Exploring Tunable Pulsatile-Release Microparticles to Enable Single-Administration Vaccination for Rabies Postexposure Prophylaxis

Introduction: Although completely treatable, approximately ~60,000 die annually due to rabies infections as a result of limited healthcare access and patient non-adherence. Treatment for potential rabies exposure involves immediately administering rabies immunoglobulin followed by four separate vaccine doses spaced over two weeks. Logistical factors such as travel distance, lost wages, cost of treatment, and forgetfulness result in poor adherence to post-exposure treatment for rabies. Recent advances in microfabrication have enabled the generation of biodegradable microparticles exhibiting a burst release of therapeutic after a predetermined delay. The modularity achieved by mixing microparticles that release their payloads after different delays enables the platform to deliver multiple doses of vaccines in a single injection.

Materials and

Methods: Microscale open-faced hollow cylinders of poly(lactic-co-glycolic acid) (PLGA) were fabricated using multi-photon 3D printing and soft lithography. These structures were then filled with a dextran-linked fluorescent dye or inactivated rabies vaccine using a piezoelectric dispensing instrument and then thermally sealed. In vivo release kinetics were determined using an In Vivo Imaging System to monitor the release of fluorescently conjugated dextran from particles injected subcutaneously into mice. The rabies vaccine stability was determined using an enzyme-linked immunoassay specific for the immunity-conferring conformation of the antigen.

Results, Conclusions, and Discussions: Results
We successfully fabricated, filled, and sealed microparticles having a central depot of material fully encompassed by PLGA (Figure 1A). We showed the ability to make microparticles as small as 100 μm (Figure 1B,C), allowing them to be injected through needles commonly used for vaccination. The timing of release can be controlled by selecting PLGAs with different molecular weights, copolymer ratios, and end groups. This is demonstrated in vivo with four PLGAs releasing over 50% of cargo on days 10±1, 15±1, 17±2, and 36±2. The release was pulsatile as each group released over 75% of cargo over a span of three or fewer days with nearly no leakage prior to release (Figures 1D and E). Encapsulating the rabies vaccine through thermal sealing resulted in an approximate 19±6% loss in antigen stability. However, the addition of stabilizing excipients fully mitigated thermal degradation of the vaccine (Figure 1F). Finally, using microparticles containing antigen and a stabilizing excipient formulation, the rabies vaccine can be released in an immunity-conferring state on day 3 and day 7 (Figure 1G) to recapitulate time points of the standard-of-care rabies vaccination schedule.

Discussion
These studies demonstrate the fabrication of fully biodegradable and injectable microparticles with tunable pulsatile release kinetics. Although not inherently stable during encapsulation and release, we show that formulation with the proper excipients can ameliorate these losses, resulting in the release of antigen in its immune conferring conformation. By combining multiple microparticle populations that release at different times, we can recapitulate the multi-injection vaccination schedules currently used in rabies postexposure prophylaxis. Further as a platform these microparticles could potentially revolutionize vaccination by enabling single injection delivery for many other vaccines.

Conclusion
These studies represent a major step forward toward developing a single-injection formulation for rabies postexposure prophylaxis. These results are also exciting because of their potential applications for rabies treatment and beyond, where this platform could reduce the burden to prophylactic vaccines worldwide, significantly reducing healthcare in low-resource settings and improving immunization rates to reduce the 1.7 million vaccine-preventable deaths that occur annually due to limited patient access.

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