Encapsulation-induced aggregation and loss in activity of γ-chymotrypsin and their prevention
Encapsulation-induced aggregation and loss in activity of γ-chymotrypsin and their prevention
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DOI：
10.1016/s0168-3659(02)00073-1
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发表时间：
2002-06-17
影响因子：
10.8
通讯作者：
Griebenow, K
中科院分区：
文献类型：
--
作者：
Castellanos, IJ;Cruz, G;Griebenow, K
由于蛋白质稳定性问题，需要开发常用的水包油包水技术的替代程序，将蛋白质封装在聚合物中。在此，使用水包油包固体 (s/o/w) 封装技术将模型蛋白 γ-胰凝乳蛋白酶封装在聚（D,L-乳酸-乙醇酸）酸 (PLGA) 微球中。选择模型蛋白是因为它具有可测量的生物活性并且其解折叠是不可逆的。后者使蛋白质成为封装过程中展开事件的优秀传感器。虽然冻干不会导致任何不可逆的聚集或活性损失，但通过 s/o/w 技术封装冻干酶被证明对其完整性有害。具体而言，34%的封装蛋白发生聚集，24小时内释放的酶比活性降低至约10%。封装前的 50%。 FTIR 光谱表明，γ-胰凝乳蛋白酶的二级结构存在大量封装引起的扰动。为了在封装过程中实现γ-胰凝乳蛋白酶的稳定性，在最初的冻干过程中使用了赋形剂。当γ-胰凝乳蛋白酶与聚乙二醇 (PEG) 共冻干时，微球内非共价聚集体的形成显着减少至 8%。 FTIR 数据表明 PEG 可以防止封装引起的结构扰动。相反，当γ-胰凝乳蛋白酶与海藻糖共冻干时，聚集体的量仍然很高（34%）。体外释放 1 周期间没有形成额外的不溶性聚集体。此外，封装后微球中不溶性聚集体的量与未释放的蛋白质的量相关。因此体外释放不会引起聚集。在保留比酶活性方面也发现了类似的结果，其中 PEG 提供了优异的稳定性。 (C) 2002 Elsevier Science B.V. 保留所有权利。
Development of alternative procedures to the commonly employed water-in-oil-in-water technique to encapsulate proteins in polymers is needed due to protein stability issues. Herein the model protein gamma-chymotrypsin has been encapsulated in poly(D,L-lactic-co-glycolic)acid (PLGA) microspheres using the solid-in-oil-in-water (s/o/w) encapsulation technique. The model protein was chosen because it has a measurable biological activity and its unfolding is irreversible. The latter make the protein an excellent sensor for unfolding events in the encapsulation procedure. While lyophilization did not cause any irreversible aggregation or loss in activity, encapsulation of the lyophilized enzyme by the s/o/w technique proved detrimental to its integrity. Specifically, 34% of the encapsulated protein was aggregated and the specific activity of enzyme released within 24 h was reduced to ca. 50% of that prior to encapsulation. FTIR spectra demonstrated substantial encapsulation-induced perturbations of the secondary structure of gamma-chymotrypsin. To achieve stabilization of gamma-chymotrypsin during encapsulation, excipients were employed during the initial lyophilization process. When gamma-chymotrypsin was co-lyophilized with poly(ethylene glycol) (PEG) the formation of non-covalent aggregates inside the microspheres decreased significantly to 8%. FTIR data showed that PEG prevented encapsulation-induced structural perturbations. In contrast, the amount of aggregates remained high (34%) when gamma-chymotrypsin was co-lyophilized with trehalose. No additional non-soluble aggregates were formed during 1 week of in vitro release. Furthermore, the amount of non-soluble aggregates in the microspheres after encapsulation correlated with the amount of non-released protein. Therefore in vitro release did not cause aggregation. Similar results were found with respect to the retention of the specific enzyme activity where PEG afforded excellent stability. (C) 2002 Elsevier Science B.V. All rights reserved.