湿热灭菌对聚(3-羟基丁酸-co-4-羟基丁酸)微球结构及复合凝胶可注射性能的影响

Effects of moist-heat sterilization on the structure of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) microspheres and injectability of the composite hydrogels

摘要:

本研究旨在评估终末湿热灭菌对聚(3-羟基丁酸-co-4-羟基丁酸) [poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P34HB]微球结构及其与交联透明质酸(hyaluronic acid, HA)复合凝胶可注射性能的影响，界定适宜的工艺参数窗口。以121 ℃为灭菌温度，设置10、20、30 min这3种时间并以未灭菌样品为对照，将P34HB微球与交联HA凝胶复配后采用“先复配、后灭菌”的共灭菌路径处理。通过凝胶渗透色谱(gel permeation chromatography, GPC)、差示扫描量热法(differential scanning calorimetry, DSC)、傅里叶变换红外光谱(Fourier transform infrared spectroscopy, FTIR)、粒径分析及扫描电镜(scanning electron microscopy, SEM)表征微球的分子量、热性能、化学结构与形貌变化；并结合流变学测试、推挤力、内聚力和离心加速稳定性评价制剂可注射性与使用稳定性，同时进行生物负载和无菌检查。结果发现，在121 ℃湿热10-20 min条件下，P34HB微球重均分子量仅轻度下降，熔融温度基本稳定，形貌和粒径分布保持良好，复合凝胶具有适宜的黏弹性，稳态推挤力约为12-15 N；当灭菌时间延长至30 min时，微球分子量明显下降，DSC出现冷结晶增强和双熔峰，复合凝胶的弹性模量(elastic modulus, G′)和推挤力波动加大并出现一定程度分相。所有灭菌组无菌检查结果均符合要求。在本研究条件下，121 ℃湿热灭菌10-20 min可在保证无菌的同时维持P34HB微球结构稳定性及复合凝胶的可注射性能，是适宜的终末湿热灭菌工艺窗口。结果表明P34HB微球具有较好的结构耐受性，可作为预灌封即用型软组织填充注射制剂的候选材料，为相关工艺和剂型选择提供了参考。

Abstract:

We assessed the effects of terminal moist-heat sterilization on the structure of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) microspheres and the composite hydrogels with hyaluronic acid (HA), aiming to define an acceptable process window. According to pharmacopeial and sterilization standards, we selected 121 ℃ as the sterilization temperature and set holding time of 10, 20, and 30 min, with non-sterilized samples as controls. P34HB microspheres were first blended with HA and then subjected to moist-heat sterilization (co-sterilization). Gel permeation chromatography (GPC), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), particle size analysis, and scanning electron microscopy (SEM) were employed to characterize the changes in molecular weight, thermal properties, chemical structure, and morphology of microspheres. The injectability and stability of the composite hydrogels were assessed based on oscillatory rheology, extrusion force, cohesiveness, and centrifugation stability. In addition, bioburden and sterility tests were carried out. After sterilization at 121 ℃ for 10-20 min, P34HB microspheres showed a slight decrease in molecular weight and basically unchanged melting temperature, particle size, and morphology, and the composite hydrogels retained suitable viscoelasticity and a steady extrusion force of 12-15 N. After sterilization for 30 min, the microspheres showed a significantly decreased molecular weight and enhanced cold crystallization and double melting peaks in DSC, and the composite hydrogels presented increased fluctuations in elastic modulus and extrusion force as well as signs of phase separation. All sterilized groups passed sterility testing. We conclude that moist-heat sterilization at 121 ℃ for 10-20 min is effective while retaining the complete structure of P34HB microspheres and good injectability of the composite hydrogels, and it is thus suitable as the condition for terminal moist-heat sterilization. Compared with literature data for polycaprolactone (PCL) and poly(L-lactide) (PLLA), P34HB shows superior tolerance to moist-heat and appears to be a promising material for prefilled ready-to-use soft tissue filler injections. The findings provide practical guidance for formulation and process design.