SRI SRIDHAR, Ph.D.

NCI/NSF NANOMEDICINE SCIENCE AND TECHNOLOGYELECTRONIC MATERIALS RESEARCH INSTITUTE

PHYSICS DEPARTMENTNORTHEASTERN UNIVERSITY

SUPPORTED BY NSF/NCI and AFRL

Collaborators:Dattatri NageshaEvin GultepeMansoor Amiji, Pharma Sciences Robert Campbell, Pharm SciVladimir Torchilin, Pharma Sciences Charles DiMarzio, ECE Latika Menon, Physics Alain Karma, PhysicsDon O’Malley, BiologyCarol Warner, Biology Ahmed Busnaina, MIE Shashi Murthy, Chemical EngineeringLaura Lewis, Chem Engg Nikos Soukos, Forsyth Dental InstituteMike Makrigiorgos & Robert Cormack, BWH-DFCI

NANOMEDICINE RESEARCH PLAN

Therapy

Diagnosis

Nanoparticle Platforms

Delivery

Sensing

Imaging

Control

Harvesting

DNA

Drugs

Hyperthermia

Magnetic

Electronic

Intra-cellular tracking

In Vivo imaging

Proteomics

Metal NP

Micelles

Nanoassemblies

Polymeric NP

Liposomes

FunctionMethods and Phenomena Disease

Cancer

Infectious Diseases

Cardio-vascular Diseases

Sickle Cell Disease

Embryonic Stem Cell

Multi-functional Nanoplatforms

PEGSpacer

TAT

Y

pH-SensitiveFluorescentLabel

Radioactive Label

Antibody EndosomeBufferingAgent

HIV TATPeptide

pDNA orOligonucleotide

Magnetic core

Au or Fe-Au NP1-100nm

Micelles10-50nm

Liposomes100-250 nm

Polymeric NP~ 20nm – 10 m

40 nm 30 nm40 nm40 nm40 nm 30 nm30 nm30 nm

TITANIA AND ALUMINA NANOTEMPLATES

SENSING, TARGETING, DELIVERY

SENSING, DRUG ELUTING PLATFORMS, SCAFFOLDS AND

TEMPLATES, NEUROCHIP

Y

Y

Y

Y

Fe

Au orFe-Au

Au + PEGAu + PEG +Fluorescent label

Au + PEG +LM609 Antibody

TARGETINGMRI CONTRAST ENHANCEMENTMICROWAVE HYPERTHERMIAGENE DELIVERY

Y

Y

Y

Y

Intracellular experimentsCellular Trafficking

Au + PEG +LM609 Antibody + pDNA

MULTI FUNCTIONAL NANOPARTICLES

OH-PEG-OHAg2O, KI

CH2Cl2, 0oC, 30 min

HO-PEG-OTs

Scheme 1

n = 1.5 K

'desymmetrization'

S Me,

O

K+ -

TsCl (10 equiv.)

pyr, 60oC

NaOMe / MeOHthen, Dowex DR 2030

OO

Me

NH

(95%)

MeOH, reflux, 48 hr

TsO-PEG-OTs

(96%)

(90%)

O-PEG-S Me

OO

OO

Me

NH

O-PEG-SH

O

HO-PEG-S Me

O

coumarin-isocyanatetoluene, reflux(85%)

(76%)

Au NANOPARTICLES WITH HETERO-BIFUNCTIONAL PEG FOR BIOMEDICAL APPLICATIONSWei Fu, Dinesh Shenoy, Curtis Crasto, Matt Bouchard, Jane Liu

Mansoor Amiji, Graham Jones, Sanjeev Mukerjee, Charles di Marzio, Sri Sridhar

AuAu + PEG

Au + PEG +Fluorescent label

Intracellular experimentsCellular Trafficking

Int. J. Nanomedicine (2005), Bioorganic & Medicinal Chemistry Letters (2005)

Nucleus

MultifunctionalNanoparticle

Protein

Release

mRNA

Early Endosome

Cell Membrane

Protein Expression

Nuclear Uptake

EndocytosisNon-SpecificUptake

TAT-MediatedUptake

Receptor-MediatedUptake

Free DNA

LIFE-CYCLE OF A NANOPARTICLE VECTOR

Mechanisms and PathwaysBinding

EndocytosisEndosomal Escape

Cytosolic TransportDegradation

Nuclear UptakeTransfection

Protein ExpressionMagnetic control with MagNP

2-Photon Luminescence – Gold nanoparticles in Mouse Embryonic Stem Cells

Int. J. of Nanomedicine (2007)

Gold nanoparticles when excited by a 2-photon femto second laser source, exhibits photoluminescence

Photoluminescence of 20 l gold NPs dried on glass slide

Collaboration: Dr. Charles Dimarzio and Dr. Gary Laevsky (ECE)Paula Lampton, Carol Warner (Biology), Int. J. Nanomedicine (2007)

Figure 3.

Imaging without a fluorophore

Texas Red (568nm) Alexa 488 conj. to FNG Au (760nm) from FNG

20X lens

IMAGING OF LARVAL ZEBRAFISH USING MULTI-PHOTON LUMINESCENCE OF Au NANOPARTICLESFluor-gold nanoparticles and Texas RedFluorophores for validation of technique (IGERT project)

Collaboration with Sucharita Saha, Don O’Malley, Chuck DiMarzio

New technique for studying gene delivery for spinal cord regeneration

Brightfield Scale Bar 100µm

Two Photon λ=750nm

Confocal Reflectance λ=920nm

Second Harmonic λ=920nm

Ag NANOPARTICLES IN ANIMAL SKIN With Yogesh Patel, Chuck DiMarzio

z-stack of excised skin sample recording an image every 3µm from the surface down 30µm

MOVIE

15µm below glass coverslip

21µm below glass coverslip

Confocal ReflectanceTwo-PhotonSecond-Harmonic

MAGNETIC NANOPLATFORMS

A variety of applications• Separation Science: cells, biomarkers,

harvesting • MRI contrast enhancement• Localized energy delivery –

hyperthermia, ablation• Magnetic Manipulation- magnetic

targeting, magnetic tweezers• Magnetic biosensors

SPION in hexane at 100 Oe

0.E+00

5.E-06

1.E-05

2.E-05

2.E-05

3.E-05

3.E-05

4.E-05

4.E-05

0 50 100 150 200 250 300 350T (K)

M (

emu

/ul)

ZFC

FCBlocking

temperature (TB) of

SPIONs

M vs B

-4.E-05

-3.E-05

-2.E-05

-1.E-05

0.E+00

1.E-05

2.E-05

3.E-05

4.E-05

-0.5 -0.3 -0.1 0.1 0.3 0.5

B (T)

M (em

u/uL)

hexane

micelle

SuperparamagnetismNo hysteresis in M-H curves

SuperParamagnetic Iron Oxide Nanoparticles (SPIONS)

Surface Modification Techniques for Magnetic Nanoparticles

-

- ----------

---

-

---

Iron oxide nanoparticles

+vely chargedpolyelectrolyte

+

+vely charged Iron oxide nanoparticles

+++ +

++

+ ++

+

a) Surface coating of magnetic NPs for direct attachment to cells

Cell membrane is –vely charged and will bind to +vely charged particles through electrostatics.

OH + EtO-Si-(CH2)3-R

OEt

OEt

O -Si-(CH2)3-R

OH

OH

R

R

R

R

R = NH2, SH, CHO

b) Attachment of molecules for conjugation of antibodies

Antibodies are conjugated through the R- functional group

c) Gold coating to form core-shell morphology

Iterative addition

HAuCl4 +Hydroxylamine

Gold surface can be readily conjugated with various biomolecules

Iron oxide NPs Iron oxide-goldCore-shell NPs

MAGNETIC NANOPARTICLES FOR MRI CONTRAST ENHANCEMENT

TEM image shows encapsulation of iron oxide NPs in the core of the micelle

SPION Encapsulated PEG-PE Micelle System

2C5-Iron oxide NP-Micelles incubated with BT20 cells.Nuclei stained with Hoechst (blue) and 2C5-Iron oxide NP-Micelle stained with Rhodamine B (Red).

Nanosized Cancer Cell-Targeted Polymeric Immunomicelles Loaded withSuperparamagnetic Iron Oxide NanoparticlesRishikesh M. Sawant a, Rupa R. Sawant a, Evin Gultepe b, Dattatri Nagesha b, BrigittePapahadjopoulos-Sternberg c, Srinivas Sridhar b, Vladimir P. Torchilin a

MULTI-MODAL USE OF MAGNPIMAGE GUIDED MAGNETO THERAPY

• MRI CONTRAST ENHANCEMENT

• MAGNETIC TARGETING– MAGC LIPOSOMES : CAMPBELL

collaboration– SPION-IMMUNOMICELLES :

TORCHILIN colaboration• MAGNETIC HYPERTHERMIA

NOVEL NANOPARTICLE APPROACHES TO MRI• NEW NANOPARTICLES, eg. Mn-Fe-O• MIX T1 AND T2 AGENTS• EXPLOIT NEW PULSE SEQUENCES FOR DIFFERENT TIME SCALES

• BOUND VS. FREE NANOPARTICLES

SPION-Micelles

SPION

SPIONs encapsulated in micelle specific anti-body attached SPION-

Micelle packages

intra-cellular experimentsin vivo studies

NMR studies

• Targeting

• MRI Contrast Agents

• Magnetic Hyperthermia

5 50Diameter (nm)

5 50Diameter (nm)

mPEG2000-DSPE Iron oxide NPs

Lipid film hydration

Sonication

Encapsulation in micelles system

-100000

-50000

0

50000

100000

-2.00E+06 0.00E+00 2.00E+06

Iron oxide NPs

Iron oxide NPsPEG-PE micelle

T = 260K

H (A/m)

M(A

/m)

SQUID Magnetization Measurement

With Sawant, Torchilin

TEM image shows encapsulation of iron oxide NPs in the core of the micelle

SPION Encapsulated PEG-PE Micelle System

2C5-Iron oxide NP-Micelles incubated with BT20 cells.Nuclei stained with Hoechst (blue) and 2C5-Iron oxide NP-Micelle stained with Rhodamine B (Red).

+NH2

Aqueous buffer

pH 8-8.5, 4˚C

Tumor-cell specific antinuclear antibody 2C5 was attached onto surface of SPION-

micelles via para-nitrophenyl-PEG-PE

mAb2C5 SPION-micelles

RHODAMINE filter

Bovine IgG SPION-micelles-

Bare SPION-micelles

HOECHST filter

Rhodamine-labeled-SPION-micelles were incubated with human breast tumor

MCF-7 cells in vitro. Associated fluorescence was observed using epifluorescence

microscopy.

Anti-body conjugation

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Flu

ores

cen

ce a

t 55

0/59

0 n

m“Plain” SPION-micelles

mAb2C5 SPION-micelles

Bovine IgG SPION-micelles

0.35 mM PEG-PE 0.70 mM of PEG-PE0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Flu

ores

cen

ce a

t 55

0/59

0 n

m“Plain” SPION-micelles

mAb2C5 SPION-micelles

Bovine IgG SPION-micelles

“Plain” SPION-micelles

mAb2C5 SPION-micelles

Bovine IgG SPION-micelles

0.35 mM PEG-PE 0.70 mM of PEG-PE

Associated rhodamine fluorescence was also quantified using fluorescence spectroscopy.

Fluorescence spectroscopy

Guided Drug DeliverySolid tumor

Apply magnetic field to concentrate particles

Modulate field to release drug from particles

Inject NMPs IV,NMP will circulate through the blood stream

Other options for targeting:1 - Direct injection into tumor site2 - Coating NMP with antibodies to target tumor

Magnetic Targeting

Iron oxide NPs encapsulated PEG-PE micelle systemShow significant R2 enhancement

Torchilin, Sawant (Pharmaceutical Sciences ), Kautz (Barnett)

NMR of Iron oxide NPs PEG-PE micelle

0

0.04

0.08

0.12

0.16

0.2

0 0.025 0.05 0.075 0.1 0.125 0.15

Concentration of iron oxide NPs (mg/mL)

1/T2

(sec

-1)

Iron oxide NPs

Iron oxide NPs-micelles Animal MRI

studies are ongoing at MGH

Relaxivity R2 (1/T2) signal associated with MCF-7 cells was measured using Varian INOVA 500MHz NMR Spectrometer.

Tumor-cell-specific-2C5-SPION micelles interact better and thus deliver more signal to the human breast tumor MCF-7 cells compared to the “plain” or non-specific IgG-SPION micelles.

R2 enhancement validated in in vitro cell studies

Animal MRI studies are

ongoing at MGH

mAb2C5-SPION-micelle

• SPION-micelle retains all the magnetic properties of the nanoparticles

• Magnetic targeting leads to 2x increase in tumor accumulation

• R2 relaxation significantly increased, leading to potential MRI contrast enhancement advantages

MAGNETIC CATIONIC LIPOSOMES FOR MRI AND HYPERTHERMIA

With Robert Campbell (NU)

Key advantages of MagC• Targets tumor vasculature• Very high cargo loading of nanocarrier• Can be combined with drugs, genes, etc

Healthy Mouse 1hr After Injection (H2)

Healthy Mouse 24hr After Injection (H1)

1hr after: the kidneys are dark because of the accumulation of iron oxide.

24hr after: kidneys are clear

Healthy Mouse (H2)

Tumor Mouse 24hr After Injection (T1)

Tumor Mouse 24hr After Injection + Magnet for 1hr (T2)

Heavily T2 weighted images

Angiography Pre-Injection Angiography Post-Injection

Arteries are invisible in post-injection images because of the circulating iron oxide.

Conclusion

• No animal death because of the injection• The concentration used is enough for the

contrast• Immediately after injection iron-oxides

circulate in the blood for at least one hour• 24hr after injection the kidneys are cleared• 1hr magnet placement has a positive

effect on the accumulation in the tumor even after 24 hours.

Magnetic nanoparticles heating up due to the oscillating magnetic field.

Gram-positive bacterium, E. faecalis, is exposed to ac magnetic field after incubating with nanoparticles for 5 minutes.

% killing vs exposure time

MAGNETIC HYPERTHERMIA FOR CANCER AND INFECTIOUS DISEASES

With Nikos Soukos, Forsyth Dental

100% kill achieved in 5-10 minsPromising results, further studies needed to assess clinical potentialPotential application to Biofilm disruption

Killing seems to be non-thermalMechanism needs to be understood

LOCALIZED ENERGY DELIVERY

EM induced drug release from liposomesWith Campbell, Torchilin

Cancer Hyperthermia and Thermal Ablation

BIOMOLECULAR CONTROLManipulating genetic structure by localized energy delivery

EM Microbicide

EM induced cell membrane disruption, eg. hemolysis

40 nm 30 nm40 nm40 nm40 nm 30 nm30 nm30 nm

Cross section SEM images of Titania nanohole arrays. The average height of thefilm is ~ 250nm and pore diameter 25-35nm.

Nanoporous Titania and Alumina Arrays Nanoassembly, Drug delivery Applications

Nanobead Assembly

More than 85% coverage over

1cm2 area.

• 50nm beads• 80nm nanoholes• 10V, electrophoresis• plate counter electrode.

Applied Physics Letters (2007)

Carbon Nanotube Assembly

SEM micrographs of assembled SWCNTs in anodic alumina array.

Applications:BiosensorsHigh Density Memory

Elution from Nanotemplates

www.csmc.edu

Example: Drug eluting stents

T. C. Woods, A. R. Marks Ann. Rev. Med., 55, 2004

Coronary artery stenosis

Balloon angioplasty

Stent Implantation

Drug eluting stent bare stent

Patent artery Restenosis

Elution from Nanotemplates

Concentration of the drug is measured in the release medium over the time.

A model drug, Doxorubicin, is loaded into alumina nanotemplates to observe release kinetics from a porous medium.

excitation wavelength

in-situ measurement

532 nm spectrometer

Anodic Alumina Nanoholes

Pore diameter: 190nm Pore thickness: 10um

Templates after loading with DOX.E: empty template

Titania NanotubesPore diameter: 125nm Pore thickness: 2.5um

Drug Elution from Nanotemplates

t<10min, best fit is a power. C(t) ∝ t1/2 ⇛ Fickian diffusion

t>10min, best fit is logarithmic. C(t) ∝ log(t) ⇛ CC(t)/ e dt

dC(t)

t1/2

log(t)

www.igert.neu.edu

A NEW INTERDISCIPLINARY IGERT PH.D. PROGRAM FUNDED BY THE

NATIONAL CANCER INSTITUTEAND NATIONAL SCIENCE FOUNDATION

$3.3M 2005-2010

PI & Dir: Professor Sri SridharMansoor Amiji, Sanjeev Mukerjee, Mary Jo Ondrechen, Gilda Barabino, Ph.D.

A NEW MODEL FOR GRADUATE EDUCATIONDRIVEN BY REAL WORLD RESEARCH PROBLEMS

Approaches and methods

ReportsPublicationsPresentationsResearch IntegrityEthics of Nanomedicine

Applications of Nanosystems in MedicineNanosystems at Biological InterfacesScientific Skills, Ethics, and CommercializationNanomedical Technology Seminar

Introduction to Nanomedical TechnologyNanosystems Design for Biomedical Applications

CommercializationProduct development Patent writingIntellectual propertyCultural diversityInternational outlook

•NP Preparation & functionalization•Electromagnetic energy delivery•Magnetic Nanoparticles for sensing and bio-control

•Cellular trafficking and modeling•Mitochondrial gene therapy• Cellular Bio-sensing

Industrial and international internships

Multi-disciplinary courses

Education and training

Communication Skills and Professional Conduct

Inter-disciplinary research groups

Interdisciplinary graduate Interdisciplinary graduate education, research, and education, research, and

training at the interface of training at the interface of nanotechnology, biotechnology nanotechnology, biotechnology

and medicineand medicine

Vision

IGERT Nanomedicine Science and Technology

Timeline and Requirements

Year 1 Year 2 Year 3 Year 4 Year 5

Subtitle

10/14/2004

Fellows Participating inCoursework and Seminars

Admission ofIGERT Fellows

Active Industrial Participation Including Internship,Serving on Advisory Committee, and Mentorship

Year 2Qualifying Exams

Subtitle

10/14/2004NanomedicineDissertation Research

Year 5Thesis Defenseand Graduation

PhD inNanomedtech

Year 1 Year 2 Year 3 Year 4 Year 5

Subtitle

10/14/2004

Fellows Participating inCoursework and Seminars

Admission ofIGERT FellowsAdmission ofIGERT Fellows

Active Industrial Participation Including Internship,Serving on Advisory Committee, and Mentorship

Year 2Qualifying Exams

Subtitle

10/14/2004NanomedicineDissertation Research

Year 5Thesis Defenseand Graduation

PhD inNanomedtech

OUTREACH K-12 Coordinated by Claire Duggan (www.ret.neu.edu)

HOSPITAL / INDUSTRY INTERNSHIPCo-mentoring by a scientist outside NU

Admission to home department

Ph.D. in core discipline with specialization in Nanomedicine

New Nanomedicine courses (12 Semester Hours)

IGERT FUNDING

New Nanomedicine coursesFirst of its kind

1. Nanomedicine Science and Technology Seminar (Jan 2006)

2. Intro to Nanomedicine S&T ( started Fall 2006)3. Nanosystems Design for Biomedical

Applications (started Spring 2007)

Guest lecturers from Mass Gen, Dana Farber, Beth Israel, Advance Nanotech, …

IGERT NANOMEDICINEBouve College

of Health SciencesCollege of Artsand Sciences

College of Engineering

BiologyPaula LamptonAdvisor: Carol Warner

Anthony D’OnofrioAdvisor: Kim Lewis

Sucharita SahaAdvisor: Don O’Malley

ChemistryHeather BroadkinAdvisor: Mary Jo Ondrechen

Adam HendricksAdvisor: Robert Hanson

Tatyana ChernenkoAdvisor: Max Diem

Electrical and Computer EngineeringYogesh Patel Advisor: Charles DiMarzio

Mechanical and Industrial EngineeringRobert CampAdvisor: Jeff Ruberti

Chemical EngineeringSavidra LucateroAdvisor: Rebecca Carrier

Pharmaceutical SciencesLilian van VlerkenAdvisor: Mansoor Amiji

Mattia MiglioreAdvisor: Barbara Waszczak

Lara JabrAdvisor: Volkmar Weissig

Luis BritoAdvisor: Mansoor Amiji

Padmaja MagadalaAdvisor: Mansoor Amiji

Lilian van VlerkenModulation of Intracellular Ceramide

using Polymeric Nanoparticles to Overcome Multidrug Resistance (MDR) of Breast Cancer

Advisor: Dr. Mansoor M. Amiji

Mattia M. Migliore Advisor: Dr. Barbara Waszczak

Intranasal delivery of GDNF using nanoparticle technology for the treatment of Parkinson’s disease

occurance of apoptosis in MDR breast cancer cells, previously resistant to cell death, with experimental therapy

Substantia nigra dopamine neurons ( 40x view) of a rat that was administered cationic liposomes loaded with Alexa-488 ovalbumin intranasally and sacrificed after 24hrs

IGERT NANOMEDICINE + PHARMA SCI

Padmaja MagadalaDepartment: Pharmaceutical Sciences

Advisor: Mansoor Amiji

"HER2/neu Receptor-Targeted Gelatin-Based Nanovectors for Pancreatic Cancer Gene Therapy"

C)

Blank Gel NPs Unmodified Gel NPs

PEG-Gel NPs Pep-PEG-Gel NPs

Quantitative and qualitative tranfection efficiency study of control and modified gel-NPs in panc-1 cells by FACS (A), ELISA (B) and fluorescence microscopy (C)

Paula LamptonAdvisor: Dr.Carol WarnerDepartment of Biology

Nanoparticles for analysis of

immune system molecules in

mouse embryonic stem cells

IGERT NANOMEDICINE + BIOLOGY

Sucharita Saha

Advisor: Donald O'Malley

Visualization and Enhancement of Spinal Cord Regeneration using Nanoparticles

Application of Nanoarray Technologies to Spinal Stimulation and Regeneration: the Nano-BMI (brain-machine interface)

See poster at this meeting

IGERT K-12 outreach• Research Experiences for Teachers (RET)• Young Scholars Program• Lesson Development• Field Trips/Presentations• Science Fair Mentoring and Support

www.igert.neu.edu