Premises• Since the human body is basically an extremely complex system of interacting molecules (i.e., a molecular machine), the technology required to truly understand and repair the body is the molecular machine technology : NANOTECHNOLOGY• A natural consequence of this level of technology will be the ability to analyze and repair the human body as completely and effectively as we can repair any conventional machine today.
Nanomedicine:EC European Technology Platform (ETP)
E.C.-ETP“Nanomedicine, is defined as the application ofnanotechnology to achieve breakthroughs inhealthcare. It exploits the improved and often novelphysical, chemical and biological properties ofmaterials at the nanometer scale. Nanomedicinehas the potential to enable early detection andprevention, and to essentially improve diagnosis,treatment and follow-up of diseases.……………………….Diagnostics, targeted drug delivery andregenerative medicine constitute the coredisciplines of nanomedicine.”
Nanomedicine:European Science Foundation (ESF) “The field of Nanomedicine is the science and technology of diagnosing, treating and preventing disease and traumatic injury, of relieving pain, and of preserving and improving human health, using molecular tools and molecular knowledge of the human body. It embraces sub- disciplines which are in many ways overlapping and are underpinned by common technical issues.”
The numbers of nanomedicine The total market for nanobiotechnology products is $19.3 billion in 2010 and is growing at a compound annual growth rate (CAGR) of 9% to reach a forecasted market size of $29.7 billion by 2015.
Topics in nanomedicine• Therapy:Drug Delivery: Use nanodevices specifically targeted to cells, to guide delivery of drugs, proteins and genesDrug targeting : Whole body, cellular , subcellular deliveryDrug discovery : Novel bioactives and delivery systems
Topics in nanomedicine• Diagnosis: Prevention and Early Detection of diseases: Use nanodevices to detect specific changes in diseased cells and organism.
Nanoparticles (NP):Smart Nanostructures for diagnosis and therapy
Why Nanoparticles1) Drugs, contrast agents,paramagnetic or radiolabeledprobes can be vehiculated by NPs2) NPs can be multi-functionalizedto confer differents features onthem
Why Nanoparticles• Vehiculation: Drug-encapsulating nanoparticles offer extensive control over delivery.• Drugs are protected inside NPs and are not degraded.
• Targeting: nanoparticles control over delivery.• Drugs are concentrated to target. Less systemic toxicity.• Less drug is necessary
• Multi-functionalization: Control over delivery location, drug dosage, and drug release characteristics is possible
An ideal Multi-functional nanoparticle vector Anticorpo Polietilenglicol Evita che NP venga digerita nei lisosomi Indirizza la NP verso un la NP venga Evita che antigene specifico sulla dal circolo rimossa cellula da colpire Tat peptide Determina Fusione e Probe magnetico ingresso della NP nella cellula Permette imaging tramite MRI
Carbon-based: Buckyballs and Nanotubes C60 1nm
Carbon nanotubes can be thought of as a sheet of graphiterolled into a cylinderNanotubes have a very broad range of electronic, thermal, andstructural properties that change depending on diameter,length,). They exhibit extraordinary strength and uniqueelectrical properties, and are efficient conductors of heat.Used as sensors
Proposed as a vessel for transporting drugs into the body.The drug can be attached to the side or trailed behind, or the drug can actually be placed inside the nanotube Nanotube Nanocap
Funzionalizzazione covalenteFunzionalizzazione non covalente
Their final usage, however, may be limited by theirpotential toxicity.Under some conditions, nanotubes can cross membranebarriers and can induce harmful effects: inflammation,epithelioid granulomas (microscopic nodules),fibrosis,and biochemical/toxicological changes in thelungs.
gold nanoparticles (1-20 nm) are produced by reductionof chloroauric acid (H[AuCl4]), To the rapidly-stirred boiling HAuCl4 solution, quickly add 2 mL of a 1% solution of trisodium citrate dihydrate, Na3C6H5O7.2H2O. The gold sol gradually forms as the citrate reduces the gold(III). Remove from heat when the solution has turned deep red or 10 minutes has elapsed.
In cancer research, colloidal gold can be used to targettumors and provide detection using SERS (SurfaceEnhanced Raman Spectroscopy) in vivo.They are being investigated as photothermal convertersof near infrared light for in-vivo applications, as ablationcomponents for cancer, and other targets since nearinfrared light transmits readily through human skin andtissue
Polymeric/Dendrimers(e.g.PLGA, PAA, PACA) spherical polymers of uniform molecular weight made from branched monomers are proving particularly adapt at providing multifunctional modularity.
Dendrimers are repetitively branched molecules. PAA= POLI AMMINO AMMIDEPLGA PAA
HYDROGELSPolymers or co-polymers (e.g. acrylamide and acrylic acid) createwater-impregnated nanoparticles with pores of well-defined size.Water flows freely into these particles, carrying proteins and other smallmolecules into the polymer matrix.By controlling the pore size, huge proteins such as albumin andimmunoglobulin are excluded while smaller peptides and othermolecules are allowed.The polymeric component acts as a negatively charged "bait" that attracts positivelycharged proteins, improving the particlesperformance.
Mesoporous silica particles: nano-sized spheres or rods filled with a regulararrangement of pores with controllable pore size from 3 to 15nm and outer diameterfrom 20nm to 1000 nm .The large surface area of the pores allows the particles to be filled with a drug or with a fluorescent dye that would normally be unable to pass through cell walls.The MSN material is then capped off with a molecule that is compatible with thetarget cells. When are added to a cell culture, they carry the drug across the cellmembrane. These particles are optically transparent, so a dye can be seen throughthe silica walls. The dye in the particles does not have the same problem with self-quenching that a dye in solution has. The types of molecules that are grafted to theoutside will control what kinds of biomolecules are allowed inside the particles tointeract with the dye. EM
A quantum dot is a semiconductor whose excitons areconfined in all three spatial dimensions.An immediate optical feature of colloidal quantumdots is their colorationFirst attempts have been made to use quantum dotsfor tumor targeting under in vivo conditions.Generically toxic
Quantum Dots• Raw quantum dots, 2-8 nm are toxic, CdSe or CdTe cores with ZnS shell• But they fluoresce brilliantly, better than dyes (imaging agents)• Only way of clearance of protected QDs from the body is by slow filtration and excretion through the kidneyhttp://www.azonano.com/Details.asp?ArticleID=1726
Quantum DotsQD technology helps cancer researchers to observe fundamentalmolecular events occurring in the tumor cells by tracking theQDs of different sizes and thus different colors, tagged tomultiple different biomoleules, in vivo by fluorescentmicroscopy.QD technology holds a great potential for applications innanobiotechnology and medical diagnostics where QDs could beused as labels.
Nano-particulate pharmaceuticalsBrand name DescriptionEmend Nanocrystal (antiemetic) in a capsule(Merck & Co. Inc.)Rapamune Nanocrystallized Rapamycin (immunosuppressant) in a(Wyeth-Ayerst Laboratories) tabletAbraxane Paclitaxel (anticancer drug)- bound albumin particles(American Biosciences, Inc.)Rexin-G A retroviral vector carrying cytotoxic gene(Epeius Biotechnologycorporation)Olay Moisturizers Contains added transparent, better protecting nano zinc(Procter and Gamble) oxide particlesTrimetaspheres (Luna Nanoworks) MRI imagesSilcryst Enhance the solubility and sustained release of silver(Nucryst Pharmaceuticals) nanocrystalsNano-balls Nano-sized plastic spheres with drugs (active against(Univ. of South Florida) methicillin-resistant staph (MRSA) bacteria) chemically bonded to their surface that allow the drug to be dissolved in water.
Company ProductCytImmune Gold nanoparticles for targeted delivery of drugs to tumorsNucryst Antimicrobial wound dressings using silver nanocrystalsNanobiotix Nanoparticles that target tumor cells, when irradiated by xrays thenanoparticles generate electrons which cause localized destruction of the tumor cells.Oxonica Disease identification using gold nanoparticles (biomarkers)Nanotherapeutics Nanoparticles for improving the performance of drug delivery byoral, inhaled or nasal methodsNanoBio Nanoemulsions for nasal delivery to fight viruses (such as the flu andcolds) and bacteriaBioDelivery Sciences Oral drug delivery of drugs encapuslated in a nanocrystallinestructure called a cochleateNanoBioMagnetics Magnetically responsive nanoparticles for targeted drugdelivery and other applicationsZ-Medica Medical gauze containing aluminosilicate nanoparticles which help boodclot faster in open wounds.
Open ProblemsManufacturing NPs for medical SOLUTION: use: Assessment of NPs:• Putting the drug on the particle Dynamic structural• Maintaining the drug on the features in vivo particle Kinetics of drug• Making the drug come off the release particle once application is Triggered drug release done• Purity and homogeneity of nanoparticles
Open ProblemsToxicity:short term - no toxicity in animalslong term- not knownToxicity for both the host and the environment should be addressed
Open ProblemsDelivery: SOLUTION:• Ensuring Delivery to target detection of NPs organ/cell at target, organs ,• Removal of nanoparticles from cells , subcellular the body location et al. Tissue distribution
Open Problems: Targeting the brain• Brain micro-vessel endothelial cells build up the blood brain barrier (BBB)• The BBB hinders water soluble molecules and those with MW > 500 from getting into the brain
Open Problems• GMP Challenges• No standards for:• Purity and homogeneity of nanoparticles• Manufacturing Methods• Testing and Validation
Summary• Toxicities of nanomaterials are unknown• How to best target the nanomaterials so that systemic administration can be used ?• How to uncage the drug so it gets out at the desired location ?• Is there a way to “re-cage” the drug when it is no longer desired ?• How are nanoparticles removed from the body ?• Mathematical modeling of nanostructures is in its infancy• Barrier crossing (BBB, G.I., et al.)
Nanomedicine programs atDepartment of Experimental Medicine (DIMS) Università di Milano-Bicocca
Nanoparticles for therapy and diagnosis of Alzheimer Disease(NAD)- European Community September 2008-August 2013• Synthesis of new NPs with improved biocompatibility, targeting and drug delivery features - Cariplo Bank Foundation Nanoparticles safety
A Nanochip to test the activity of candidate anti- amyloidogenic drugsOverlay (reflection and fluorescence) confocalimages of labelled Abeta aggregates innanochannels.1 cross-section 260 nm × 300 nm. 2 cross section 310 nm × 300.Sordan R. et al, Vertical arrays of nanofluidic channels fabricated withoutnanolithography Lab on a Chip,2009
NAD: NANOPARTICLES FOR THERAPY AND DIAGNOSIS OF ALZHEIMER DISEASE 2008-2012 Funding scheme: COLLABORATIVE PROJECT Large-scale integrating project (IP) proposal Work programme topic addressed: FP7-NMP-2007-LARGE-1NMP-2007-4.0-4 Substantial innovation in the European medical industry: development of nanotechnology-based systems for in-vivo diagnosis and therapy
No Partner Country Role Person1 University of Milano-Bicocca, Milano Italy Ligand generation, Binding Massimo Masserini BiocompatibilityCharacterization2 Universidad Autonoma de Madrid, Madrid Spain Transgenic models of AD Francisco Wandosel3 University of Brighton, Brighton UK Biomimetics Moghimi S Moein4 University of Paris-Sud XI, Chatenay- France Polymeric NPs Karine Andrieux Malabry5 Slovak Academy of Sciences, Bratislava Slovakia Computational models Igor Tvaroska6 Karolinska Institutet, Stockholm Sweden Neurobiology of AD Jin Jing Pei7 Academic Medical Center, Amsterdam The Neurobiology of AD Wiep Scheper Netherlands8 Biotalentum Ltd , Gödöllő Hungary Management Andras Dinnyes9 Turku PET Center, Turku Finland PET Juha Rinne10 Nanovector Srl, Torino Italy SLN Gasco Mariarosa11 University Of Patras, Patras Greece Liposomes Sophia G. Antimisiaris12 University of Antwerp, Antwerp Belgium MRI Annemie Van der Linden13 Universidad del País Vasco, Bilbao Spain Biophysics Felix Goni14 Lancaster University, Lancaster UK Neurobiology David Allsop15 I.R.F. "Mario Negri”, Milan Italy Pharmacokinetics Mario Salmona16 Stab Vida, Oieras Portugal Antibodies Bruno Mateus17 Université Pierre et Marie Curie, Paris France Post-mortem samples Charles Duyckaerts18 GUERBET SA, Villepinte France Imaging Marc Port19 Bial Industrial Farmacéutica S.A., Bilbao Spain Immunogenicity Alberto Martínez
ALZHEIMER DISEASE NEL : 24 milioni di casi di demenza 4.6 milioni nuovi casi/anno (1 ogni 7 secondi). EU: Circa 5 milioni di casi di demenza Più di 3 milioni di Alzheimer (AD). 2x entro il 2040 in Europa occidentale 3x nell’ Europa dell’est
La produzione progressiva e l’accumulo di b-amiloide (Ab), un frammento della Amyloid Precursor Protein (APP), svolgono un ruolo centrale nella malattia di Alzheimer. APP
Ab aggrega formandoOligomeri, fibrille, placche
IL PROGETTO “NAD” 1- DISGREGAZIONE e IMAGING di placche di Ab nel cervello: terapia e diagnostica combinate (theranostics) in modelli animali di AD.2- CLEARANCE of Ab dal sangue, richiamando l’eccessoanche dal cervello (“sink” effect) in modelli animali di AD. NANOPARTICELLE
FASI 1: Sintesi di NPs2: Selezione e Sintesi di ligandi per Ab /funzionalizzazione di NP 3: Funzionalizzazione di NP per attraversare la barriera emato-encefalica (BBB) 4: Funzionalizzazione di NP per diagnostica (MRI, PET) 5: Biocompatibilità 6: Prove in vitro della efficienza del sistema (cell cultures) 7: Prove in vivo della efficienza del sistema (TG mice)
1- DISGREGAZIONE e IMAGING di placche di AbLe nanoparticelle saranno funzionalizzate per legare Ab e per MRI or PET imaging Ab NPs aggregates
NANOPARTICELLE E BARRIERA EMATOENCEFALICA Ab AGGREGATES Capillari cerebrali blood FLUSSO DEL SANGUE Le Nanoparticelle saranno funzionalizzate per superare la barriera emato encefalica (BBB).Entrando nel cervello per endo/transcitosi
2- RIMOZIONE DI Ab DAL SANGUE ( “SINK EFFECT” )BRAIN b-AMYLOID AGGREGATES EQUILIBRIUM BRAIN/BLOOD SHIFT Blood-brain barrier AbNANOPARTICLES BLOOD CIRCULATION RES + LIVER
“NAD” OBJECTIVES1- DISGREGATION and IMAGING of brain Ab : combined therapy e diagnostics (theranostics) in animal models of AD.