Nanomedicine Michael D. Buschmann Department of Chemical Engineering Institute of Biomedical Engineering Ecole Polytechnique, Montreal, CanadaTHE 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 definition* Nanomedicine uses nano-sized tools for the diagnosis, prevention and treatment of diseaseand 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 “Nanobiotechnology” is a broader concept : fundamental cellular mechanisms, molecular forces, molecular motors, cellularelectrochemical phenomena in nonhuman, plant and animal models.
Nanomedicine Products•Iron Oxide or Gd Imaging Agents Constrast agents for MRI•Iron Oxide Supplements To treat anemia•Drug Nano Crystals Increase oral bioavailability•Liposomes Layered Micelle Nanoparticle Drug delivery containers•Polymer Therpauetics Prolong and target action•Nanoparticles Multifuncitonal delivery systems
Marketed Nanomedicines Duncan Molecular Pharmaceutics 2011 Several marketed for > 20 years
Application Domains of NanomedicineTherapeutics – 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 billionDiagnostics – 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
Point of Care Diagnostics - Target Applications Ref: POC 2010 March 2010, Yole Développement SA.
Nanobiotechnology Market & Application Areas (Includes Recombinant Proteins/Antibodies) Ref: Genetic Engineering and Biotechnology News Oct. 15, 2010.
Why NANO ? Conner Nature 2003 Internalisation to Cells is Nano-size Dependent
Why NANO ? Size controlsRoutes of Drug Administration Biodistribution Gaumet E J Pharm Biopharm 2008
Why NANO ? Gaumet E J Pharm Biopharm 2008Endothelial Fenestration Fenestration space is Critical to Nanosized : 1-1000nm Biodistribution Desirable size range 50-200nm avoids rapid renal excretion and avoids liver and spleen
Why NANO ?Targeting can be passive (size) and/or active (ligand)
Dobrovolskaia Nanoletters 2008Why NANO ?Surface chemistry (PEG) determines proteinbinding (opsonisation) and phagocytosis by macrophages versus delivery to the target
Nanomedicines under Clinical Development Duncan Molecular Pharmaceutics 2011
Successful Research & DevelopmentRequires •Efficacy of Treatment •Safety of Treatment •Manufacturing Capability •Cost-effectiveness •Competitive Advantage •Patent Protection •Regulatory Approval •Reimbursement Strategy
Safety – Cell-based Toxicity•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
Groupe de recherche en sciences ettechnologies biomédicales (GRSTB du FRQ- S) Chercheurs réguliers 30 Chercheurs-boursiers 11 Chercheurs associés 20 Étudiants (MScA & PhD) 183 Stagiaires post-doc 32
Regroupements de recherche du FRQ GRSTB Biomatériaux Imagerie & & Biomécanique Traitement de signal Transfert Transfert detechnologique Nano-médecine & connaissanc aux Médecine régénératrice es à la entreprises clinique Pôle sectoriel SGV Plans stratégiques universitaires
Nanoparticles for Gene Delivery(Buschmann) Viral Vectors: Nonviral Vectors: Retrovirus, adenovirus... Cationic lipids High efficiency Cationic polymers (polycations) Toxicity Greater safety Immunogenicity Low efficiency • Cationic polymers → low toxicity and ease of preparation (ex: chitosan, polyethyleimine, poly-L-lysine, dendrimers..) Intracellular Condense DNA into transport Nuclear 3 localization nanoparticles 4 1 Need strategies to Binding to target increase cells 2 Uptake into cell 5 DNA releasetransfection efficiency endosomes Gene expression 25
>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.”
Chitosan • A frequently used natural polymer for gene delivery • Biodegradable, biocompatible, non-toxic OH OH Chitin O O O HO HO NHAc NHAc Deacetylation OH OH O O Chitosan O HO HO NH 2 NH2 + DDA NH3 NHAc NH 2 NHAc Linear chain of glucosamine and N-acetyl-glucosamine units linked by (1,4)-glycosidic bonds 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®”
Formation of DNA-chitosan complexes Protonated amine Phosphate groups groups (negative charges) (positive + charges) + - - Plasmid DNA + + - + - + + + + + + + - - + Chitosan - - (in excess) - - DNA condensed intoImportant factors: nanoparticles• Chitosan molecular + + + weight and DDA + +• pH +• N/P ratio (chitosan + + amine to DNA DNA/Chitosan phosphate ratio) complexes 28
Influence of MW & DDA on Transfection• Transfection efficiency as good or higher then state-of-the-a• Strong chitosan structural –dependence of efficiency
Influence of MW & DDA on Transfection * *Transfection efficiency as good or higher then state-of-the-art
In vivo Delivery of Growth Factors FGF2 &PDGF M 1 2 3 pVax-1 ( 3kb) 4sFGF-2 ( 470 bp) PDGFbb ( 327bp) pVax1 vector HindIII 4sFGF-24sFGF -2 or PDGFbb XhoI PDGFbb - pVax1/4sFGF-2 (3 465 pb) ou pVax1/PDGF-bb (3 327 pb)
4sFGF-2 Recombinant Protein and Anti-4sFGF-2Antibodies in plasma following subcutaneous injectionof chitosan/pVax-4sFGF-2 nanocomplexes 92-10-5 increases protein and reduces antibody 80-10-10 reduces protein and increases antibody
Chitosan Properties SignificantlyModulate 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
Type II DiabetesInsulin, GLP-1, and Glucagon Levels Insuline
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 normoglycemia two hours• No significant difference between SC and IM routes of administration
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 methods Freeze-dried, Room Temperature Stable Pharmaceutical Product
Gold Nanoshell Probes for Atherosclerosis Diagnosis and Personalized Medicine Professor Fréderic Lesage Optical absorbance at NIR wavelength -Easily tunable -Monodisperse -Non-toxic 39
No visible sign of toxicity afterinjections of large doses A Kidney Heart Spleen Liver B Saline control AuNS (OD=200) LiverControlSaline(OD=200) Spleen AuNS
Optical projection tomographyVCAM targeting in ApoE miceA B A diagnostic technique to identify fragile plaques and guide personalised treatment C Control ApoE-/- D
Drug Targeting using Magnetic Systems Professor Sylvain MartelA clinical platform designed to agglomerate and control flagellated Magnetotactic Bacteria (type MC-1) acting as micro-carriers Magnetotaxis SYSTEM
Self-assembled and self-reproducing computer-controlled biological bacterial carriers Drug Delivery to Colorectal Cance Flagella acting as the avoiding propulsion system Radiotherapy and MC-1 flagellated magnetotactic bacterium Surgery that can be directed towards the tumorue to a chain of nanoparticles synthesized in the cell and acting like a miniature driven wheelN-38 drug molecules encapsulated in liposomes and attached to the bacterial cell
TMMC Drug inside TMMCDrug Targeting inVessels Driven by TMMCMRI controlledMagnetic Published in Biomaterials 2011
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
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