurate, cheap, quick and easy
spectrophotometric activity assay suitable for measuring both chABC activity and
kinetics. This newly developed assay was compared to previous methods used for the
detection of chABC activity to show its enhanced capabilities and was also used for the
work presented in the final chapter. Chapter Six investigates the addition of
stabilization agents with chABC. Unfortunately, the significant stabilization seen with
the industrially relevant enzymes and the nanoparticles (presented in Chapter Four) did
not translate to imparting thermal stability on chABC. A range of other additives were
also trialed, however to no avail. Covalent attachment of the enzyme to the nanoparticle
was also assessed. However, due to the instability of the enzyme with regards to pH
fluctuations away from physiological pH this was also abandoned. Finally, data
presented show that the chABC without additives can actually persist with activity on
time scales out to six weeks. The chapter finishes with discussion about the future use
of this enzyme, the potential for translation to the clinic and also the need for its
stabilization.
Finally, Chapter Seven explores the use of nanoparticles as gene delivery vehicles in
models of both colon and breast cancer. The advent of RNA interference technology for
the treatment of cancer, where short interfering RNA (siRNA) is delivered to
specifically knockdown genes, has revolutionized how scientists look to treat the
41
disease. However, siRNA alone has poor transfection efficiencies and hence requires
some form of delivery vehicle to aid its cellular uptake. In this chapter, investigation
into the use of PEI coated multifunctional PGMA nanoparticles for the delivery of
shRNA for gene regulation in colon and breast cancer is presented. These data include
the in vitro transfection of the nanoparticles in a range of cell lines, in vitro
confirmation of target gene knockdown with shRNA delivered by the nanoparticles and
finally in vivo assessment in relevant mouse models of both breast and colon cancer
where the tumour burden and survivability have both been monitored accordingly. It
was anticipated that the delivery of shRNA was more biologically stable then siRNA
and able to induce longer target gene suppression.
Each chapter will contain a specific introduction and review of the literature required to
give the reader relevant background to the chapter. This will be followed by the results
and discussion and then conclusions and future directions of the work presented in that
chapter as well as the appropriate methods used will be provided. Finally at the
conclusion of the chapters an overall conclusion and future directions of the general
strategy of polymeric nanoparticles for the application as therapeutic delivery vehicles
will be presented.
42
43
Chapter 2
Poly(Glycidyl Methacrylate) (PGMA) nanoparticle synthesis and characterization
Statement of Contribution: TDC played an integral part in the synthesis, characterization and
toxicity assessments of both multifunctional nanoparticle systems presented. The
nanoparticles with a polymer core of PGMA (Mw 200 kDa) were developed within the
laboratory during the term of 2010-2012 inclusive. Although the nanoparticles were
developed primarily for drug delivery from within the nanoparticle core, through my work,
strong evidence has been found to suggest that therapeutic attachment to the nanoparticle
surface and subsequent transfection is also a viable delivery mechanism. Below is a summary
of the PGMA nanoparticle system, highlighting the rationale behind the nanoparticle
composition, the characterization and general testing of these nanoparticle systems in
preparation for use as therapeutic delivery vehicles.
2.1 An introduction to polymeric nanoparticles for drug delivery
Polymeric nanoparticles made of natural or artificial polymers in which therapeutic agents can
be absorbed, dissolved, entrapped, encapsulated or covalently attached have been used
extensively in animal trials as drug delivery systems, with some of these more recently
undergoing clinical trials.132-135 The polymeric nature of the particle permits the attainment of
desired properties such as controlled and sustained drug release, allowing drug release at the
targeted site over a period ranging from hours through to days or even weeks.132, 134 Drug
release from particles can be mediated by desorption, diffusion through the polymeric wall,
polymeric wall degradation or any combination of these processes.133 The choices of polymer
for the synthesis of the nanoparticles are also vast and varied throughout the literature.
Considerations that need to be made include polymer biocompatibility, polymer degradation,
toxicity, chemical functionalities for further modifications and also ease of manufacturing and
44
synthesis. The most common choice of polymer for nanoparticle formulations is the FDA
approved biodegradable PLGA. This polymer has been successful for the development of
polymeric nanoparticles because it undergoes hydrolysis in the body to produce the
biodegradable metabolite monomers lactic acid and glycolic acid, both of which the body can
effectively remove.136
The incorporation of ‘smart polymers’ which have the potential to alter characteristics such as
swelling, pore size or degradation in the event of an external stimuli or even more importantly
a change in the pathophysiological conditions at the site of injury or disease has lead to some
exciting advances in this field.137-139 Compared to the relatively neutral pH found in many
healthy tissues (pH approximately 7.4), changes in pH are often seen with a different location
within the body whether it be the digestive tract (pH 1.0-8.2) or cellular packaging into
endosomes (endosomal pH 5.0-6.5). Even the presence of tumour cells or ischemia can result
in a reduction in pH (pH 6.5-7.2) from physiological values.140 One strategy employed for
triggered delivery from polymer nanoparticles is to take advantage of changes in polymer
protonation states that can occur at differing pH values. Such a change can transform an
insoluble and predominantly hydrophobic polymer into a hydrophilic charged and completely
water soluble polymer which will in turn readily release its therapeutic payload.140 An
example of this is the incorporation of carboxylic acid (RCOOH) functionalities. In neutral or
basic pH carboxylic acid functionalities often present as their conjugate base (RCOO-) and
will begin to lose this charge as the media becomes more acidic. Oppositely, the use of amino
groups which present with a positive charge in acidic conditions will be rendered neutral as
the media becomes more basic, a phenomenon which has great application in targeted drug
delivery.140 Potineni et al. prepared 100-150 nm paclitaxel loaded poly(beta-amino ester)
polymeric nanoparticles which exhibited pH responsive changes in solubility in the range of
pH 6.5-7.4 and demonstrated a high paclitaxel loading efficiency coupled with rapid delivery
of this payload to the slightly acidic cytoplasm of BT-20 human breast cancer cells.141
Similarly a study by Rehor et al. published in 2004 demonstrated the effectiveness of reactive
oxygen species as a potential trigger for drug release. In this study nanoparticles synthesized
from poly-(propylene sulfide) were shown to be solubilized in the presence of reactive
oxygen species such as hydrogen peroxide.142 This allowed for the direct release of cargo
from the nanoparticles in the presence of oxidative stress, a significantly prevalent marker of
injury and disease across all organs.142 Throughout this thesis, a range of multifunctional
polymeric nanoparticles were prepared. Herein Chapter Two, the synthesis of two of these
45
systems will be outlined. Although these nanoparticles are similar in structure they were
designed with very different applications in mind.
2.2 Multimodal PGMA nanoparticles with a PEI functionalized surface
PGMA is an ideal polymer for the core structure of polymeric nanoparticles. This is a result
of the ease with which it can be manufactured into nanoparticles making use of the ‘oil in
water’ emulsion technique as well as the ease of which the core can be modified due to the
high presence of the epoxide functionality in the polymer chain (one epoxide per monomer
unit). Epoxide ring opening reactions have been extensively used for the linkage of other
polymers and fluorophores, for substrate binding and in biochemistry for the linkage of
proteins and ligands to epoxide rich surfaces through immobilization, making it an ideal
candidate for a polymeric nanoparticle delivery system.143, 144 Before the nanoparticle was
produced by the ‘oil in water’ emulsion process, the polymer was pre-modified by covalently
attaching a fluorescent dye, Rhodamine B (RhB), to PGMA according to previously reported
procedures (detailed methods of nanoparticle preparation are included at the end of this
chapter).143
The covalent attachment of the RhB to the nanoparticle core rendered the nanoparticles
fluorescent, providing a cheap and highly accurate mode of imaging to track the nanoparticles
both in vivo and in an in vitro setting. The choice of fluorescent probe was based on the
ability to bind RhB directly to the PGMA through a ring opening reaction between the
epoxide groups of the PGMA and the free carboxyl group of the RhB. The attachment of RhB
to the nanoparticle core resulted in the excitation peak narrowing and being red shifted in
comparison to free RhB in solution. Similarly the emission peak also narrowed upon
conjugation to the nanoparticle cores however it did not appreciably move with regards to
peak position (Figure 2.1). RhB is also relatively cheap in comparison to other fluorescent
dyes with suitable chemical functionalities for PGMA attachment making this compound an
economical and ideal choice for the polymer modification. The modified polymer was
purified through a series of precipitations before being used in the synthesis of polymeric
nanoparticles by making use of the ‘oil in water’ emulsion method. The RhB modified
polymer, magnetite and other agents required for encapsulation (drugs or therapeutics) are
dissolved in an organic mixture which is added drop wise to a vortexing aqueous solution.
The aqueous phase contains Pluronic F108, a block copolymer of PEG-poly(propylene
glycol)-PEG to act as a stabilizing agent to aid in the formation of a stable emulsion.
46
After the addition of the organic phase, ultrasonic waves of high power are passed through the
solution using an ultrasonic hand held probe tip to further disperse the oil emulsion,
producing nanoparticles of the preformed polymer containing the encapsulated magnetite and
potentially any hydrophobic therapeutic if desired. These nanoparticles are then slightly
heated and stirred under a steady stream of nitrogen to evaporate the organic solvents away
from the mixture leaving the fluorescent and magnetic nanoparticles within an aqueous
solution of pluronic F108.
Figure 2.1. Comparison of the fluorescent excitation (red dashed) and emission (blue dashed)
spectra for rhodamine B dye conjugated to PGMA nanoparticles, which contained both
magnetite and PEI modification, suspended in water to that of the fluorescent excitation
(purple) and emission (black) of native rhodamine B (RhB) in water.
In order to produce nanoparticles with multimodal imaging characteristics magnetite (Fe3O4)
nanoparticles are encapsulated within RhB modified PGMA polymer spheres during the
emulsion process to provide a magnetic core to the nanoparticles. The presence of magnetite
serves two purposes. The first is for imaging as an MRI contrast agent for in vivo tracking of
the nanoparticles. The magnetite produced for polymer entrapment was synthesized following
a procedure outlined by Sun et. al. for the production of magnetite nanoparticles with an
approximate size of 6 nm.112 Through the reaction of iron(III) acetylacetonate with surfactants
47
at high temperatures it was possible to produce monodispersed Fe3O4 nanoparticles which
could be easily purified and collected from the high boiling point solvent used for reflux.112
Sun et. al. found that reflux at lower temperatures produced smaller Fe3O4 nanoparticles
suggesting that high reaction temperatures produce larger nanoparticles.112 The use of iron
oxides as T2 MRI contrast agents is well documented in the literature with commercial
contrast agents such as Resovist® and Endorem® already available on the market for clinical
use.145
Characterization of the magnetic properties of the encapsulated magnetite within the RhB
modified PGMA nanoparticles was achieved using Superconducting Quantum Interference
Device (SQUID) magnetometry. SQUID magnetometry is considered the most sensitive
technique for the measurement of changes in magnetic flux, making it an ideal instrument for
the magnetic characterization of the encapsulated magnetite within the nanoparticles.146
SQUID magnetometry measurements found the encapsulated magnetite retained its
superparamagnetic properties, with no magnetic hysteresis at 300 K (with specific saturation
magnetization of 6 emu g-1) (Figure 2.2). Although entrapped within a polymer sphere the
magnetite still possessed all the same magnetic characteristics expected of a
superparamagnetic Fe3O4 nanoparticle with hysteresis evident at 5 K (Figure 2.2A) but not at
room temperature (Figure 2.2B).112 The zero field cooled/field cooled curves show an
estimated blocking temperature of approximately 50 K which is also consistent with values
found in the literature for magnetite nanoparticles in similarly applied magnetic fields (Figure
2.2C).147 The transverse relaxivity (r2) of the nanoparticle was determined based on the iron
content inside the polymer, to be 340 s-1 mM-1 Fe.65
Besides MRI contrast enhancement the second purpose of the magnetite in the polymeric
nanoparticles is that it provides an alternative means to separate, wash and concentrate the
nanoparticles using a magnetic fractionation column instead of the more conventional
centrifugation techniques.148-150 This allows for faster collection of clean nanoparticles
suitable for further modification or application. The incorporation of the Fe3O4 nanoparticles
also provides the potential for magnetic maneuverability of the nanoparticles, an attribute well
suited to the site-specific delivery of therapeutics.
48
Figure 2.2. Magnetite characterization. SQUID magnetometry of magnetite particles
portraying superparamagnetic behavior. A) Hysteresis loop at 5 K. B) Hysteresis loop at 300
K, displaying no hysteresis. C) Zero field cooled/field cooled (ZFC/FC) curves are coincident
at temperatures above 50 K. D) TEM image of magnetite sample, scale bar 50 nm.
The final addition in the nanoparticle synthesis is the surface modification of the polymer
spheres. PEI is an amino functionalized polymer well known for its ability to complex DNA
and act as a suitable transfection vector to transport a range of therapeutic cargoes. The amino
functionality of PEI allows for covalent attachment of PEI to PGMA through a ring opening
reaction of the PGMA epoxide group. This was achieved at elevated temperatures (70 °C) in
aqueous solution with an excess of PEI added to ensure good surface coverage.
After PEI attachment the nanoparticles were washed to ensure removal of excess unreacted
PEI and the pluronic F108 stabilizing agent by firstly magnetically trapping the nanoparticles
on a magnetic separation column, washing with milli-Q water and eluting the nanoparticles in
milli-Q water for storage and further characterization. These nanoparticles when characterized
by transmission electron microscopy (TEM) clearly show the magnetite nanoparticles
49
encapsulated within the PGMA polymer sphere (Figure 2.3A). The nanoparticles were further
assessed for size by dynamic light scattering (sometimes referred to as Photon Correlation
Spectroscopy) and for surface charge by zeta potential measurements. Dynamic light
scattering measures Brownian motion of the particles in a solution and relates this to the size
of the particles. This is achieved through illumination of the particles with a laser and then
measuring fluctuations in the intensity of the light scattered from the particles in the
system.151 Zeta potential measurements are made firstly by measuring the electrophoretic
mobility of a particle sample, which in turn can be used to calculate the zeta potential of the
particles from theoretical considerations.151 The nanoparticles formed from this emulsion
process have an average size of 160 nm (95% confidence interval 85-342 nm) as assessed by
dynamic light scattering (Figure 2.3B) and have a net positive surface charge resulting from
the PEI surface modification (Figure 2.3B and C). Due to the strength of amines as a
nucleophile and the large excess of amines available for attachment it was possible to get
good surface coverage of the nanoparticles with PEI post nanoparticle formation in aqueous
solution. This ensured that the attached PEI was surface bound to the nanoparticles, a result
supported by the dramatic shift in nanoparticle zeta potential before and after PEI attachment
(Figure 2.3C). Elemental analysis conducted on the PGMA-Mag-RhB nanoparticles both
before and after PEI attachment was conducted to determine the elemental composition and
amount of PEI bound to the polymeric nanoparticles. From these results it was found that the
nanoparticles with PEI attached to the surface contained 3.4% by mass PEI coating on the
nanoparticle surface (see Appendix A for calculations).
50
Figure 2.3. A) TEM assessment of nanoparticles, scale bar 500 nm with high magnification
inset (scale bar 50 nm), B) size distribution of polymeric nanoparticles as assessed by
dynamic light scattering and C) zeta potential of nanoparticles before (red) and after (blue)
PEI functionalization.
PEI is considered as the gold standard for synthetic polymers suitable for cellular transfection
and it was this fact that formed the basis of the initial reasoning behind PEI incorporation into
the nanoparticle system. However further work addressed in this thesis outlines the benefits in
using this positively charged exterior for the electrostatic attachment of negatively charged
cargoes and therapeutics suitable for transfection and in turn rapid release from the
nanoparticles as opposed to drug loading in the polymer core and subsequent slow drug
release by diffusion through the polymer walls.
51
2.3 Multimodal PGMA nanoparticles with a PEGylated surface
The PEGylation of nanoparticles to improve biocompatibility is well documented within the
literature, with the neutrally charged chains being able to mask the hydrophobic nature of
polymeric nanoparticles from recognition by opsonin proteins, and in doing so avoid
premature recognition and clearance from the body by the mononuclear phagocyte system
(MPS).90, 91 This strategy provides a nanoparticle system which, although will have poor cell
penetrating properties, will have enhanced circulation times in blood and can even still be
internalized by passive processes such as the enhanced permeability and retention (EPR)
effect seen in most cancerous tumour environments. Similarly to the PEI functionalization of
the PGMA nanoparticle core described in the previous section, the attachment of a carboxylic
acid functionalized PEG chain to PGMA can be achieved through an epoxide ring opening
reaction.
The attachment of PEG chains to the PGMA was to address a common concern seen with
cationic nanoparticles in that despite rapid cell internalization, they also have the potential to
experience rapid clearance and removal from the body through the MPS. The incorporation of
PEG into the nanoparticle construct produces a neutral nanoparticle surface which aids the
nanoparticles in avoiding recognition by macrophages and in turn undergoing phagocytosis
and clearance.51 The PEGylation of nanoparticles and other drug formulations has long been
shown to improve circulation times of nanoparticles based on this finding and hence the basis
for incorporation into our PGMA nanoparticle system92 (the experimental details for
achieving both PEG and PEI functionalization of the nanoparticles is outlined in full at the
end of this chapter). Due to the PEG chain functionalization with a carboxylic acid (weak
nucleophile) it was decided to pre-attach this to PGMA using methyl ethyl ketone as solvent.
The attachment of PEG to PGMA to produce PEG decorated PGMA polymer was quantified
by 1H-NMR (see Appendix B for 1H-NMR spectra). The attachment of PEG was confirmed
due to the evolution of the NMR signal at δ3.6 ppm consistent with the addition of PEG, with
the remaining signals in the spectra in agreement with those expected from PGMA.152 Once
this attachment was confirmed, the polymer was further modified with RhB according to
established procedures and the nanoparticles were synthesized once again by the ‘oil in water’
emulsion process making use of the RhB and PEG functionalized PGMA polymer as a
starting material and encapsulating once again magnetite within the nanoparticle core to
render the nanoparticles magnetic.
52
Figure 2.4. Comparison of the fluorescent excitation (red dashed) and emission (blue dashed)
spectra for rhodamine B dye conjugated to PGMA nanoparticles, which contained both
magnetite and PEG modification, suspended in water to that of the fluorescent excitation
(purple) and emission (black) of native rhodamine B (RhB) in water.
Once again the conjugation of the RhB dye to PGMA in the presence of magnetite and PEG
resulted in small differences in the rhodamine excitation and emission characteristics
analogous to what was evident with the PEI coated nanoparticles (Figure 2.4). Spherical
nanoparticles with the magnetite encapsulated within the polymer sphere were evident from
TEM analysis (Figure 2.5A). The size of the PEGylated nanoparticles was approximately the
same as the PEI coated nanoparticles with an average size of 148 nm (95% confidence
interval 81-246 nm) as assessed by dynamic light scattering (Figure 2.5B). PEGylation of the
nanoparticles resulted in a very slight decrease in zeta potential in comparison to the non-
modified PGMA nanoparticles (Figure 2.5C).
53
Figure 2.5. A) TEM assessment of nanoparticles, scale bar 500 nm with high magnification
inset (scale bar 50 nm), B) size distribution of polymeric nanoparticles as assessed by
dynamic light scattering and C) zeta potential of nanoparticles without (red) and with (blue)
PEG incorporation.
54
2.4 In vitro toxicity and cellular internalization studies
With the aforementioned synthesis and subsequent physiochemical characterization of both
nanoparticle systems it was important to assess the interaction of these nanoparticles in cell
culture. To assess the toxicity of both nanoparticle systems the immortalized PC12 cell line, a
cell line which is derived from a pheochromocytoma of the rat adrenal medulla was used.153
Both the PEI and PEG-coated PGMA nanoparticles did not elicit a toxic response in the
concentrations assessed, with the PEG-coated PGMA nanoparticles showing no significant
toxicity to concentrations as high as 1000 µg ml-1 after 24 hours of incubation (Figure 2.6).
Further experiments investigated cellular transfection of the nanoparticles and showed that
over a period of 24 hours the cationic PEI-coated PGMA nanoparticles were extensively
internalized within cells while the PEG-coated PGMA nanoparticles did not appreciably
appear to be internalized (Figure 2.7). It was anticipated that despite the PEG-coated PGMA
nanoparticles still exhibiting a slight positive surface charge (see figure 2.5C) that this was
insufficient to produce significant cellular membrane attachment and in turn resulted in
minimal cellular uptake. These findings support the hypothesis with regards to the high order
transfection abilities of PEI and the known ability of PEG to avoid cellular uptake.154, 155
Figure 2.6. Toxicity assessments of the A) PEI and B) PEG-coated PGMA polymeric
nanoparticle systems with no significant difference measured between controls (0 µg ml-1)
and those with nanoparticles after 24 h of incubation. Data are mean ± SE with n ≥ 500 cells
per nanoparticle concentration tested, per well (averaged across 5 wells per treatment).
Significance tested by a one way ANOVA and Bonferroni/Dunn post hoc tests, requiring a
significance of p ≤ 0.05.
55
Figure 2.7. A) PC12 cells with no nanoparticles (control) DIC image overlayed with Hoescht
nuclear staining, B) PC12 cells incubated with PGMA-Mag-RhB-PEI nanoparticles for 24 h
20 µg-ml-1 before washing and fixation, C) PC12 cells incubated with PGMA-Mag-RhB-PEG
nanoparticles for 24 h 20 µg-ml-1 before washing and fixation, all scale bars are 10 µm.
2.5 Conclusion
In conclusion we have successfully synthesized and characterized two polymeric nanoparticle
systems suitable for drug delivery. It is clearly evident that changing the polymer coating on
the surface of the PGMA nanoparticles by either the addition of the cationic PEI or the neutral
PEG altered the interactions of these nanoparticles with PC12 cells. Future experiments
described in this thesis made use of the PEI coated multimodal PGMA nanoparticles
described herein, Chapter Two. The PEG coated PGMA nanoparticles are currently under
further investigation with collaborators for efficacy in drug delivery and did not play a
significant role in the applications presented as a part of this thesis. The novelty of the PGMA
core of the nanoparticle system renders a highly adaptable and easily modifiable nanoparticle
construct with the ease of surface modification demonstrated herein. However, through
similar chemistry the potential to add targeting ligands, different imaging probes or
encapsulation of a range of contrast agents are all real possibilities yet to be explored.
2.6 Detailed methods of nanoparticle synthesis and characterization
2.6.1 Nanoparticle synthesis Materials
All chemicals were purchased from Sigma-Aldrich unless otherwise stated: benzyl ether
56
(99%), carboxylic acid terminated polyethyleneglycol (PEG-COOH, 5000 Da gift from
Igor Luzinov and Bogdan Zydyrko, Clemson University, Clemson), iron(III)
acetylacetonate (97%), oleic acid (BDH, 92%), oleyl amine (70%), Pluronic F108,
polyethylenimine (PEI 50% w/w solution, Mn 1200, Mw 1300), poly(glycidyl
methacrylate) (PGMA, Mn=230 kDa, Mw=470 kDa, PDI=1.96 was a gift from Igor
Luzinov and Bogdan Zydyrko, Clemson University, Clemson) rhodamine B (RhB,
Kodak, 95%), and 1,2-tetradecanediol (90%) were used as received.
Magnetite synthesis
Magnetite was prepared in accordance with the method described by Sun et al.112
Briefly, iron(III) acetylacetonate (2 mmol), 1,2-tetradecanediol (10 mmol), oleic acid (6
mmol), oleylamine (6 mmol), and benzyl ether (20 ml) were mixed with a magnetic
stirrer and gradually heated under a constant flow of N2. The mixture was held at
100 °C for ≈1 hour before being ramped to 200 °C, held for a further 2 hours and finally
heated to reflux (300 °C) and held for 1 hour under a blanket of N2. The sample was
allowed to cool to room temperature over night under N2 flow. The sample was
collected and purified through a series of precipitations with ethanol, collection via
centrifugation and then resuspension in hexane.
PGMA modification with RhB
PGMA (100 mg) and RhB (20 mg) were dissolved in ethyl methyl ketone (MEK) (30
ml) and heated to reflux under N2 atmosphere for 18 hours. The PGMA-Rhodamine
modified polymer was precipitated with diethyl ether and dried before use in
nanoparticle production.
PGMA modification with PEG-COOH and RhB
PGMA (100 mg) and PEG-COOH (60 mg) were dissolved in ethyl methyl ketone
(MEK) (30 ml) and heated to reflux under N2 atmosphere for 18 hours. The PGMA-
PEG modified polymer was cooled and RhB (30 mg) was added to the reaction mixture
before being heated again to reflux under N2 atmosphere for 18 hours. The PGMA-
PEG-RhB modified polymer was precipitated with diethyl ether and dried before use in
nanoparticle production.
Multimodal polymeric nanoparticle synthesis and PEI modification
Nanoparticles were prepared by an ‘oil in water’ emulsion process. The organic phase
57
contained magnetite nanoparticles (20 mg), dissolved PGMA-RhB (80 mg) in a 1:3
mixture of CHCl3 and MEK (6 ml). The organic phase was added drop wise to a
vortexing aqueous solution of Pluronic F108 (1.25% w/v, 30 ml) with the resulting
microemulsion homogenized with a probe-type ultrasonicator for 1 minute. Organic
solvents were allowed to evaporate under moderate stirring and N2 flow overnight.
Magnetite aggregates and unreacted polymer was removed via centrifugation (3000 x g,
45 min), with the supernatant being collected and incubated with PEI (50% w/w
solution, 100 mg) at 70 °C for 20 hours. (Note: nanoparticles produced from the
PGMA-PEG-RhB modified polymer did not undergo PEI addition). The PEI modified
polymeric nanoparticles with encapsulated magnetite were collected on a magnetic
separation column (LS, Miltenyi Biotec), washed with milli-Q water to remove excess
Pluronic F108 and unattached dye before being collected, aliquoted and stored. The
equivalent dry mass of samples was determined by freeze-drying.
2.6.2 Nanoparticle characterization Transmission electron microscopy (TEM)
Nanoparticle samples were prepared by deposition onto carbon coated grids and imaged
at 120 kV on a JEOL JEM-2100.
Dynamic Light Scattering (DLS) and zeta Potential measurements
Nanoparticle samples were thoroughly washed (3x ≈ 2 ml milli-Q water) while being
held on a magnetic separating column before being resuspended in milli-Q water for
analysis. DLS and zeta potential measurements were obtained on a Zetasizer Nano
series ZEN 3600 (Malvern Instruments).
Superconducting quantum interference device (SQUID) magnetometer
measurements
Magnetic properties of the magnetite as well as the PGMA-magnetite-RhB-PEI
composite nanoparticles were measured using a Quantum Design MPMS SQUID
magnetometer. For both samples hysteresis was measured at 5 K and 300 K; zero field
cooled and field cooled (ZFC/FC) measurements were also collected.
2.6.3 In vitro testing of nanoparticles Cell culture
Rat pheochromocytoma cells (PC12) were obtained from the Mississippi Medical
58
Center (Jackson, MS), cultured in poly-(L-lysine)-coated polystyrene flasks in a
humidified atmosphere containing 5% CO2 at 37 °C, and maintained in RPMI1640
medium containing horse serum (10% v/v), fetal bovine serum (5% v/v),
penicillin/streptomycin (100 U ml-1, 100 µg ml-1), L-glutamine (2 mM), non essential
amino acids (100 µM), and sodium pyruvate (1 mM).
Cell toxicity experiments with PC12 cells
PC12 cells were plated on a 96-well plated pre-coated with poly-(L-lysine) at a density
of 2.5 x 104 cells ml-1. Cell viability was assessed using a Live/Dead cell kit
(Invitrogen). Cells were incubated for 24 hours before the cell media was replaced with
nanoparticle suspensions at the desired concentrations in media. After a further 24 hours
of incubation the nanoparticles and media was removed, cells washed once with PBS,
and 100 µl of live/dead reagents were added (calcein AM, 1 µM; ethidium homodimer-1,
3 µM). After 30 minutes of incubation in the dark, images were recorded using an
inverted fluorescence microscope at 20x magnification (Olympus IX-71). Four images
were collected from each well at consistent locations for all wells and all experiments
and total live and dead cells were counted from these images and expressed as mean ±
SE values for each experiment.
Nanoparticle localisation studies in PC12 cells.
PC12 cells were plated onto pre poly-(L-lysine)-coated glass coverslips at a density of
2.5 x 104 cells ml-1.These cells were incubated for 24 hours before being treated with
either PEI coated nanoparticles (10 µg ml-1, in media) or PEG coated nanoparticles (10
µg ml-1, in media) or media alone (controls). These samples were left to incubate for a
further 24 hours. After incubation media and nanoparticles were removed, cells were
washed 3x with PBS before the samples were fixed in paraformaldehyde (4 %). Fixed
cells were incubated in PBS containing Triton X-100 (0.2%) before being incubated
with Hoescht 33342 (sigma, 1 µg ml-1) for 1-2 hours for nuclear staining. Samples were
then washed again with PBS and mounted using fluoromount gold onto microscope
slides for confocal microscopy (Leica TCS SP2, Nikon A1Si).
