(J-387) Microneedle Mediated Mucosal Immunization

Introduction:: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus invade the body via mucosal routes of infection [1]. Intramuscular (IM) vaccines, however, elicit varying degrees of protective mucosal immunity. IM vaccines also require -70°C storage and administration in centralized settings, creating significant barriers to distribution particularly in low-income countries, which saw Covid vaccination rates 8-fold lower than high-income countries [2]. To elicit mucosal immune responses and improve equitable vaccine distribution, we developed electrospun microneedle (MN) patches for immunization of the oral cavity. MNs were formulated with GFP plasmid DNA (pDNA) or GFP self-replicating RNA (repRNA) for in vitro studies, and influenza pDNA or SARS-CoV-2 repRNA for in vivo studies. The MNs can be self-administered and deliver cargo to the buccal mucosa, a site capable of inducing potent antibody and T cell responses in both local and distal mucosal tissues [3].

Materials and Methods:: GFP or influenza pDNA were complexed with Lipofectamine (Thermo) to form DNA polyplexes. GFP or SARS-CoV-2 repRNA were complexed with LION, a cationic oil-in-water emulsion (HDT Bio) to form RNA polyplexes. DNA or RNA polyplexes were emulsified in poly(lactic-co-glycolic acid) (PLGA, Lactel) at 0.10 µg/µL and electrospun into a custom MN mold to a dose of 1.25 µg nucleic acid. DNA or RNA polyplexes were dissolved in poly(vinyl acetate) (PVA, Spectrum Chemical) at 16.9 ng/µL and poly(vinyl pyrrolidone) (PVP, Sigma) at 56.2 ng/µL; solutions were sequentially added to MN molds under vacuum to a total dose of 8 µg nucleic acid. Baby hamster kidney (BHK) cells were transfected with aqueous polyplexes (positive control) or MNs by incubation in OptiMem for 4 hours and FBS-supplemented DMEM for 20 hours, followed by fluorescence microscopy and flow cytometry.

Results, Conclusions, and Discussions:: MN patches were fabricated with an average dose of 6.42 µg (influenza pDNA MN) and 5.65 µg (SARS-CoV-2 repRNA MN). GFP pDNA MNs were found to have low but non-negligible transfection efficiency in vitro. Notably, aqueous pDNA polyplexes stored for 48 hours at 4°C lost all transfection efficiency, while pDNA MNs stored up to 8 days at 20°C showed no decrease in transfection. GFP repRNA MNs demonstrated robust transfection efficiency which was maintained after storage up to 7 days at 4°C. This stability may result from the high viscosity of the polymer solutions, which reduces degradation pathway kinetics. Higher efficiency in repRNA MNs may be due to repRNA’s self-amplification within the cytosol and different lipophilic carrier systems. These MNs represent a self-administered vaccine platform with reduced need for cold chain storage; ongoing studies are evaluating systemic and mucosal immune responses to buccal immunization with SARS-CoV-2 repRNA MNs and influenza pDNA MNs.

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References (Optional): : [1] Batty, C. J. et al. Adv. Drug Deliv. Rev. 169 (2021). [2] Pilkington, V. et al. Front. Public Health., 10 (2022). [3] Creighton, R. L. et al. Adv. Healthc. Mater., 8, 4 (2018).