Stabilization of proteins encapsulated in cylindrical poly(lactide-co-glycolide) implants: Mechanism of stabilization by basic additives

Purpose, A previous study from our group has shown that in the acidic micro climate of paly(lactide-co-glycolide) (PLGA) implants, encapsulated BSA for ms insoluble noncovalent aggregates and is hydrolyzed during in vitro relea se. Incorporation of ME(OH)(2) strongly inhibits these mechanisms of instab ility and facilitates continuous protein release. The purpose of this study was to determine the protein stabilization mechanism in the presence of ba sic additives. Methods. BSA, as a model protein, was encapsulated in PLGA millicylinders b y a solvent extrusion method. The release of BSA from the PLGA millicylinde rs with and without basic additives (Mg(OH)(2), Ca(OH)(2), ZnCO3 and Ca-3(P O4)(2)) in a physiological buffer was carried out at 37 degrees C acid quan tified by a modified Bradford assay. The insoluble aggregates extracted Fro m the polymer with acetone were reconstituted in a denaturing (6 M urea) or denaturing/reducing solvent (6 M ureal 10 mM DTT) to determine the type of aggregation. Results. Aggregation of encapsulated BSA was inhibited with increasing amou nt of bass to-encapsulated in the polymer, irrespective of the type of base used. The pH drop in the release medium and extent of acid-catalyzed PLGA degradation were both inhibited in the presence of base. The resultant effe ct was also reflected in an increase in water uptake and porosity of the de vices. The inhibition and mechanism of BSA aggregation was correlated with the basicity of die additive. For Ca(OH)(2), at 3% loading, covalent BSA ag gregation due to thiol-disulfide interchange was observed (indicative of io nization of albumin's free thiol at high pH), whereas at 3% ZnCO3 or Ca-3(P O4)(2), a higher percentage of non-covalent aggregates was observed compare d to Mg(OH)(2). Decreasing the loading of BSA at constant Mg(OH)(2) content caused an increase in BSA aggregation. Conclusions. The mechanism by which Mg(OI-I)2 stabilizes encapsulated BSA i n PLGA implants is through neutralizing the acidic microclimate pH in the p olymer. The successful neutralization afforded by the basic additives requi res a percolating network of pores connecting both base and protein. The mi croclimate pH inside PLGA implants can be controlled by selecting the type of basic salt, which suggests a potential approach to optimize the stabilit y of encapsulated pharmaceuticals in PLGA including therapeutic proteins.