Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Chembiochem. 2010 Jun 14;11(9):1273-9. doi: 10.1002/cbic.201000125.
The structurally regular and stable self-assembled capsids derived from viruses can be used as scaffolds for the display of multiple copies of cell- and tissue-targeting molecules and therapeutic agents in a convenient and well-defined manner. The human iron-transfer protein transferrin, a high affinity ligand for receptors upregulated in a variety of cancers, has been arrayed on the exterior surface of the protein capsid of bacteriophage Qbeta. Selective oxidation of the sialic acid residues on the glycan chains of transferrin was followed by introduction of a terminal alkyne functionality through an oxime linkage. Attachment of the protein to azide-functionalized Qbeta capsid particles in an orientation allowing access to the receptor binding site was accomplished by the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. Transferrin conjugation to Qbeta particles allowed specific recognition by transferrin receptors and cellular internalization through clathrin-mediated endocytosis, as determined by fluorescence microscopy on cells expressing GFP-labeled clathrin light chains. By testing Qbeta particles bearing different numbers of transferrin molecules, it was demonstrated that cellular uptake was proportional to ligand density, but that internalization was inhibited by equivalent concentrations of free transferrin. These results suggest that cell targeting with transferrin can be improved by local concentration (avidity) effects.
来源于病毒的结构规整且稳定的自组装衣壳可用作细胞和组织靶向分子和治疗剂的多个拷贝的展示支架，其方式既方便又具有良好的定义性。人类铁传递蛋白转铁蛋白是多种癌症中上调受体的高亲和力配体，已被排列在噬菌体 Qβ的蛋白衣壳的外表面。转铁蛋白糖链上唾液酸残基的选择性氧化，接着通过肟键引入末端炔基官能团。通过铜（I）催化的叠氮-炔烃环加成（CuAAC）点击反应，将蛋白附着在带有叠氮基的 Qβ衣壳颗粒上，以允许与受体结合位点的方式附着。通过绿色荧光蛋白标记的笼形蛋白轻链在表达 GFP 的细胞上的荧光显微镜，证实了转铁蛋白与 Qβ颗粒的缀合允许通过网格蛋白介导的内吞作用进行特定的转铁蛋白受体识别和细胞内化。通过测试带有不同数量转铁蛋白分子的 Qβ颗粒，证明细胞摄取与配体密度成正比，但游离转铁蛋白的等效浓度会抑制内化。这些结果表明，通过局部浓度（亲合力）效应可以改善转铁蛋白的细胞靶向性。
