Optimizing zinc-HisTag coordination remote loading of proteins in PLGA microspheres

Metal-HisTag coordination remote loading (MHCRL) of proteins in PLGA microspheres was previously developed to provide a useful tool for discovery and preclinical development of controlled release protein formulations. Here we describe optimization of MHCRL, including (1) reducing thermal stress, (2) decreasing the complexity and duration of the procedure, (3) increasing loading capacity, (4) increasing the penetration depth of protein, and (5) improving the release profile. Directly encapsulating ZnCO₃as a Zn²⁺source for HisTag coordination, rather than remotely loading Zn²⁺, increased the Zn content ∼6-fold. Microspheres with directly encapsulated ZnCO₃more deeply encapsulated green fluorescent protein and more efficiently encapsulated human serum albumin at protein loading solutions concentrations ≥100 μg/mL than remotely loaded Zn²⁺microspheres. Tributyl acetylcitrate plasticized microspheres in terms of decreasingT_g, but led to a decrease in protein encapsulation efficiency. As such, the plasticizer was not deemed useful. The loading/healing cycles were reduced in time and temperature from 48 h/42 h at 43 °C to 2 h/6h at 37 °C while maintaining strong encapsulation efficiency, resulting in significantly improved protein stability. Immunoreactive protein was slowly released for months following a modest burst release. The improved microspheres and shorter, low-temperature encapsulation could be a valuable asset to drug discovery scientists interested in controlled release of delicate and/or costly biologic candidates.