PROJECT SUMMARY/ABSTRACT Every year an estimated 19.4 million children do not receive the set of vaccines recommended by the World Health Organization, leading to 1.5 million vaccine-preventable deaths.1,2 A majority of undervaccinated children live in low- and middle-income countries and often have limited access to healthcare.2,3 Nearly 6 million of these children receive at least one vaccine dose, but remain at risk because they have not completed the full dosing regimen.4,5 A vaccination method that delivers all doses of a vaccine, or multiple vaccines, in a single injection would enable children with even one-time access to healthcare to be fully protected from the corresponding infectious disease. Unfortunately, most controlled-release drug delivery systems exhibit continuous release kinetics, which is vastly different from traditional soluble vaccines administered in multiple discrete doses over a course of months. One recent study has described the development of biodegradable microparticle platform with a polymer shell encapsulating a vaccine-loaded core that exhibits delayed, pulsatile release after a period determined by the polymer degradation rate.6 By injecting patients with a mixed population of particles with different degradation rates, vaccine can be released as discrete pulses, thereby mimicking traditional vaccination schedules known to be safe and effective. Unfortunately, the original microparticle production method negatively affects antigen stability, requires the use of large-gauge needles, and is low-throughput. This project seeks to overcome these challenges by preparing microparticles using coaxial electrospraying, a single-step fabrication process that can produce a single aqueous, vaccine-loaded core surrounded by a shell of polymer. This proposal first aims to create small core-shell microparticles with dense polymeric shells that demonstrate the delayed, pulsatile release of macromolecules in vitro and in vivo. Fluorescently tagged proteins will be used as model vaccines to study the effects of particle size, shell density, relative wall thickness, and post-processing on release kinetics. After identifying formulations that achieve pulsatile release, we will then optimize processing conditions to maximize encapsulated antigen stability. An enzymatic reporter and a pH-sensitive dye will be added to the core and tested at several stages of the particle life cycle to monitor microenvironmental conditions during fabrication, storage, and release. Electrospraying materials and parameters will be adjusted to minimize changes to protein conformation that could result from solvent interactions, thermal instability, and particle acidification, which may affect the immune system's ability to create neutralizing antibodies. Although further optimization will be required to fine-tune conditions for specific vaccines, this project will provide a framework for quickly developing controlled-release vaccine formulations. Ultimatel...