Pérez-Rodriguez Caroline, Montano Nashbly, Gonzalez Karilys, Griebenow Kai
University of Puerto Rico, Río Piedras Campus, Department of Chemistry, P.O. Box 23346, San Juan, PR 00931-3346, USA.
J Control Release. 2003 Apr 14;89(1):71-85. doi: 10.1016/s0168-3659(03)00074-9.
Protein inactivation and aggregation are serious drawbacks in the encapsulation of proteins in bioerodible polymers by water-in-oil-in-water (w/o/w) encapsulation. The model protein alpha-chymotrypsin was employed to investigate whether its stabilization towards the major stress factors in the w/o/w encapsulation procedure would allow for the encapsulation and release of non-aggregated and active protein. Due to the formation of amorphous aggregates alpha-chymotrypsin is an excellent sensor to probe unfolding events. Furthermore, its enzymatic activity is highly sensitive towards the presence of organic solvents. alpha-Chymotrypsin in aqueous solution showed substantial aggregation and activity loss when it was homogenized with CH(2)Cl(2) due to adsorption to the interface. Its w/o/w encapsulation in poly(lactic-co-glycolic)acid (PLGA) microspheres caused formation of 35% non-covalent aggregates and reduced the specific activity by 14%. Screening for efficient excipients revealed that co-dissolving the protein with maltose and polyethylene glycol (PEG, M(w) 5000) in the first aqueous phase reduced interface-induced protein aggregation and inactivation. Employing these excipients during encapsulation led to a reduction in alpha-chymotrypsin inactivation (10%) and aggregation (12%). Optimizing the effect of PEG by also dissolving the excipient in the organic phase prior to encapsulation further decreased the amount of non-covalent aggregates to 7% and loss in activity to 5%. The data obtained demonstrate that the w/o emulsification step is the main stress-factor in the w/o/w encapsulation procedure but subsequent encapsulation steps also cause some protein aggregation.
在通过水包油包水（w/o/w）包封法将蛋白质包封于可生物降解聚合物的过程中，蛋白质的失活和聚集是严重的缺陷。采用模型蛋白α-胰凝乳蛋白酶来研究其在w/o/w包封过程中对主要应激因素的稳定性是否能够实现非聚集且有活性的蛋白质的包封与释放。由于形成无定形聚集体，α-胰凝乳蛋白酶是探测去折叠事件的优良传感器。此外，其酶活性对有机溶剂的存在高度敏感。水溶液中的α-胰凝乳蛋白酶与CH₂Cl₂匀浆时，因吸附于界面而出现大量聚集并丧失活性。其在聚乳酸-乙醇酸共聚物（PLGA）微球中的w/o/w包封导致形成35%的非共价聚集体，并使比活性降低14%。对有效辅料的筛选表明，在第一水相中将蛋白质与麦芽糖和聚乙二醇（PEG，分子量5000）共溶解可减少界面诱导的蛋白质聚集和失活。在包封过程中使用这些辅料可使α-胰凝乳蛋白酶的失活（10%）和聚集（12%）减少。通过在包封前也将该辅料溶解于有机相中优化PEG的作用，可进一步将非共价聚集体的量降至7%，活性损失降至5%。所获得的数据表明，w/o乳化步骤是w/o/w包封过程中的主要应激因素，但随后的包封步骤也会导致一些蛋白质聚集。
