Krems2013

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Krems2013

  1. 1. NADNANOPARTICLES FOR THERAPY AND DIAGNOSIS OF ALZHEIMER’S DISEASE 2008-2013 FP7-NMP-2007-LARGE-1 Massimo Masserini Department of Health Sciences University of Milano-Bicocca
  2. 2. ALZHEIMER DISEASE (AD)AD is a neurodegenerative disorder that begins with deficits in short-term memory and culminates in total 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 2040 Sporadic (majority) Small number of cases are familial forms [Honjo K. et al. J. Neurol. Sci. 2012]
  3. 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 ANTAGONISTS↓NMDA 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. 4. NAD STARTING PREMISESProgressive production in brain and accumulation of Aβ, a fragment of Amyloid Precursor Protein (APP), is central in Alzheimer Disease. Beta-Amyloid Oligomers Beta-Amyloid Fibrils [Honjo K. et al. J. Neurol. Sci. 2012]
  5. 5. THE OBJECTIVE OF NAD PROJECT: TO REMOVE ABETA FROM THE BRAIN UTILIZING NANOPARTICLESLIPOSOMES
  6. 6. 1 st PHASETo find a ligand for Aβ peptide
  7. 7. SCREENING OF Aβ/LIPIDS INTERACTIONThin Layer Chromatography (TLC) immunostaining
  8. 8. Standard TLC Immuno- TLC Cardiolipin (CL) Phosphatidic Acid (PA) gangliosides 0.5 nmoles each lipid + 2.5 mg/mL of AβStrong interaction with Acidic lipids: phosphatidic acid (PA) and cardiolipin (CL) (+ gangliosides) [Re F. et al. Biomaterials 2010]
  9. 9. LIPOSOMES CARRYING PHOSPHATIDIC ACID (PA) Liposomes composition: Sphingomyelin/Cholesterol (1:1 M/M) 5-20mol% of PA by extrusion procedure Ø = 100nmDiameter = 102 ± 3 nm PDI = 0,098 Z-pot = -24,3 ± 5 mV Stable > 1 week
  10. 10. LIPOSOMES FUNCTIONALIZED WITH ACIDIC LIPIDS SHOW HIGH BINDING CAPACITY TOWARDS Aβ Kd 50-60 nM (Surface Plasmon Resonance) [Re F. et al. Biomaterials 2010]
  11. 11. LIPOSOMES FUNCTIONALIZED WITH PA RESCUE Aβ TOXICITY IN N2A CELLS 5µM Aβ5µM Aβ PLAIN +CL +PA PLAIN +CL +PA Wt N2a Neuroblastoma N2a Neuroblastoma overexpressing APP [Bereczki E. et al.Nanomedicine NBM 2011]
  12. 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. 13. 2nd PHASE: To find a ligand for the blood brain barrier (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. 14. SYNTHESIS OF LIPOSOMES WITH BBB-LIGAND Thiol-maleimide Thiol-maleimide linkage linkage Thiolated Anti-TfR antibody (OX-26 or RI-7217) Ø = 141 nm Ø = 175,3 nm PDI = 0,223 or TAT-1 peptide PDI = 0,170Diameter = 135 ± 9 nm Diameter = 125 ± 2 nmPDI = 0,141 PDI = 0,122Stability > 1 week Stability > 1 weekZ-pot = -15,30 ± 1,75 mV Z-pot = -33,12 ± 1,25 mV
  15. 15. CROSSING OF A BBB MODELTranswell system for BBB model in vitro hCMEC/D3 cells CELLULAR UPTAKE MONOLAYER PERMEABILITY (cm/min)
  16. 16. PERMEABILITY OF LIPACROSS hCMEC/D3 MONOLAYER 14 C-PA / 3H-Sphingomyelin Lipid dose = 200 nmols of total lipids 0,5-2h incubation
  17. 17. ARE THESE NPs SUITABLE FOR TREATING ALZHEIMER DISEASE IN VIVO ?
  18. 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 old liposomes intra-peritoneal injection I.P. APP/PS1 Tg mice containing 130ug of PA (weight 22-25g)(total lipids = 2,5mg/injection) 25 days (3 times a week) BRAIN 1- immunohystochemical analysis of brain slices 2- Aβ quantification by ELISA assay BLOOD on brain homogenate and plasma
  19. 19. TG MICE TREATED WITH PA-LIPOSOMES FUNCTIONALIZED WITH ApoEBRAIN Abeta content after treatment (ELISA test) * -40% *p<0,01 ELISA assays
  20. 20. BEHAVIOURAL TEST:OBJECTS RECOGNITION TRAINING PHASE In each phase, the time spent exploring each of the objects is quantified, and calculation of a novelty or «discrimination index» is based on these measurements. TEST PHASE
  21. 21. ALMOST TOTAL RECOVERY OF COGNITIVE DEFICIT AFTER TREATMENT WITH ApoE-PA-LIPOSOMES
  22. 22. MECHANISM OF ACTION ?
  23. 23. WHERE HAS BRAIN Aβ GONE?1. The excess of Aβ is recovered in liver and spleen
  24. 24. IN VIVO BIODISTRIBUTION OF LIPOSOMES IN HEALTHY MICE 14 C-PA / 3H-Sphingomyelin [PA]brain ~ 1,5µM Sm/ molar ratio PA 2. ApoE-PA LIPOSOMESin original liposomes = 12 in the blood = 12± 10 CROSS THE BBB IN VIVO in the brain = 12± 6
  25. 25. …OUR HYPOTHESIS… PREVENT AGGREGATION PA PA DISAGGREGATIONliver spleen PA SINK EFFECT PA PA PA PA PA
  26. 26. PUBLICATIONSMORE THAN 20 PUBLICATIONS ABOUT THESE LIPOSOMESON INTERNATIONAL JOURNALS PATENTS1. M. Masserini, F. Re, S. Sesana. Liposomes efficientlybinding beta-amyloid. PCT International patent.2. M. Masserini, F. Re, G. Sancini, G. Forloni, M.Salmona. Liposomes active in-vivo on neurodegenerativediseases (in particular Alzheimers disease). USA patent.
  27. 27. Department of Health Sciences University of Milano-BicoccaFrancesco FrancescaMantegazza Re Laura Elisa Bana Salvati Giulio Sancini Silvia Sesana Adriana Monti Maria Gregori

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