Vrikkis Regina M, Fraser Kevin J, Fujita Kyoko, Macfarlane Douglas R, Elliott Gloria D
Department of Mechanical Engineering, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte, NC 28223, USA.
J Biomech Eng. 2009 Jul;131(7):074514. doi: 10.1115/1.3156810.
Ionic liquids (ILs) have shown excellent promise as both solutes and solvents for stabilizing proteins at room temperature. Because many modern drugs are protein-based, these stabilizing characteristics have great potential to provide advances in the field of liquid formulation of therapeutic proteins. However, before these developments can be translated into clinical solutions it is essential to establish data related to the biocompatibility of these ILs. The current work investigates the cytotoxicity of several ILs that were rationally synthesized from natural biomolecules and compounds that have already been approved as excipients for drug formulations. The effect of choline dihydrogen phosphate (choline dhp), choline saccharinate, and 1-butyl 3-methyl imidazolium lactate (bmim lactate) on the metabolic activity of a mouse macrophage cell line (J774) was assessed using the reduction in resazurin as an indicator of activity and, by extension, viability. Two formulations of lysozyme (10 mg/ml and 100 mg/ml) in 80 wt % choline dhp (aq) were prepared and the proteins were evaluated for structural stability immediately following formulation and again at 1 month. Equivalent formulations in 0.1 M Na acetate aqueous buffer were evaluated as controls. A differential scanning microcalorimeter (DSC) was used to evaluate the structural stability on the basis of the unfolding temperature and the enthalpy of unfolding, and a micrococcus lysodiekticus activity test was used to evaluate functional activity. All compounds were found to be relatively benign, with toxicity increasing in the order choline dhp<choline saccharinate<bmim lactate. At 1 month lysozyme that had been stored in choline dhp had a higher activity and folded fraction than lysozyme that had been stored in aqueous buffer. These results suggest that biocompatibility and protein stabilization characteristics can be rationally designed into ionic liquids.
离子液体（ILs）作为在室温下稳定蛋白质的溶质和溶剂已展现出卓越的前景。由于许多现代药物都是基于蛋白质的，这些稳定特性在治疗性蛋白质液体制剂领域具有极大的潜力带来进展。然而，在这些进展能够转化为临床解决方案之前，建立与这些离子液体生物相容性相关的数据至关重要。当前的工作研究了几种由天然生物分子和已被批准作为药物制剂辅料的化合物合理合成的离子液体的细胞毒性。使用刃天青还原作为活性指标进而作为活力指标，评估了磷酸二氢胆碱（胆碱二氢磷酸盐，choline dhp）、胆碱糖精和1-丁基-3-甲基咪唑鎓乳酸盐（bmim乳酸盐）对小鼠巨噬细胞系（J774）代谢活性的影响。制备了80 wt%胆碱二氢磷酸盐（水溶液）中两种溶菌酶制剂（10 mg/ml和100 mg/ml），并在制剂后立即以及1个月后评估蛋白质的结构稳定性。将0.1 M醋酸钠水溶液缓冲液中的等效制剂作为对照进行评估。使用差示扫描量热仪（DSC）基于解折叠温度和解折叠焓来评估结构稳定性，并使用溶壁微球菌活性测试来评估功能活性。发现所有化合物相对无害，毒性按胆碱二氢磷酸盐＜胆碱糖精＜bmim乳酸盐的顺序增加。在1个月时，储存在胆碱二氢磷酸盐中的溶菌酶比储存在水性缓冲液中的溶菌酶具有更高的活性和折叠分数。这些结果表明，可以将生物相容性和蛋白质稳定特性合理地设计到离子液体中。
