Acidification of the internal poly(lactide-co-glycolide) (PLGA) microenvironment is considered one of the major protein stresses during controlled release from such delivery systems. A model protein, bovine serum albumin (BSA), was incubated at 37 degrees C for 28 days to simulate the environment within the aqueous pores of PLGA during the release phase and to determine how acidic microclimate conditions affect BSA stability. Size-exclusion high performance liquid chromatography (SE-HPLC), SDS-PAGE, and infrared spectroscopy were used to monitor BSA degradation. BSA was most stable at pH 7, but rapidly degraded via aggregation and hydrolysis at pH 2. These simulated degradation products were nearly identical to that of unreleased BSA found entrapped within PLGA 50/50 millicylinders. At pH 2, changes in BSA conformation detected by various spectroscopic techniques were consistent with acid denaturation of the protein. By contrast, at pH 5 and above, damage to BSA was insufficient to explain the instability of the protein in the polymer. Thus, these data confirm the hypothesis that acid-induced unfolding is the basis of BSA aggregation in PLGA and the acidic microclimate within PLGA is indeed a dominant stress for encapsulated BSA. To increase the stability of proteins within PLGA systems, formulations must protect against potentially extreme acidification such that native structure is maintained.

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The purpose of this paper was to investigate the phenomena of pore closing and opening in microspheres of poly(lactic-co-glycolic acid) (PLGA) and PLGA-glucose star copolymer (PLGA-Glu) and their effects on protein release. We used scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) to visually characterize the pore state and the uptake of dextran labeled with pH-insensitive probes by microspheres, as an indicator of pore connectivity. The effect of temperature on initial protein release from microspheres was also investigated. It was found that (1) pore closing occurs in both PLGA and PLGA-Glu; (2) pore closing can take place at later time during incubation at physiological condition (37 degrees C) as well as during the initial stage; (3) pore closing is much more significant at elevated temperatures; (4) previously isolated pores can become open by, for example, osmotic-mediated events; and (5) pore closing/opening correlates with the release rate of biomacromolecules from PLGA or PLGA-Glu microspheres. The pore closing/opening appeared potentially a universal event throughout the release period dictating the kinetics of protein release from PLGA microspheres. Hence, these results strongly suggest that open and isolated pores are able to toggle back-and-forth periodically during PLGA degradation while controlling protein release; these observations imply a novel new hypothesis concerning erosion-controlled release of biomacromolecules from PLGA and related polymers.

The diffusion coefficient of a small hydrophobic probe in poly(lactide-co-glycolide) (PLGA) microparticles was determined by laser scanning confocal microscopy (LSCM). PLGA microparticles preincubated in a physiological buffer for various times were immersed in an aqueous solution of the pH-insensitive fluorescent probe bodipy at 37 °C. Probe concentration gradients inside individual microparticle matrices were then recorded by LSCM, which were accurately fit by the solution to Fick's second law of diffusion to determine an effective probe diffusion coefficient (D) in the polymer matrix. Values of D varied less than expected from the blank eroding polymer and were in the range (3−10) × 10-12 cm2/s. The apparent polymer-water partition coefficient of the probe was also determined to be roughly 20, indicative of strong partitioning into the polymer phase. The diffusion of bodipy in microparticles varied over 3 orders of magnitude between 22 and 43 °C, indicative of transport control in the polymer phase as opposed to a pore diffusion mechanism. The diffusion model also predicted exceptionally well the release of probe encapsulated in microparticles when a time-averaged D, which had been determined in blank microparticles by LSCM, was used. Values of D for bodipy in PLGA microparticles encapsulating bovine serum album were of similar value ((3−24) × 10-12 cm2/s) but better reflected the multiphasic behavior characteristic of PLGA erosion. The LSCM method described here is simple, nondestructive, and accurate and can be used to study the diffusion inside a single polymer microparticle.