Stability of proteins encapsulated in injectable and biodegradable poly(lactide-co-glycolide)-glucose millicylinders

Abstract

Purpose: To characterize protein stability in poly(lactide-co-glycolide) 50/50-glucose star (PLGA-Glu) injectable millicylinders and to compare results with linear PLGA 50/50.

Methods: Bovine serum albumin (BSA), a model protein, was encapsulated in PLGA-Glu and linear PLGA millicylinders by solvent-extrusion and incubated under physiological conditions. Important system properties were characterized, including: polymer molecular weight distribution, soluble acidic residues, polymer morphology, polymer water uptake, microclimate pH, protein content and release, and protein aggregation. The polymer microclimate late in the release incubation was simulated and protein recovery was analyzed by UV280, size exclusion chromatography, amino acid analysis, and a modified Bradford assay.

Results: PLGA-Glu contained higher levels of low molecular weight oligomers, more rapidly biodegraded, and exhibited a lower microclimate pH than the linear 50/50 PLGA, which is the most acidic type in the PLGA family. BSA, when encapsulated in PLGA-Glu millicylinders, underwent extensive noncovalent insoluble aggregation over 2 weeks in vitro release, which was almost completely inhibited upon co-encapsulation of Mg(OH)2. However, by 5 weeks release for base-containing formulations, although insoluble aggregation was still suppressed, the soluble fraction of protein in the polymer was unrecoverable by the modified Bradford assay. Polymer microclimate simulations with extensive protein analysis strongly suggested that the low recovery was mostly caused by base-catalyzed hydrolysis of the oligomeric fraction of BSA.

Conclusions: In PLGA-Glu, the acidic microclimate was similarly responsible for insoluble aggregation of encapsulated BSA. BSA aggregation was inhibited in millicylinders by co-incorporation into the polymer an insoluble base, but over a shorter release interval than linear PLGA likely because of a more acidic microclimate in the star polymer.

Figure 1

GPC chromatograms of regular PLGA (A) and PLGA-Glucose polymer (B)

Figure 2

Scanning electron micrographs of PLGA-Glucose millicylinders containing both 15% BSA and 3% base. (A) Surface; (B) Cross-section.

Figure 3

Cumulative release of BSA from PLGA-Glucose millicylinders with (■) or without (●) 3% Mg(OH)₂ (Mean ± S.E.M., n = 3).

Figure 4

Size exclusion chromatogram of BSA in saturated Mg(OH)₂ solution, monitored at 280 nm.

Figure 5

pH change of the release medium of PLGA-Glucose millicylinders (Mean ± S.E.M., n = 3). Polymer only (●); with 15% BSA (■); with 15% BSA + 3% Mg(OH)₂ (▲).

Figure 6

Water uptake kinetics of PLGA-Glucose millicylinders (Mean ± S.E.M., n = 3). Polymer only (●); with 15% BSA (■); with 15% BSA and 3% Mg(OH)₂ (▲)

Figure 7

Microclimate pH (µpH) in PLGA-Glucose and PLGA measured by coating electrode method.