Enhanced protein internalization and efficient endosomal escape using polyampholyte-modified liposomes and freeze concentration
Enhanced protein internalization and efficient endosomal escape using polyampholyte-modified liposomes and freeze concentration
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DOI：
10.1039/c6nr03940e
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发表时间：
2016-09-21
期刊：
影响因子：
6.7
通讯作者：
Matsumura, Kazuaki
中科院分区：
文献类型：
--
作者：
Ahmed, Sana;Fujitab, Satoshi;Matsumura, Kazuaki
在这里，我们展示了一种新的策略，通过聚两性电解质修饰的脂质体的内体逃逸特性获得的蛋白质的有效冷冻浓缩介导的细胞质递送。冷冻浓缩方法通过简单地用蛋白质和纳米载体复合物冷冻细胞成功地诱导蛋白质的有效内化。然而，蛋白质内化的机制仍不清楚。在这里，我们通过在脂质体中掺入疏水性聚两性电解质来修饰脂质体，设计了一种新型蛋白质递送载体。这些复合物的特征在于粒径，包封效率，和细胞毒性。流式细胞仪和显微镜分析表明，蛋白质负载的两性聚电解质修饰的脂质体冷冻后的吸附和内化增强相比，观察到在未冷冻的复合物。抑制研究表明，未修饰的和聚两性电解质修饰的脂质体之间的内化机制不同。此外，聚两性电解质修饰的脂质体在促进内体逃逸以增强蛋白质递送至细胞质方面表现出高功效，同时具有低毒性。这些结果强烈表明，基于冷冻浓缩的策略不仅可以广泛用于癌症治疗，还可以用于基因治疗，用于体外有效地将货物递送到细胞质中。
Here we show a new strategy for efficient freeze concentration-mediated cytoplasmic delivery of proteins, obtained via the endosomal escape property of polyampholyte-modified liposomes. The freeze concentration method successfully induces the efficient internalization of proteins simply by freezing cells with protein and nanocarrier complexes. However, the mechanism of protein internalization remains unclear. Here, we designed a novel protein delivery carrier by modifying liposomes through incorporating hydrophobic polyampholytes therein. These complexes were characterized for particle size, encapsulation efficiency, and cytotoxicity. Flow cytometry and microscopic analysis showed that the adsorption and internalization of protein-loaded polyampholyte-modified liposomes after freezing were enhanced compared with that observed in unfrozen complexes. Inhibition studies demonstrated that the internalization mechanism differs between unmodified and polyampholyte-modified liposomes. Furthermore, polyampholyte-modified liposomes exhibited high efficacy in facilitating endosomal escape to enhance protein delivery to the cytoplasm with low toxicity. These results strongly suggest that the freeze concentration-based strategy could be widely utilised for efficient cargo delivery into the cytoplasm in vitro not only in cancer treatment but also for gene therapy as well.