Metal - HisTag Coordination for Remote Loading of Biomacromolecules into PLGA Microspheres

Abstract

Challenges to discovery and preclinical development of long-acting release systems for protein therapeutics include protein instability and use of organic solvents during encapsulation, specialized equipment and personnel, and high costs. Remote loading self-healing encapsulation has been used to gently and efficiently encapsulate proteins in controlled-release polymers, primarily through protein-specific affinity for a trapping agent. To create a universal remote loading self-healing encapsulation platform, coordination bonds between polyhistidine tags (HisTags) of proteins and divalent transition metal cations (M2+) in self-healing polymers were utilized, similar to immobilized metal affinity chromatography. Porous, drug-free self-healing poly(lactic-co-glycolic acid) (PLGA) microspheres with high molecular weight dextran sulfate (HDS) and immobilized remotely-loaded M2+ ions were placed in the presence of proteins with HisTags to bind in the polymer pores before healing the surface with modest temperature. Using human serum albumin (HSA), insulin-like growth factor 1, and granulocyte-macrophage colony-stimulating factor (GM-CSF), encapsulation efficiencies (EE) of immunoreactive protein increased with the inclusion of HisTags and Zn2+. Immunoreactive protein was continuously released over seven to ten weeks. GM-CSF showed bioactivity >95% relative to immunoreactive protein throughout. Increased EEs were found with other M2+ ions, but not with Ca2+. Ethylenediaminetetraacetic acid interfered with this process, reverting EE to Zn2+-free levels. Following this promising proof-of-concept work, areas of potential improvement were identified: (1) reducing thermal stress, (2) decreasing the complexity and duration of the protocol, (3) increasing the loading capacity, (4) increasing the penetration depth of protein, and (5) improving the release profile. Directly encapsulating ZnCO3, rather than remotely loading Zn2+, increased the Zn content in the microspheres ~6-fold. Microspheres with directly encapsulated ZnCO3 (DEZnCO3) more efficiently encapsulated HSA at protein loading solution concentrations ≥ 100 μg/mL than remotely loaded Zn2+ (RLZn2+) microspheres. HisTag green fluorescent protein was more deeply encapsulated in DEZnCO3 microspheres than in RLZn2+ microspheres. Tributyl acetylcitrate was an effective plasticizer in terms of decreasing the glass transition temperature, but also led to a decrease in EE. The loading stage was reducible to 2 hours at 4°C and the healing stage to 6 hours at 37°C while maintaining strong EE for DEZnCO3 microspheres, which slowly released immunoreactive protein for months, following a substantial burst release. Plasticization decreased the initial burst release. Next, the effects of various excipients on physiochemical properties of the formulations were studied. HDS was shown to function as a porosigen and encourage water uptake. While HDS did not significantly affect the Zn2+ loading of DEZnCO3 microspheres, it was shown be critical in remote loading of M2+ cations. Though the erosion and degradation profiles of the microspheres were not affected by HDS, replacing MgCO3 with ZnCO3 accelerated erosion and degradation significantly, potentially owed to the superior pH-modulation of MgCO3. HDS exhibited a high burst release followed by a plateau and seemingly degradation-dependent release. Zn2+ release appeared erosion-driven and was released more quickly from ZnCO3-containing microspheres than from MgCO3-containing microspheres. HisTag HSA release from HDS-free DEZnCO3 microspheres showed higher burst and faster release than HDS-containing formulations have shown previously. The self-healing polymer platform described here for remote loading of HisTag proteins could be a valuable asset to drug discovery and early development scientists interested in the controlled release of delicate biologic candidates using very small quantities of the proteins in in vitro and pre-clinical in vivo studies, providing valuable information to inform further potential development and clinical translation.