Taluja Ajay, Bae You Han
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84108, USA.
Mol Pharm. 2007 Jul-Aug;4(4):561-70. doi: 10.1021/mp060120z. Epub 2007 Apr 18.
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).
本研究的目的是调查一种阳离子聚电解质，聚（乙二醇）-b-聚（L-组氨酸）二嵌段共聚物（PEG-聚组氨酸），能否在微囊化过程中形成的水/二氯甲烷界面稳定胰岛素。在276nm处用分光光度法监测该界面处胰岛素的聚集情况。观察了蛋白质浓度、水相介质的pH值以及二氯甲烷（MC）中聚乳酸-羟基乙酸共聚物（PLGA）的存在对胰岛素聚集的影响。对于磷酸盐缓冲液（PB）中2.0mg/mL的胰岛素溶液，还在不同的胰岛素/聚合物辅料重量比下评估了添加作为阳性对照的普朗尼克F-127和在PB中添加作为新型辅料的PEG-聚组氨酸的效果。通过圆二色性（CD）光谱评估了受PEG-聚组氨酸保护并在界面暴露后回收的胰岛素的构象。随着胰岛素浓度的增加，可溶性胰岛素的损失更大。基于zeta电位和粒径研究，选择pH 6.0以实现胰岛素与PEG-聚组氨酸之间的最佳离子相互作用。选择pH 4.5和7.4（胰岛素与PEG-聚组氨酸之间无离子络合）作为对照，以比较PEG-聚组氨酸在pH 6.0时与其他条件下的稳定效果。即使存在PLGA，在pH 6.0下将PEG-聚组氨酸与胰岛素孵育也能显著减少蛋白质聚集。PEG-聚组氨酸和F-127在非络合pH条件下减少了胰岛素聚集，这表明PEG在调节胰岛素在界面的吸附中所起的作用。远紫外（205-250nm）CD研究表明，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（RP-HPLC）和尺寸排阻高效液相色谱分别显示胰岛素没有脱酰胺化或形成可溶性高分子量转化产物。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）证实了色谱分析结果。对选定样品进行的放射免疫分析表明，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，用于评估蛋白质在界面的稳定性并筛选新型蛋白质稳定剂。理解和操纵这种聚电解质-胰岛素络合可能会在通过微球制剂进行胰岛素控释中发挥作用。
