Evans Brian C, Nelson Christopher E, Yu Shann S, Beavers Kelsey R, Kim Arnold J, Li Hongmei, Nelson Heather M, Giorgio Todd D, Duvall Craig L
Department of Biomedical Engineering, Vanderbilt University, USA.
J Vis Exp. 2013 Mar 9(73):e50166. doi: 10.3791/50166.
Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.
构成内溶酶体囊泡的磷脂双层膜可能会对生物药物向细胞内靶点的递送造成障碍。为了克服这一障碍，人们设计了多种合成药物载体，以主动破坏内体膜并将货物递送至细胞质中。在此，我们描述了溶血试验，该试验可作为一种快速、高通量的筛选方法，用于检测细胞内药物递送系统的细胞相容性和内体溶解活性。在溶血试验中，将人类红细胞与测试材料在模拟细胞外、早期内体和晚期内溶酶体环境的特定pH值缓冲液中共同孵育。经过离心步骤使完整的红细胞沉淀后，用分光光度法测量释放到培养基中的血红蛋白量（最佳动态范围为405nm）。然后相对于用去污剂裂解的阳性对照样品，对红细胞破坏百分比进行量化。在这个模型系统中，红细胞膜充当包围内溶酶体囊泡的脂质双层膜的替代物。理想的结果是在生理pH值（7.4）下溶血可忽略不计，而在大约pH 5-6.8的内溶酶体pH范围内溶血明显。
