(M1030-09-55) Targeted Intranasal Delivery of pApoE2 via Exosome-Polymeric Micelle Hybrid Nanocarrier Attenuates Neuroinflammation in Aged Mice

Purpose: Gene delivery to the brain is challenging due to the presence of the blood-brain barrier (BBB). Moreover, genetic materials are prone to nuclease degradation, further complicating their effective delivery to the brain. The intranasal route provides a non-invasive and direct means of accessing the brain through the olfactory and trigeminal nerves. This study focuses on the development of a hybrid nanocarrier comprising exosomes and multi-functionalized chitosan polymeric micelles that can effectively deliver pApoE2 to the brain via the intranasal route.

Methods: Chitosan (CS, 50kDa) was conjugated with caproic acid (Cap), penetratin (PEN), and mannose (MAN). The synthesized polymeric micelles were complexed with Human iPSC-derived exosomes to develop the hybrid Exo/Cap-g-CS-PEN-MAN nanocarrier. The prepared hybrid nanocarrier system was characterized, including size, Zeta, Gene loading, DNase protection, and biocompatibility. The cytotoxicity of the developed hybrid nanocarrier platform was evaluated against primary astrocytes. In-vitro transfection efficiency of Exo/Cap-g-CS-PEN-MAN/pApoE2 hybrid polyplex was investigated in primary neurons and astrocytes. In vivo brain transfection and therapeutic efficacy against neuro-inflammation were evaluated in 3 and 24-month-old mice for a comparative analysis after intranasal administration.

Results: The Exo/Cap-g-CS-PEN-MAN hybrid showed uniform distribution with a diameter of ≤ 250 nm. The gene loading of the developed hybrid nanocarrier was ≥ 80%. In-vitro toxicity assessment revealed that the hybrid nanocarrier was non-toxic against primary astrocytes at different concentrations. Qualitative in-vitro transfection studies showed significantly higher transfection in hybrid polyplex-treated cells (Fig. 1). Furthermore, the Exo/Cap-g-CS-PEN-MAN /pApoE2 polyplex demonstrated elevated expression of ApoE in the 24-month-old mouse brain. Additionally, aged mice have shown significantly reduced levels of TNF-α and IL-6 after the treatment with Exo/Cap-g-CS-PEN-MAN /pApoE2 polyplex (Fig. 2).

Conclusion: The multi-functional hybrid nanocarrier, based on polymeric micelles and exosomes, was successfully developed and characterized. Our hybrid nanocarrier system did not exert any toxicity against primary astrocytes. The hybrid nanocarrier system has shown higher in vivo transfection in primary astrocytes, neurons, and in the brains of 24-month-old mice. Our findings suggest that multi-functionalized chitosan polymeric micelles and exosome-based hybrid nanocarriers could be a more effective approach for the intranasal delivery of genetic material to the brain, potentially leading to the development of an effective treatment for brain disorders.

Acknowledgements: This research was supported by the National Institutes of Health (NIH) Grants R01 AG068034-02, RF1 AG068034, R01AG083981, and RF1AG068034-01A1S1.

Fig. 1: Qualitative in-vitro transfection in bEnd.3, primary astrocytes and neurons after treating with pGFP, Exosome/pGFP, Cap-g-CS-PEN-MAN/pGFP and Exo/Cap-g-CS-PEN-MAN/pApoE2 polyplexes.


Fig 2: (A) TNF-α (B) IL-6 levels in the brains of 24 months old C57BL6/j mice after the intranasal administration of saline, pApoE2, exosomes/pApoE2, Cap-CS-PEN-MAN/pApoE2, and exosome/Cap-CS-PEN-MAN/pApoE2 polyplexes. The dosing was administered on 1st, 3rd, and 5th day; the animals were sacrificed at the 7th day after the last administration. The data is represented as mean ± SD (n=6)