Effect of the covalent modification with poly(ethylene glycol) on α-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic) microspheres
Effect of the covalent modification with poly(ethylene glycol) on α-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic) microspheres
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DOI：
10.1002/jps.20243
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发表时间：
2005-02-01
影响因子：
3.8
通讯作者：
Griebenow, K
中科院分区：
文献类型：
--
作者：
Castellanos, IJ;Al-Azzam, W;Griebenow, K
研究了用甲氧基聚乙二醇（PEG）共价修饰α-胰凝乳蛋白酶（α-chymotrypsin，α-chymotrypsin），使其在聚乳酸-羟基乙酸共聚物（PLGA）微球中稳定化的有效性。α-胰凝乳蛋白酶用PEG（M-w = 5000）化学修饰，PEG与胰凝乳蛋白酶的摩尔比为0.4 - 96。获得各种缀合物，并通过毛细管电泳测定PEG修饰的量。在该研究中，仅考虑PEG/凝乳酶摩尔比在约1和8之间的那些缀合物，因为即使在包封之前，更高水平的修饰也会导致蛋白质不稳定。在包封之前通过测量酶动力学、热稳定性和三级结构完整性以及在初始冻干过程之后通过测定二级结构含量来研究胰凝乳蛋白酶制剂的稳定性和功能性。这些稳定性参数与包封在PLGA微球中后选择的参数相关（具体来说，不溶性聚集体的量、残留酶活性和蛋白质结构扰动的幅度）。结果表明，包封前蛋白质构象越稳定，包封后的比活性保留率越高。与此相反，没有发现之间的关系，封装前的蛋白质的稳定性和封装诱导的蛋白质聚集的幅度。即使是最低水平的修改（PEG-胰凝乳蛋白酶的摩尔比为0.7）大幅减少不溶性聚集体的量从18%的未修饰的蛋白质到4%。结果表明，PEG修饰能够在很大程度上防止糜蛋白酶聚集和活性损失后，固体-油-水包包封在PLGA微球中。它表明，它是必不可少的，以优化蛋白质修饰的程度，以确定蛋白质的稳定性封装后。(C)2004 Wiley-Liss，Inc.和美国药剂师协会
The effectiveness of the covalent modification of alpha-chymotrypsin with methoxy poly(ethylene glycol) (PEG) to afford its stabilization during encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres by a solid-in-oil-in-water method was investigated. alpha-Chymotrypsin was chemically modified with PEG (M-w = 5000) using molar ratios of PEG-to-chymotrypsin ranging from 0.4 to 96. Various conjugates were obtained and the amount of PEG modification was determined by capillary electrophoresis. In this investigation, only those conjugates with PEG/chymotrysin molar ratios between approximately 1 and 8 were considered because higher lever of modification caused protein instability even before encapsulation. The stability and functionality of the chymotrypsin formulations were investigated before encapsulation by measuring enzyme kinetics, thermal stability, and tertiary structure intactness, and after the initial lyophilization process by determining the secondary structure content. These stability parameters were related to select ones after encapsulation in PLGA microspheres (specifically, the amount of insoluble aggregates, residual enzyme activity, and magnitude of protein structural perturbations). The results show that the more stable the protein conformation before encapsulation was, the higher was the retention of the specific activity after encapsulation. In contrast, no relationship was found between the protein stability before encapsulation and the magnitude of encapsulation-induced protein aggregation. Even the lowest level of modification (PEG-to-chymotrypsin molar ratio of 0.7) drastically reduced the amount of insoluble aggregates from 18% for the nonmodified protein to 4%. The results demonstrate that PEG modification was able to largely prevent chymotrypsin aggregation and activity loss upon solid-in-oil-in-water encapsulation in PLGA microspheres. It is demonstrated that it is essential to optimize the degree of protein modification to ascertain protein stability upon encapsulation. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.