NanomedicineMichael D. Buschmann

Department of Chemical Engineering

Institute of Biomedical Engineering

Ecole Polytechnique, Montreal, Canada

THE RESPONSIBLE DEVELOPMENT OF NANOTECHNOLOGY CHALLENGES AND PERSPECTIVES

Ne3LS Network International Conference 2012 Nov 1, 2012

Overview

► Nanomedicine Products

► Why Nano for Medicine ?

► Requirements for Successful R&D

► New Technologies from Ecole

Polytechnique

► Conclusion & Perspectives

Nanomedicine uses nano-sized tools for the diagnosis, prevention and treatment of disease

and to gain increased understanding of the complex underlying patho-physiology of

disease.

The ultimate goal is improved quality-of-life.

*European Science Foundation’s Forward Look Nanomedicine, 2005

Nanomedicine definition*

“Nanobiotechnology” is a broader concept : fundamental

cellular mechanisms, molecular forces, molecular motors,

cellular electrochemical phenomena in nonhuman, plant

and animal models.

• Iron Oxide or Gd Imaging Agents

Constrast agents for MRI• Iron Oxide Supplements

To treat anemia• Drug Nano Crystals

Increase oral bioavailability• Liposomes

Drug delivery containers• Polymer Therpauetics

Prolong and target action• Nanoparticles

Multifuncitonal delivery systems

Nanomedicine Products

Layered Micelle Nanoparticle

Nanomedicine Products Layered Micelle Nanoparticle

Marketed NanomedicinesDuncan Molecular Pharmaceutics 2011

Marketed NanomedicinesDuncan Molecular Pharmaceutics 2011

Several marketed for > 20 years

Therapeutics– Nanovectors for drug delivery – small molecules, proteins,

antibodies, DNA, RNA– Targeted smart systems – hit the right tissue, the right cell– Next generation medicines – RNAi– Market > $50 billion

Diagnostics– Imaging & contrast agents – magnetic, metallic, fluorescent– Molecular sensors – arrays, chips, SPR etc– Microfluidics – separation and analyses on a chip– Personalized Medicine – make the treatment fit patient

symptoms and molecular profiling by diagnostics – emerging area

– Market > $5 billion

Application Domains of Nanomedicine

Ref: POC 2010 March 2010, Yole Développement SA.

Point of Care Diagnostics - Target Applications

Ref: Genetic Engineering and Biotechnology News Oct. 15, 2010.

Nanobiotechnology Market & Application Areas (Includes Recombinant Proteins/Antibodies)

Why NANO ?

Internalisation to Cells is Nano-size Dependent

Conner Nature 2003

Why NANO ?

Routes of Drug Administration

Size controls Biodistribution

Gaumet E J Pharm Biopharm 2008

Why NANO ?Endothelial

Fenestration Critical to

Biodistribution

Fenestration space is

Nanosized : 1-1000nm

Gaumet E J Pharm Biopharm 2008

Desirable size range 50-200nm avoids rapid renal excretion and avoids liver

and spleen accumulation

Targeting can be passive (size) and/or active (ligand)

Why NANO ?

Surface chemistry (PEG) determines protein binding (opsonisation) and

phagocytosis by macrophages versus delivery to the target

Why NANO ?Dobrovolskaia Nanoletters

2008

Nanomedicines under Clinical Development

Nanomedicines under Clinical Development

Nanomedicines under Clinical Development

Duncan Molecular Pharmaceutics 2011

Successful Research & Development Requires

• Efficacy of Treatment

• Safety of Treatment

• Manufacturing Capability

• Cost-effectiveness

• Competitive Advantage

• Patent Protection

• Regulatory Approval

• Reimbursement Strategy

• Cell Viability, Proliferation, Metabolism

MTT (cellular respiration) and related • Cell membrane damage and necrosis

LDH release, direct membrane perturbation• Apoptosis (programmed cell death)

Annexin V (early), TUNEL (late)• DNA damage and genotoxocity

Strand breaks by COMET• Oxidative Stress

O and N radicals damage multiple cell components• Gene screening

Elucidate networks related to toxic responses

Safety – Cell-based Toxicity

• Clinical Signs

Behavior, Weight loss• Hematology & Serum Chemistry

Cell counts, Hemolysis, Complement Activation • Inflammation

Inflammatory Cytokines• Biodistribtion, Organ Damage

Histology, Immunohistochemistry, Liver Enzymes

Safety – Animal-based Toxicity

Cytotoxicity depends on Chemistry (& size)

5 chemistries =

5 distinct toxicities

Beyerle Mol Therap 2009

Groupe de recherche en sciences et technologies biomédicales (GRSTB du

FRQ-S)

Chercheurs réguliers 30Chercheurs-boursiers 11Chercheurs associés 20Étudiants (MScA & PhD)183Stagiaires post-doc 32

Regroupements de recherche du FRQ

Plans stratégiques universitaires

GRSTB

Imagerie&

Traitement de signal

Nano-médecine

&Médecine

régénératrice

Biomatériaux&

Biomécanique

Transfert technologiq

ue aux entreprises

Transfertde

connaissances à la clinique

Pôle sectoriel SGV

25

Viral Vectors:Retrovirus, adenovirus...

High efficiency Toxicity Immunogenicity

Nonviral Vectors:Cationic lipids

Cationic polymers (polycations)

Greater safety Low efficiency

• Cationic polymers → low toxicity and ease of preparation

(ex: chitosan, polyethyleimine, poly-L-lysine, dendrimers..)

Need strategies to increase

transfection efficiency

Condense DNA into nanoparticles

1

Uptake intoendosomes

Intracellular transport

3Nuclear

localization4

DNA releaseGene expression

5

Binding to

target cells

2 cell

Nanoparticles for Gene Delivery (Buschmann)

>1500 Clinical Trials in Gene Therapy

“FDA has not yet approved any human gene therapy product for sale.”“EU has approved Glybera in 2012 for lipoprotein lipase (LPL) deficiency in patients with severe or multiple pancreatitis attacks.”

NHAcNH

NHDDA

2

2

Linear chain of glucosamine and N-acetyl-glucosamine units linked by b(1,4)-glycosidic

bonds

HO

OH

NHAc

O

OHO

OH

NHAc

OChitin

• A frequently used natural polymer for gene delivery

• Biodegradable, biocompatible, non-toxic

ChitosanHO

OH

NH2

O

OHO

OH

NHAc

O

Deacetylation

Chitosan

We have previously developed a chitosan-based biomaterial to repair cartilage : April 11 2012 : “Piramal Healthcare announces approval of its first innovative product for regenerative

medicine, BST-CarGel®”

NH3+

28

+ ++ +

+

+ + + +

Protonated amine groups

(positive charges)

Chitosan

Phosphate groups

(negative charges)

-

-

-

--

- --

-

-200 nm200 nm

Plasmid DNA

+++

+

Important factors:• Chitosan

molecular weight and DDA

• pH• N/P ratio

(chitosan amine to DNA phosphate ratio)

(in excess)

150 nm

DNA condensed into nanoparticles

+

+

+

++

+

+

+DNA/Chitosan

complexes

Formation of DNA-chitosan complexes

Influence of MW & DDA on Transfection

• Transfection efficiency as good or higher then state-of-the-art• Strong chitosan structural –dependence of efficiency

Transfection efficiency as good or higher then state-of-the-art

* *

Influence of MW & DDA on Transfection

In vivo Delivery of Growth Factors FGF2 & PDGF

1 2 3

pVax-1 ( 3kb)

4sFGF-2 ( 470 bp)PDGFbb ( 327bp)

M

HindIII 4sFGF-2 XhoI

PDGF-bbHindIII 4sFGF-2 or PDGFbb XhoI

pVax1/4sFGF-2 (3 465 pb)ou

pVax1/PDGF-bb (3 327 pb)

pVax1 vector

4sFGF-2 Recombinant Protein and Anti-4sFGF-2 Antibodies in plasma following subcutaneous injection of chitosan/pVax-4sFGF-2 nanocomplexes

92-10-5 increases protein

and reduces antibody

80-10-10 reduces protein

and increases antibody

Chitosan Properties Significantly Modulate Protein and Antibody Levels

• Higher DDA and lower MW (92%DDA-10kDa) efficiency expresses protein, minimises inflammatory cell recruitment, lowers antibody response and generates systemic circulating recombinant protein

• Lower DDA (80%DDA) augments inflammatory cell recruitment and antibody levels, reducing circulating protein, possibly useful for DNA vaccines

Insuline

Type II Diabetes

Insulin, GLP-1, and Glucagon Levels

Glucose tolerance

Insulin release

Inflammatory reaction

Weight variation

GLP-1 Expression

Therapeutic nanocomplexe (NCT): 165 µg

Days: 0, 7, 14, 21, 35, 49 et 63

Gene Therapy for Diabetes

Maximal GLP-1 Expression with 92-10-5

92-10-5 is the most efficient formulation in both routes of administration

80-10-10 also effective

80-80-5 ineffective due to immunostimulatory and poor release properties as demonstrated in the PDGF/FGF study

GLP-1 expression is maintained for 24 days following end of treatment greatly exceeding the lifetime of current daily injected GLP-1 peptides

Glucose Tolerance Normalised with 92-10-5

• Formulation 92-10-5 is the most effective

• Rapid return to normoglycemia within two hours

• No significant difference between SC and IM routes of administration

normoglycemia

Nanoparticle Nucleic Acid Delivery

• siRNA delivery in Atherosclerosis (ApoB) and Cancer (Helicase)

– Cell and animal studies

• Arthritis and inflammation

– pDNA/chitosan delivery of TNFα inhibitors (Etanercept , Enbrel)

• Manufacturing and Scale-up

– High volume mixing technologies, concentration, freeze-dry

– Transfer to cGMP facility with Quality Control methodsFreeze-dried, Room Temperature Stable Pharmaceutical Product

39

Gold Nanoshell Probes for Atherosclerosis Diagnosis and

Personalized Medicine Professor Fréderic Lesage

Optical absorbance at NIR wavelength

-Easily tunable-Monodisperse-Non-toxic

Synthesis and Characterization of Gold Nanoshells

OD=1

OD=0.1

OD=10

500 600 700 800 9000.0

0.2

0.4

0.6

0.8

1.0

Wavelength (nm)

Ab

sorb

ance

(O

D)

15 20 25 30 35 40 45 50 550

10

20

30

Diameter (nm)

Nu

mb

er o

f A

uN

S

2 3 4 5 6 7 80

10

20

30

40

Thickness (nm)

Nu

mb

er o

f A

uN

S

AuNS AuNS-PEG-Ab

0

2

4

6Ab-AuNS

TCEP 2.75x 2.75x 10x 10xAb (ug/mL) 2 20 2 20

PEG-AuNS

***

*

Ab

sorb

ance

Rat

io(O

D4

50

nm

/OD

71

0n

m)

500 600 700 800 9000.0

0.2

0.4

0.6

0.8

1.0

AuNS-PEGAuNS-PEG-AbAuNS

Wavelength (nm)

Ab

sorb

ance

(O

D)

680 700 720 740

0.96

0.98

1.00

No visible sign of toxicity after injections of large doses

Saline control AuNS (OD=200)

Sple

en

Live

r

BA

AuN

S

Sal

ine

(OD

=200

)

Con

trol

Kidney Heart Spleen Liver

Optical projection tomography

BA

Control ApoE-/- DC

VCAM targeting in ApoE mice

A diagnostic technique to

identify fragile plaques and guide

personalised treatment

Drug Targeting using Magnetic Systems Professor Sylvain Martel

A clinical platform designed to agglomerate and control

flagellated Magnetotactic Bacteria (type MC-1)

acting as micro-carriers

MagnetotaxisSYSTEM

Self-assembled and self-reproducingcomputer-controlled

biological bacterial carriers

MC-1 flagellated magnetotactic bacteriumthat can be directed towards the tumor

due to a chain of nanoparticles synthesized in the cell and acting like a miniature driven wheel

SN-38 drug molecules encapsulated in liposomesand attached to the bacterial cell

Flagella acting as the propulsion system

Drug Delivery to Colorectal Canceravoiding Radiotherapy and Surgery

Drug inside TMMCTMMC

TMMC

Published in Biomaterials 2011

Drug Targeting in

Vessels Driven by

MRI controlled

Magnetic

Field Gradients

Nanomedicine Summary

• Nano & Biology are a Natural Fit

• Nano = chemistry, physics, engineering

requires multidisciplinary teams to address

complex issues of bio-efficacy and safety

• A relatively mature field with established

markets and products, but in rapid expansion

• Need for a balanced approach to regulate

uniformly between countries and foster

development