Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K.
Department of Materials, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K.
ACS Appl Mater Interfaces. 2020 Jul 1;12(26):28941-28951. doi: 10.1021/acsami.0c03260. Epub 2020 Jun 16.
Cell cryopreservation plays a key role in the development of reproducible and cost-effective cell-based therapies. Trehalose accumulated in freezing- and desiccation-tolerant organisms in nature has been sought as an attractive nontoxic cryoprotectant. Herein, we report a coincubation method for very rapid and efficient delivery of membrane-impermeable trehalose into ovine erythrocytes through reversible membrane permeabilization using pH-responsive, comb-like pseudopeptides. The pseudopeptidic polymers containing relatively long alkyl side chains were synthesized to mimic membrane-anchoring fusogenic proteins. The intracellular trehalose delivery efficiency was optimized by manipulating the side chain length, degree of substitution, and concentration of the pseudopeptides with different hydrophobic alkyl side chains, the pH, temperature, and time of incubation, as well as the polymer-to-cell ratio and the concentration of extracellular trehalose. Treatment of erythrocytes with the comb-like pseudopeptides for only 15 min yielded an intracellular trehalose concentration of 177.9 ± 8.6 mM, which resulted in 90.3 ± 0.7% survival after freeze-thaw. The very rapid and efficient delivery was found to be attributed to the reversible, pronounced membrane curvature change as a result of strong membrane insertion of the comb-like pseudopeptides. The pseudopeptides can enable efficient intracellular delivery of not only trehalose for improved cell cryopreservation but also other membrane-impermeable cargos.
细胞冷冻保存在可重复且经济高效的基于细胞的治疗方法的发展中起着关键作用。在自然界中，冷冻和干燥耐受生物中积累的海藻糖已被寻求作为有吸引力的无毒冷冻保护剂。在此，我们报告了一种共孵育方法，通过使用 pH 响应的梳状假肽可逆地透化膜，将不可渗透膜的海藻糖非常快速有效地递送至绵羊红细胞中。含有相对长的烷基侧链的假肽聚合物被合成以模拟膜锚定融合蛋白。通过操纵具有不同疏水性烷基侧链的假肽的侧链长度、取代度和浓度、pH、孵育温度和时间以及聚合物与细胞的比例以及细胞外海藻糖的浓度，优化了细胞内海藻糖的递送效率。用梳状假肽处理红细胞仅 15 分钟，即可获得 177.9 ± 8.6mM 的细胞内海藻糖浓度，这导致冻融后 90.3 ± 0.7%的存活率。发现这种非常快速且高效的传递归因于梳状假肽的强烈膜插入导致的可逆的、显著的膜曲率变化。这些假肽不仅可以有效地将海藻糖递送至细胞内，从而改善细胞冷冻保存，还可以递送至其他不可渗透膜的载物。
