Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, No. 1800, Lihu Avenue, Wuxi, 214122, China.
Chembiochem. 2024 Nov 18;25(22):e202400512. doi: 10.1002/cbic.202400512. Epub 2024 Oct 24.
Bovine milk exosomes (BmExo) have been identified as versatile nanovesicles for anti-cancer drugs delivery due to their natural availability and biocompatibility. However, tumor-specific delivery based on BmExo often requires post-isolation modifications of the membrane surface with active-targeting ligands. In this study, we report an alternative approach to functionalize BmExo with nanobody combining facile chemical modification and Sortase A-mediated site-specific ligation, as demonstrated by the development of an epidermal growth factor receptor (EGFR)-targeted drug delivery system. The BmExo membrane was first coated with a diglycine-containing amphiphile molecule, NH-GG-PEG2000-DSPE, by hydrophobic insertion. The diglycine as nucleophiles displayed on the membrane enabled the subsequent ligation of the EGFR nanobody (7D12) by Sortase A (SrtA)-mediated site-specific transpeptidation. The successful construction of BmExo-7D12 was confirmed by Western blotting analysis, electron microscopy, and dynamic light scattering (DLS). As a demonstration model, BmExo-7D12 loaded with the chemotherapeutic drug doxorubicin (Dox) was shown to be able to deliver Dox to cancer cells in response to the expression of EGFR as manifested by immunocytochemistry and flow cytometry analysis. Finally, the cytotoxicity assay showed that BmExo-7D12-Dox was more effective in killing tumor cells with high EGFR expression while significantly reduced the non-specific toxicity to EGFR negative cells. In conclusion, these results demonstrate that 7D12-functionalized BmExo can serve as a target-specific delivery system for Dox to selectively kill EGFR-expressing tumor cells. This approach should prove to be versatile and efficient for the generation of protein-ligands modified BmExo.
牛源外泌体（BmExo）由于其天然存在和生物相容性，已被鉴定为用于抗癌药物递送的多功能纳米囊泡。然而，基于 BmExo 的肿瘤特异性递送通常需要通过主动靶向配体对膜表面进行分离后的修饰。在这项研究中，我们报告了一种替代方法，通过简单的化学修饰和 Sortase A 介导的定点连接，使纳米体与 BmExo 结合，从而实现表皮生长因子受体（EGFR）靶向药物递送系统的开发。首先，通过疏水插入，将含有二甘氨酸的两亲分子 NH-GG-PEG2000-DSPE 涂覆在外泌体膜上。膜上显示的二甘氨酸作为亲核试剂，可通过 Sortase A（SrtA）介导的定点转肽反应，随后连接 EGFR 纳米体（7D12）。Western blot 分析、电子显微镜和动态光散射（DLS）证实了 BmExo-7D12 的成功构建。作为示范模型，负载化疗药物阿霉素（Dox）的 BmExo-7D12 能够根据 EGFR 的表达将 Dox 递送到癌细胞中，这表现在免疫细胞化学和流式细胞术分析中。最后，细胞毒性测定表明，BmExo-7D12-Dox 对高 EGFR 表达的肿瘤细胞更有效，而对 EGFR 阴性细胞的非特异性毒性显著降低。总之，这些结果表明，7D12 功能化的 BmExo 可作为 Dox 的靶向递药系统，用于选择性杀伤 EGFR 表达的肿瘤细胞。该方法应该为生成蛋白-配体修饰的 BmExo 提供一种通用且高效的方法。
