Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho nam 2, 162 06 Prague 6, Czech Republic.
Biomacromolecules. 2011 Oct 10;12(10):3645-55. doi: 10.1021/bm200897b. Epub 2011 Aug 31.
We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.
我们设计、合成并表征了含有四个 VAALEKE（肽 E）或 VAALKEK（肽 K）序列重复的肽。虽然这些肽在水溶液中单独采用无规卷曲构象，但它们的等摩尔混合物通过 CD 光谱证实形成异二聚体卷曲螺旋。5-叠氮戊酸通过短聚乙二醇间隔臂连接到肽 E 的 N 末端。末端叠氮基团使肽能够与基于含有炔丙基基团的 N-(2-羟丙基)甲基丙烯酰胺共聚物的合成药物载体通过“点击”化学进行缀合。当掺入聚合物药物载体中时，肽 E 与属于 M75 抗体的重组单链片段 (scFv) 的肽 K 形成稳定的非共价复合物。该复合物从而介导聚合物药物载体和蛋白质之间的非共价连接。重组 scFv 抗体片段被选为针对碳酸酐酶 IX 的靶向配体，碳酸酐酶 IX 是各种人类癌的肿瘤细胞过表达的标志物。通过 ELISA 证实了聚合物-scFv 复合物的抗原结合亲和力。这种方法为设计用于细胞特异性递药的蛋白（抗体）靶向聚合物药物和基因载体提供了一种明确、特异且非破坏性的通用方法。
