Effect of salts on lysozyme stability at the water-oil interface and upon encapsulation in poly(lactic-co-glycolic) acid microspheres.
Effect of salts on lysozyme stability at the water-oil interface and upon encapsulation in poly(lactic-co-glycolic) acid microspheres.
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DOI：
10.1002/bit.10632
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发表时间：
2003-06
影响因子：
3.8
通讯作者：
C. Pérez;K. Griebenow
中科院分区：
文献类型：
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作者：
C. Pérez;K. Griebenow
由于蛋白质固有的物理不稳定性，采用油包水（w/o/w）技术将蛋白质包封在聚乳酸-羟基乙酸（PLGA）微球中是非常具有挑战性的。特别是，将蛋白质暴露于第一个油包水乳状液中会导致不必要的界面诱导蛋白质失活和聚集。我们测试了盐是否可以稳定模型蛋白，然后是蛋清溶菌酶，以防止在w/o界面发生的有害事件以及随后的w/o/w封装。首先，我们研究了盐对溶菌酶活性的影响，以及在二氯甲烷乳化溶菌酶水溶液时可溶性沉淀和不溶性聚集体的产生。发现在乳化过程中溶菌酶析出。盐浓度在10-100 mM之间形成的沉淀物的数量与阴离子在电选择性系列中的位置(SO(4) (2-) > SCN(-) > Cl(-) > H(2)PO（4)(-)）有关，而高盐浓度（1M）导致> 80%的溶菌酶沉淀，与盐无关。沉淀由缓溶性蛋白质沉淀和不溶于水的非共价聚集物组成。在50 mM KH(2)PO(4)的存在下，溶菌酶的沉淀、聚集和乳化失活在很大程度上被阻止了，而KSCN导致了这些有害事件的增加。其次，检测溶菌酶在w/o界面处结构完整性的改善是否会提高其在PLGA微球中w/o/w封装后的稳定性。某些条件确实提高了稳定性，特别是用50 mM KH(2)PO(4)共溶溶菌酶减少了比活性和聚集的损失。综上所述，盐的种类和浓度是PLGA微球包封蛋白的关键参数。
Encapsulation of proteins in poly(lactic-co-glycolic) acid (PLGA) microspheres by the water-in-oil-in-water (w/o/w) technique is very challenging because of the inherent physical instability of proteins. In particular, exposure of proteins to the first water-in-oil emulsion causes unwanted interface-induced protein inactivation and aggregation. We tested whether salts could afford stabilization of a model protein, hen egg-white lysozyme, against the detrimental events occurring at the w/o interface and subsequently upon w/o/w encapsulation. First, we investigated the effect of salts on the specific enzyme activity and generation of soluble precipitates and insoluble aggregates upon emulsification of an aqueous lysozyme solution with methylene chloride. It was found that lysozyme precipitation occurred upon emulsification. The amount of precipitate formed at salt concentrations between 10-100 mM was related to the position of the anion in the electroselectivity series (SO(4) (2-) > SCN(-) > Cl(-) > H(2)PO(4) (-)) while high salt concentrations (1M) led to > 80% of lysozyme precipitation regardless of the salt. The precipitates consisted of buffer-soluble protein precipitates and water-insoluble noncovalent aggregates. Lysozyme precipitation, aggregation, and inactivation upon emulsification were largely prevented in the presence of 50 mM KH(2)PO(4) while KSCN caused an increase in these detrimental events. Second, it was tested whether the improved structural integrity of lysozyme at the w/o interface would improve its stability upon w/o/w encapsulation in PLGA microspheres. Some conditions indeed led to improved stability, particularly codissolving lysozyme with 50 mM KH(2)PO(4) reduced loss in the specific activity and aggregation. In conclusion, the type and concentration of salts is a critical parameter when encapsulating protein in PLGA microspheres.