(T1230-04-21) Synthesis and Characterization of Multi-Functionalized Chitosan Polymeric Micelles for Intranasal Delivery of pApoE2 to PS19 Tauopathy Mice

Purpose: Alzheimer’s disease (AD) is a disorder that progresses with neurodegeneration in the brain, destroys thinking skills, memory and eventually ability to perform daily tasks. In AD, accumulation of amyloid beta (Aβ) plaques outside of the neurons and tau tangles inside the neurons takes place. The Aβ restricts the neuron-to-neuron communication whereas tau tangles block flow of the nutrients and other essential molecules inside the neurons for normal functioning and survival. These two hall mark changes lead to neurodegeneration and it progresses over the course of time. The presence of blood brain barrier (BBB) restricts the transportation of drug/gene into the brain. Intranasal route is an effective route of administration to the brain due to the presence of the olfactory nerves in the upper nasal cavity, which connects the brain. The present study aims to develop multi-functionalized chitosan polymeric micelles that can effectively deliver pApoE2 to the brain through the intranasal route for the management of Alzheimer’s disease.

Methods: Chitosan (CS, 50kDa) was conjugated with caproic acid (Cap), followed by penetratin (PEN) and mannose (MAN) conjugation. The synthesized conjugates were subjected to physical characterization, including N/P ratio optimization, pDNase protection, and biocompatibility. In-vitro transfection efficiency of Cap-g-CS-PEN-MAN/pApoE2 polyplex was investigated in primary neurons and astrocytes. Therapeutical effect of the polyplexes were investigated in transgenic PS19 tauopathy mice.

Results: Multi-functionalized chitosan polymeric micelles were developed by grafting caproic acid (Cap) on the chitosan (CS) skeleton followed by penetratin (PEN) and mannose (MAN) conjugation. All the conjugates were characterized using ¹H-NMR and FT-IR spectroscopy (Fig 1). The Cap-g-CS-PEN-MAN graft polymer formed cationic nano-micelles in an aqueous medium and polyplexed with pApoE2 at an N/P ratio of 20:1, which were able to protect the gene from nuclease degradation. Hemotoxicity and cytotoxicity were evaluated against erythrocytes and bEnd.3 cells, respectively (Fig 2). Qualitative in vitro transfection efficiency of Cap-g-CS-PEN-MAN/pApoE2 polyplex was investigated, primary neurons, and astrocyte cells (Fig 3). Total tau and phosphorylated tau (p181) levels were analyzed in PS19 mice after intranasal administration for 7 days. The pApoE2 expression levels in primary astrocytes and neurons after Cap-g-CS-PEN-MAN/pApoE2 treatment were 8.7 ± 0.34 and 3.58 ± 1.10 ng/µg of protein, respectively. Total tau and phosphorylated tau (p181) levels in PS19 mice brain after intranasal administration for 7 days were 6067.97 ± 858.33 pg/mg and 157.91 ± 18.14 pg/mg of protein.

Conclusion: Successfully synthesized and characterized chitosan polymeric nano-micelles functionalized with caproic acid, mannose, and penetratin. The functionalized polymers formed cationic and uniformly sized polymeric nano-micelles. In-vitro transfection potential of Cap-g-CS-PEN-MAN/pApoE2 polyplex was significantly higher (p ≤ 0.01) over unfunctionalized nano-micelle-pApoE2 polyplexes. Furthermore, total tau and phosphorylated tau levels were significantly reduced in the brain of PS19 tauopathy mice with Cap-g-CS-PEN-MAN/pApoE2 polyplex in comparison to the saline and naked pApoE2 treated groups. These findings suggest that penetratin and mannose-functionalized caproic acid acid-chitosan graft polymers are an efficient non-viral gene delivery vector for brain-targeted gene delivery through the intranasal route.

Acknowledgements: This research was supported by the National Institutes of Health (NIH) Grants R01 AG051574, RF1 AG068034 and 3RF1AG068034-01A1S1


Fig 1: A) represents 1H-NMR spectra of CS, Cap-g-CS, Cap-g-CS-PEN, Cap-g-CS-MAN and Cap-g-CS-PEN-MAN conjugates, where B) represents FT-IR spectra of CS, Cap-g-CS, OA-g-CS-PEN, Cap -g-CS-MAN and Cap-g-CS-PEN-MAN conjugates.


Fig 2: A) represents percent cell viability in bEnd.3 cells and B) percent hemotoxicity in erythrocytes at different concentrations.


Fig 3: Qualitative transfection potential of pGFP, CS/pGFP, Cap-g-CS/pGFP, Cap-g-CS-pEN/pGFP, Cap-g-CS-MAN/pGFP and Cap-g-CS-PEN-MAN/pGFP polyplexes in primary neurons and astrocytes.