Role of a novel excipient poly(ethylene glycol)-b-poly(L-histidine) in retention of physical stability of insulin at aqueous/organic interface.
Role of a novel excipient poly(ethylene glycol)-b-poly(L-histidine) in retention of physical stability of insulin at aqueous/organic interface.
复制标题
DOI：
10.1021/mp060120z
复制
发表时间：
2007-04
影响因子：
4.9
通讯作者：
Ajay Taluja;Y. Bae
中科院分区：
文献类型：
--
作者：
Ajay Taluja;Y. Bae
本研究的目的是调查是否一种阳离子的双嵌段共聚物，聚（乙二醇）-b-聚（L-组氨酸）二嵌段共聚物（PEG-聚组氨酸），可以稳定胰岛素，在水/二氯甲烷界面形成的微胶囊化过程中。在276 nm处用荧光光度法监测该界面处的胰岛素聚集。观察了蛋白质浓度、水相介质pH值以及二氯甲烷（MC）中存在的聚乳酸-羟基乙酸共聚物（PLGA）对胰岛素聚集的影响。对于磷酸盐缓冲液（PB）中的2.0 mg/mL胰岛素溶液，还在各种胰岛素/聚合物赋形剂重量比下评价了添加Pluronic F-127作为阳性对照和添加PEG-聚His作为PB中的新型赋形剂的效果。通过圆二色性（CD）光谱法评价了由PEG-聚组氨酸保护的胰岛素的构象，并在界面暴露后恢复。随着胰岛素浓度的增加，观察到可溶性胰岛素的损失更大。基于zeta电位和粒度研究，选择pH 6.0用于胰岛素和PEG-聚His之间的最佳离子相互作用。选择pH 4.5和7.4（胰岛素和PEG-聚His之间无离子络合）作为对照，以比较PEG-聚His与pH 6.0下的稳定效果。PEG-聚His与胰岛素在pH 6.0下的孵育大大减少了蛋白质聚集，即使在PLGA存在下也是如此。PEG-聚组氨酸和F-127在非络合pH条件下减少胰岛素聚集，这表明PEG在界面处调节胰岛素吸附中所起的作用。远紫外（205-250 nm）CD研究显示界面暴露后对可溶性胰岛素二级结构的定性影响可忽略不计。反相高效液相色谱法（RP-HPLC）和分子排阻高效液相色谱法（HPLC）均未显示胰岛素脱酰胺或形成可溶性高分子量转化产物。MALDI-TOF质谱法证实了色谱法的结果。对选定样品进行的放射免疫测定表明，回收的可溶性胰岛素保留了其免疫反应性。设计了一种模拟蛋白质界面变性的实验方法，用于评价蛋白质在界面处的稳定性和筛选新型蛋白质稳定剂。对这种聚电解质-胰岛素络合的理解和操作将可能在经由微球制剂的胰岛素控制递送中发挥作用。
The aim of this study was to investigate whether a cationic polyelectrolyte, poly(ethylene glycol)-b-poly(L-histidine) diblock copolymer (PEG-polyHis), can stabilize insulin, at the aqueous/methylene chloride interface formed during the microencapsulation process. Insulin aggregation at this interface was monitored spectrophotometrically at 276 nm. The effects of protein concentration, pH of the aqueous medium, and the presence of poly(lactic-co-glycolic acid) (PLGA) in methylene chloride (MC) on insulin aggregation were observed. For the 2.0 mg/mL insulin solutions in phosphate buffer (PB), the effect of addition of Pluronic F-127 as a positive control and addition of PEG-polyHis as a novel excipient in PB was also evaluated at various insulin/polymeric excipient weight ratios. The conformation of insulin protected by PEG-polyHis and recovered after interfacial exposure was evaluated via circular dichroism (CD) spectroscopy. Greater loss in soluble insulin was observed with increasing insulin concentrations. pH 6.0 was selected for optimal ionic interactions between insulin and PEG-polyHis based on zeta potential and particle size studies. pH 4.5 and 7.4 (no ionic complexation between insulin and PEG-polyHis) were selected as controls to compare the stabilization effect of PEG-polyHis with that at pH 6.0. Incubation of PEG-polyHis with insulin at pH 6.0 drastically reduced protein aggregation, even in the presence of PLGA. PEG-polyHis and F-127 reduced insulin aggregation in noncomplexing pH conditions pointing to the role played by PEG in modulation of insulin adsorption at the interface. Far-UV (205-250 nm) CD study revealed negligible qualitative effects on soluble insulin's secondary structure after interfacial exposure. RP-HPLC and size-exclusion HPLC showed no deamidation of insulin or formation of soluble high molecular weight transformation products respectively. MALDI-TOF mass spectrometry confirmed the results from chromatographic procedures. Radioimmunoassay carried out on select samples showed that recovered soluble insulin had retained its immunoreactivity. An experimental method to simulate interfacial denaturation of proteins was designed for assessment of protein stability at the interface and screening for novel protein stabilizers. Understanding and manipulation of such polyelectrolyte-insulin complexation will likely play a role in insulin controlled delivery via microsphere formulation(s).