Evaluation of succinylated pullulan for long-term protein delivery in poly(lactide-co-glycolide) microspheres

Two types of pullulan with different succinylations were synthesized for an evaluation as additives for long-term protein delivery in poly(lactide-co- glycolide) microspheres (PLGA MS). Negatively charged succinylated pullulan (SP) forms an ionic complex with cationic protein (lysozyme; Lys) via an ionic interaction. SP2 (2 succinyl groups per 10 glucose units in pullulan) constructed a better nano-size complex with lysozyme (Lys) in distilled water (DW) than SP1 (1 succinyl group per 10 glucose units in pullulan). To assess the long-term delivery capability, PLGA MS complexes with different Lys:SP2 ratios (1:1 (LMS 1), 1:2 (LMS 2) and 1:3 (LMS 3), as wt% of Lys:SP2) were prepared with water in oil in water (W/O/W). The complex loaded PLGA MS showed a higher loading efficiency and a lower insoluble Lys content than PLGA without SP (LMS 0), indicating that SP helps stabilize Lys at the organic/water (O/W) interface. In evaluating the release pattern of Lys, LMS 1, 2, and 3 all demonstrated a low initial burst and complete release behavior (reaching almost 100% after 27 days), whereas the total amount of Lys released from LMS 0 did not reach 80% during the same time period. During a 14-day release test, the stability of Lys was confirmed by RP-HPLC. In the case of LMS 0, an unexpected peak (retention time 9.2 min) was created, which was not observed in LMS 1, 2, and 3. This suggests that SP suppresses the denaturation of Lys in PLGA MS. These results show that SP, as an additive in PLGA MS, has potential for the long-term delivery of therapeutic proteins.