Pharmaceutical Biotechnology, Center for System-based Drug Research, Ludwig-Maximilians-University Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
J Am Chem Soc. 2012 Jun 20;134(24):10169-73. doi: 10.1021/ja302705v. Epub 2012 Jun 6.
Intracellular delivery of active proteins presents an interesting approach in research and therapy. We created a protein transduction shuttle based on a new traceless click linker that combines the advantages of click reactions with implementation of reversible pH-sensitive bonds. The azidomethyl-methylmaleic anhydride (AzMMMan) linker was found compatible with different click chemistries, demonstrated in bioreversible protein modification with dyes, polyethylene glycol, or a transduction carrier. Linkages were stable at physiological pH but reversible at the mild acidic pH of endosomes or lysosomes. We show that pH-reversible attachment of a defined endosome-destabilizing three-arm oligo(ethane amino)amide carrier generates an effective shuttle for protein delivery. The cargo protein nlsEGFP, when coupled via the traceless AzMMMan linker, experiences efficient cellular uptake and endosomal escape into the cytosol, followed by import into the nucleus. In contrast, irreversible linkage to the same shuttle hampers nuclear delivery of nlsEGFP which after uptake remains trapped in the cytosol. Successful intracellular delivery of bioactive ß-galactosidase as a model enzyme was also demonstrated using the pH-controlled shuttle system.
细胞内活性蛋白的递呈是一种很有前景的研究和治疗方法。我们构建了一种基于新型无痕点击连接子的蛋白转导穿梭体，它结合了点击反应的优点和可逆 pH 敏感键的应用。叠氮甲基-马来酸单甲酯（AzMMMan）连接子与不同点击化学方法兼容，在染料、聚乙二醇或转导载体的生物可逆蛋白修饰中得到了验证。在生理 pH 下连接稳定，但在内涵体或溶酶体的温和酸性 pH 下可逆转。我们证明，通过定义的内涵体破坏三臂寡（乙二胺）酰胺载体的 pH 可逆连接，可以生成有效的蛋白转导穿梭体。当连接子通过无痕的 AzMMMan 连接时，携带蛋白 nlsEGFP 经历有效的细胞摄取和内涵体逃逸进入细胞质，随后进入细胞核。相比之下，不可逆连接到相同的穿梭体阻碍了 nlsEGFP 的核内递呈，摄取后 nlsEGFP 仍滞留在细胞质中。使用 pH 控制的穿梭系统，还成功地递呈了生物活性的β-半乳糖苷酶作为模型酶。
