(M1430-01-06) Multi-functionalized Cationic Chitosan Polymeric Micelles for pVGF Delivery to the Brain through the Intranasal Route

Purpose: The 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 pVGF to the brain through the intranasal route for the management of Alzheimer’s disease.

Methods: Chitosan (CS, 50kDa) was conjugated with oleic acid (OA), followed by penetratin (PEN) and mannose (MAN) conjugation. The synthesized conjugates were subjected to physical characterization, including N/P ratio optimization, pDNA loading efficiency as well as critical micelle concentration (CMC), and biocompatibility. In-vitro and in-vivo transfection efficiency of OA-g-CS-PEN-MAN/pVGF polyplex was investigated.

Results: Multi-functionalized chitosan polymeric micelles were developed by grafting oleic acid (OA) 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 OA-g-CS-PEN-MAN graft polymer formed cationic nanomicelles in an aqueous medium and polyplexed with pVGF at an N/P ratio of 10:1, which were able to protect the gene from nuclease degradation. The polymeric micelles were nontoxic and cationic in charge and had an average hydrodynamic diameter of 199.8 ± 15.73 nm. Qualitative in vitro transfection efficiency of OA-g-CS-PEN-MAN/pGFP polyplex was investigated in bEnd.3, primary neurons, and astrocyte cells. In-vivo transfection efficiency of OA-g-CS-PEN-MAN/pVGF polyplexes was analyzed in C57BL6/J mice after intranasal administration for 7 days. The VGF expression levels in primary astrocytes and neurons after OA-g-CS-PEN-MAN/pVGF treatment were 2.4 ± 0.24 and 1.49 ± 0.02 pg/μg of protein, respectively (Fig 2). The VGF expression in the OA-g-CS-PEN-MAN/pVGF polyplex-treated animal group was 64.9 ± 12.7 pg/mg of protein, significantly higher (p < 0.01) than that of the unmodified polymeric micelles (Fig 3).

Conclusion: Successfully synthesized and characterized chitosan polymeric nano-micelles functionalized with oleic acid, mannose, and penetratin. The functionalized polymers formed cationic and uniformly sized polymeric nano-micelles. In-vitro transfection potential of OA-g-CS-PEN-MAN/pVGF polyplex was significantly higher (p ≤ 0.01) over unfunctionalized nano-micelle-pVGF polyplexes. Furthermore, significantly higher (p ≤ 0.01) VGF expression was observed in the brain of mice with OA-g-CS-PEN-MAN/pVGF polyplex in comparison to the naked pVGF and CS/pVGF polyplexes. These findings suggest that penetratin and mannose-functionalized oleic acid-chitosan graft polymers are an efficient non-viral gene delivery vector for brain-targeted gene delivery through the intranasal route.

References: Lamptey, R.N.L.; Gothwal, A.; Trivedi, R.; Arora, S.; Singh, J. Synthesis and Characterization of Fatty Acid Grafted Chitosan Polymeric Micelles for Improved Gene Delivery of VGF to the Brain through Intranasal Route. Biomedicines 2022, 10, 493.

Acknowledgements: This research was supported by the National Institutes of Health (NIH) Grants R01 AG051574, RF1 AG068034, and 3RF1AG068034-01A1S1. The authors declare no conflict of interest.

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

Figure 2: Qualitative and quantitative VGF expression in different cell lines.

Figure 3: In vivo VGF expression in mice after noninvasive intranasal administration of saline, naked pVGF, CS/pVGF, and OAg-CS-PEN-MAN/pVGF polyplexes. Results are represented as mean ± SD (n = 6). (** represents significant (p ≤ 0.01) difference)