1. NADNANOPARTICLES FOR THERAPY AND DIAGNOSISOF ALZHEIMERS DISEASE 2008-2013 FP7-NMP-2007-LARGE-1Massimo Masserini Department of Health SciencesUniversity of Milano-Bicocca

2. ALZHEIMER DISEASE (AD)AD is a neurodegenerative disorder that begins with deficits in short-term memory and culminates intotal loss of cognition and executive functions WORLD : 36 Mln demented subjects 4,6 Mln new cases/y (1 every 7 seconds) EU: >2 Mln AD 2x by 2040Sporadic (majority)Small number of cases are familial forms[Honjo K. et al. J. Neurol. Sci. 2012] 3. No treatment today can cure ADHowever, for some people, in the early and middle stages of thedisease, drugs may help prevent some symptoms (e.g. sleepness,agitation, wandering, anxiety, and depression) from becomingworse, for a limited time.CHOLINESTERASE INHIBITORS Acetylcholine brain levels Communication between nerve cellsNMDA ANTAGONISTSNMDA receptor activity cognitive defects Drugs in the pipeline TARGETS Beta-amyloid and enzymes connected (beta and gamma-secretase) Inflammation Insulin resistance [Fazil M. et al. J. of Drug Targ. 2012] 4. NAD STARTING PREMISESProgressive production in brain and accumulation of A, a fragment of Amyloid Precursor Protein (APP), is central in Alzheimer Disease.Beta-AmyloidOligomersBeta-Amyloid Fibrils[Honjo K. et al. J. Neurol. Sci. 2012] 5. THE OBJECTIVE OF NAD PROJECT: TO REMOVE ABETA FROM THE BRAIN UTILIZING NANOPARTICLESLIPOSOMES 6. 1 st PHASETo find a ligand for A peptide 7. SCREENING OF A/LIPIDS INTERACTIONThin Layer Chromatography (TLC) immunostaining 8. Standard TLC Immuno- TLCCardiolipin (CL)Phosphatidic Acid (PA)gangliosides0.5 nmoles each lipid+ 2.5 mg/mL of AStrong interaction with Acidic lipids: phosphatidic acid (PA) andcardiolipin (CL) (+ gangliosides)[Re F. et al. Biomaterials 2010] 9. LIPOSOMES CARRYING PHOSPHATIDIC ACID (PA)Liposomes composition:Sphingomyelin/Cholesterol (1:1 M/M) 5-20mol% of PAby extrusion procedure = 100nmDiameter = 102 3 nm PDI = 0,098Z-pot = -24,3 5 mV Stable > 1 week 10. LIPOSOMES FUNCTIONALIZED WITH ACIDIC LIPIDSSHOW HIGH BINDING CAPACITY TOWARDS A Kd 50-60 nM (Surface Plasmon Resonance)[Re F. et al. Biomaterials 2010] 11. LIPOSOMES FUNCTIONALIZED WITH PA RESCUE A TOXICITY IN N2A CELLS 5M A5M A PLAIN +CL+PA PLAIN +CL +PA Wt N2a NeuroblastomaN2a Neuroblastoma overexpressing APP[Bereczki E. et al.Nanomedicine NBM 2011] 12. 1 st ConsiderationWe have synthesized liposomes with highaffinity for Abeta peptide and able torescue neurons from Abeta toxicity in vitro .but there is a blood-brain barrier 13. 2nd PHASE:To find a ligand for the blood brainbarrier (BBB) targeting1- ApoE-DERIVED PEPTIDES(aa 141-150 monomer or dimer)2- CELL-PENETRATING PEPTIDES (TAT-1 from HIV)3-ANTI-TfR ANTIBODIES (OX-26, RI-7217) 14. SYNTHESIS OF LIPOSOMES WITH BBB-LIGANDThiol-maleimideThiol-maleimidelinkagelinkage Thiolated Anti-TfR antibody (OX-26 or RI-7217) = 141 nm = 175,3 nm PDI = 0,223or TAT-1 peptidePDI = 0,170Diameter = 135 9 nmDiameter = 125 2 nmPDI = 0,141PDI = 0,122Stability > 1 week Stability > 1 weekZ-pot = -15,30 1,75 mV Z-pot = -33,12 1,25 mV 15. CROSSING OF A BBB MODELTranswell system for BBB model in vitrohCMEC/D3 cells CELLULAR UPTAKEMONOLAYER PERMEABILITY (cm/min) 16. PERMEABILITY OF LIPACROSS hCMEC/D3 MONOLAYER 14C-PA / 3H-SphingomyelinLipid dose = 200 nmols of total lipids0,5-2h incubation 17. ARE THESE NPs SUITABLE FOR TREATING ALZHEIMER DISEASE IN VIVO ? 18. DOUBLE TRANSGENIC MOUSE CONTAINING THE MUTANT VERSIONSOF APP-PS1 HUMAN GENES (STRAIN - B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J ) : A MOUSE MODEL OF ALZHEIMER DISEASE WITH SENILE PLAQUES AFTER 7-8 M (AMYLOIDOSIS)100uL of [40mM]8 months oldliposomesintra-peritoneal injection I.P. APP/PS1 Tg micecontaining 130ug of PA(weight 22-25g)(total lipids = 2,5mg/injection) 25 days (3 times a week) BRAIN1- immunohystochemical analysis of brain slices 2- A quantification by ELISA assayBLOODon brain homogenate and plasma 19. TG MICE TREATED WITH PA-LIPOSOMESFUNCTIONALIZED WITH ApoEBRAIN Abeta content after treatment (ELISA test)*-40%*p