(W0930-01-06) Intranasal Delivery of pApoE to the Brain for Developing Effective Therapeutics for Alzheimer’s disease

Purpose: The influential role of apolipoprotein E gene (ApoE’s) in Alzheimer’s disease (AD) progression is complex and multifactorial. Elevated expression of ApoE4 increases the risk of AD developing several folds. Conversely, higher expression of ApoE2 level decreases the LOAD risk. This suggests ApoE2 gene therapy could be a better therapeutic approach in AD management. The purpose of this study was to develop multi-functionalized micelles using oleic acid (OA), penetratin (PEN), and mannose (MAN) conjugated chitosan for the effective delivery of pApoE to the brain.

Methods: Chitosan (50 kDa) was conjugated with oleic acid (OA), followed by penetratin (PEN) and mannose (MAN) to obtain OA-g-CS-PEN-MAN conjugates. The synthesized conjugates were subjected to surface morphology evaluation using scanning electron microscopy (SEM). Surface topography and the non-crystalline phase of the developed conjugates were analyzed using atomic force microscopy (AFM) and selected area electron diffraction (SAED), respectively. Quantitative in vitro transfection efficiency of OA-g-CS-PEN-MAN/pApoE polyplex was investigated in primary neurons and astrocyte cells. In-vivo transfection efficiency of OA-g-CS-PEN-MAN/pApoE polyplexes was analyzed in C57BL6/J mice after intranasal administration for 7 days.

Results: The SEM results revealed globular and uniform size distribution of OA-g-CS-PEN-MAN/pApoE polymeric micelles. The smooth surface topography of OA-g-CS-PEN-MAN polymeric micelles can be visualized in the AFM images (Fig 1), the height of the sample was 25 nm. Synthesized OA-g-CS-PEN-MAN conjugate was non-crystalline as the irregular interplanar distances can be observed in the SAED pattern in Figure 1B and 1C. The developed polymeric micelles exerted < 5 % hemolysis even at the higher concentration of 600 µg/mL (Fig 2). The ApoE expression levels in primary astrocytes after OA-g-CS-PEN-MAN/pApoE treatment were 11.5 ± 1.6 ng/μg of protein. The ApoE expression in the OA-g-CS-PEN-MAN/pApoE polyplex-treated animal group was 81.28 ± 6.17 ng/mg of protein, significantly higher (p < 0.01) than that of the unmodified polymeric micelles.

Conclusion: Prepared polymeric micelles had uniform shape and size distribution. Functionalization with PEN, and MAN improved their transfection efficiency over the unmodified chitosan micelles in-vitro (in astrocytes and neurons) and in-vivo. Our findings suggest OA-g-CS-PEN-MAN polymeric micelles are effective nano-carrier systems for targeted gene delivery to the brain through the intranasal route to develop effective therapeutics for Alzheimer’s disease.

Acknowledgements: This research was supported by the National Institutes of Health (NIH) Grants RF1 AG068034 and 3RF1AG068034-01A1S1. The authors declare no conflict of interest.

A) SEM image SEM of OA-g-CS-PEN-MAN polymeric micelles, B and C) AFM images of OA-g-CS-PEN-MAN polymeric micelles, D) SAED pattern of OA-g-CS-PEN-MAN polymeric micelles.

Percent hemolysis of different concentrations of 50k chitosan and conjugates against erythrocytes, data are represented as mean ± SD (n=5).

In vivo ApoE expression in mice after noninvasive intranasal administration of saline, naked pApoE, CS/ pApoE, and OAg-CS-PEN-MAN/ pApoE polyplexes. Results are represented as mean ± SEM (n = 6). (** represents significant (p ≤ 0.01) difference)