Microspheres of hydrophilic PLGA highly atractive for protein delivery

Microspheres based on a hydroxylated aliphatic polyester, poly(D, L-lactic-co-hydroxymethyl glycolic acid) (PLHMGA), were prepared and the release of a model protein (BSA) as well as their in vitro degradation characteristics were studied. It was shown that the release of BSA was governed by the degradation of the microspheres, which in turn was dependent on the copolymer composition. The release time could be tailored from 5 till 50 days and the protein was almost quantitatively released. Introduction: Poly(lactic-co-glycolic acid) (PLGA) has been extensively investigated for the delivery of hormones, antigens, peptides and therapeutic proteins [1]. However, due to the instability of the proteins during preparation, storage and release [2], the therapeutic effects of these protein formulations are limited. A major concern for the delivery of proteins and other acid labile molecules using PLGA vehicles is the acidic microenvironment pH that is generated during degradation which leads to degradation and aggregation and subsequently incomplete release of the encapsulated substances. Also, this low pH has shown to result in highly unwanted acylation and deamidation reaction of the protein pharmaceutical [3]. To overcome these drawbacks, we hydrophilized PLGA by introduction of hydroxyl side groups Table and Figure Presented In a previous study we demonstrated that PLGA microspheres hardly showed release of lysozyme, whereas PLHMGA microspheres released this protein in a sustained manner for 30 days. The release however was not complete, likely due to the formation of semicrystalline and insoluble L-lactic acid oligomers. In the present study we used poly(D,L-lactic-co-hydroxymethyl glycolic acid) to avoid the formation of these insoluble oligomers. Further we investigated the effect of copolymer composition on the degradation and the in vitro release kinetics of a model protein (BSA). Experimental methods: BSA-loaded microspheres were essentially prepared using a w/o/ w solvent evaporation technique as described in our previous paper [5]. For the in vitro release study, BSA-loaded microspheres (20 mg) were suspended in a 1.5 ml PBS (pH 7.4, 150 mM, 0.02% NaN3) and incubated at 37 °C under mild agitation. The released samples were collected at the different time points by centrifuging the microspheres and removing 1 ml of the supernatant and replacing it with 1 ml of fresh buffer. The sampleswere injected into a ultra performance liquid chromatography (Acuity UPLC®) equipped with a BEH 300 C18 1.7 μm column for measuring their protein content. The degradation of the microspheres was studied by measuring the dry weight of the microspheres and the decrease in number average molecular weight as a function of time by gel permeation chromatography. Result and discussion: Different copolymers of D,L-lactide and hydroxymethyl glycolide (HMG) were synthesized via ring opening polymerization (Table 1). The encapsulation efficiencies of BSA in the microspheres obtained from the polymers of Table 1 were between 50 and 80% and the particles had a volume weighted diameter around 10 μm. Fig. 2 shows the BSA release from the different microspheres. Microspheres with 50 mol% HMG showed a burst (40%) and thereafter an almost quantitative release of BSA was observed in around 10 days. The PLHMGA microspheres with 35 mol% of HMG showed after a burst release of 20% a sustained release of the protein in 30 days. Microspheres prepared from the copolymer with 25 mol% HMG showed only a small burst (10%) and then a slow release for 20 days, after which approximately 70% of the protein was released during the following 20-25 days. Figure Presented Fig. 3 shows that in agreement with previous studies [6] the degradation time of the microspheres depends on the copolymer composition: the higher the HMG content, the faster the degradation. It should be stressed that in contrast to our previous study [5], no insoluble residues remained. Fig. 4 shows that the molecular mass of the copolymers decreased in time which demonstrates that the microspheres degrade by bulk degradation. Combining Figs. 2 and 3, it can be concluded that the release of BSA is largely governed by the degradation of the copolymer. Figure Presented. Conclusion: Microspheres prepared from PLHMGA showed a sustained release of BSA for 1 week to 2 months dependent on the copolymer composition. The higher the content of HMG, the faster the release. We showed that the microspheres are fully degradable and the degradation kinetics can be tailored by the copolymer composition. The release is to a large extent governed by degradation of the copolymer.