1. NAD
NANOPARTICLES 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. 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. No treatment today can cure AD
However, for some people, in the early and middle stages of the
disease, drugs may help prevent some symptoms (e.g. sleepness,
agitation, wandering, anxiety, and depression) from becoming
worse, for a limited time.
CHOLINESTERASE INHIBITORS
↑ Acetylcholine brain levels
↑ Communication between nerve cells
NMDA 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. NAD STARTING PREMISES
Progressive 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. THE OBJECTIVE OF NAD PROJECT:
TO REMOVE ABETA FROM THE BRAIN
UTILIZING NANOPARTICLES
LIPOSOMES

6. 1 st PHASE
To find a ligand for Aβ peptide

7. SCREENING OF Aβ/LIPIDS INTERACTION
Thin Layer Chromatography (TLC) immunostaining

8. Standard TLC Immuno- TLC
Cardiolipin (CL)
Phosphatidic Acid (PA)
gangliosides
0.5 nmoles each lipid
+ 2.5 mg/mL of Aβ
Strong interaction with Acidic lipids: phosphatidic acid (PA) and
cardiolipin (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 PA
by extrusion procedure Ø = 100nm
Diameter = 102 ± 3 nm
PDI = 0,098 Z-pot = -24,3 ± 5 mV
Stable > 1 week

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. LIPOSOMES FUNCTIONALIZED WITH PA
RESCUE Aβ TOXICITY IN N2A CELLS
5µM Aβ
5µM Aβ PLAIN +CL +PA PLAIN +CL +PA
Wt N2a Neuroblastoma N2a Neuroblastoma overexpressing APP
[Bereczki E. et al.Nanomedicine NBM 2011]

12. 1 st Consideration
We have synthesized liposomes with high
affinity for Abeta peptide and able to
rescue neurons from Abeta toxicity in vitro
….but there is a blood-brain barrier

13. 2nd PHASE:
To find a ligand for the blood brain
barrier (BBB) targeting
1- 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-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 = 0,170
Diameter = 135 ± 9 nm Diameter = 125 ± 2 nm
PDI = 0,141 PDI = 0,122
Stability > 1 week Stability > 1 week
Z-pot = -15,30 ± 1,75 mV Z-pot = -33,12 ± 1,25 mV

15. CROSSING OF A BBB MODEL
Transwell system for BBB model in vitro
hCMEC/D3 cells
CELLULAR UPTAKE
MONOLAYER PERMEABILITY (cm/min)

16. PERMEABILITY OF LIP
ACROSS hCMEC/D3 MONOLAYER
14
C-PA / 3H-Sphingomyelin
Lipid dose = 200 nmols of total lipids
0,5-2h incubation

17. ARE THESE NPs SUITABLE FOR TREATING
ALZHEIMER DISEASE IN VIVO ?

18. DOUBLE TRANSGENIC MOUSE CONTAINING THE MUTANT VERSIONS
OF 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. TG MICE TREATED WITH PA-LIPOSOMES
FUNCTIONALIZED WITH ApoE
BRAIN Abeta content after treatment (ELISA test)
*
-40%
*p<0,01
ELISA assays

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. ALMOST TOTAL RECOVERY OF
COGNITIVE DEFICIT
AFTER TREATMENT WITH
ApoE-PA-LIPOSOMES

22. MECHANISM OF ACTION ?

23. WHERE HAS BRAIN Aβ GONE?
1. The excess of Aβ is recovered in liver and spleen

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 LIPOSOMES
in original liposomes = 12
in the blood = 12± 10 CROSS THE BBB IN VIVO
in the brain = 12± 6

25. …OUR HYPOTHESIS…
PREVENT AGGREGATION PA
PA
DISAGGREGATION
liver spleen PA
SINK EFFECT
PA
PA PA
PA
PA

26. PUBLICATIONS
MORE THAN 20 PUBLICATIONS ABOUT THESE LIPOSOMES
ON INTERNATIONAL JOURNALS
PATENTS
1. M. Masserini, F. Re, S. Sesana. Liposomes efficiently
binding beta-amyloid. PCT International patent.
2. M. Masserini, F. Re, G. Sancini, G. Forloni, M.
Salmona. Liposomes active in-vivo on neurodegenerative
diseases (in particular Alzheimer's disease). USA patent.

27. Department of Health Sciences
University of Milano-Bicocca
Francesco Francesca
Mantegazza Re Laura
Elisa
Bana
Salvati
Giulio Sancini Silvia Sesana Adriana Monti
Maria Gregori