Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, Massachusetts 02115, USA.
Mol Pharm. 2010 Aug 2;7(4):1149-58. doi: 10.1021/mp1000229.
Earlier, we have shown that doxorubicin-loaded liposomes (Doxil) modified with a chimeric phage fusion coat protein specific toward MCF-7 breast cancer cells identified from a phage landscape library demonstrated a significantly enhanced association with target cells and an increased cytotoxicity. Based on some structural similarities between the N-terminus of the phage potein and known fusogenic peptides, we hypothesized that, in addition to the specific targeting, the phage protein may possess endosome-escaping potential and an increased cytotoxicity of drug-loaded phage protein-targeted liposomes may be explained by an advantageous combination of both, cell targeting and endosomal escape of drug-loaded nanocarrier. The use of the fluorescence resonance energy transfer (FRET) technique allowed us to clearly demonstrate the pH-dependent membrane fusion activity of the phage protein. Endosomal escape and cytosolic delivery of phage-liposomes was visualized with fluorescence microscopy. Endosome acidification inhibition by bafilomycin A 1 resulted in decreased cytotoxicity of the phage-Doxil, while the endosome disruption by chloroquine had a negligible effect on efficacy of phage-Doxil, confirming its endosomal escape. Our results demonstrated an endosome-escaping property of the phage protein and provided an insight on mechanism of the enhanced cytotoxicity of phage-Doxil.
早些时候，我们已经表明，从噬菌体景观文库中鉴定出的针对 MCF-7 乳腺癌细胞的嵌合噬菌体融合外壳蛋白修饰的多柔比星负载脂质体（Doxil）与靶细胞的结合显著增强，细胞毒性增加。基于噬菌体蛋白的 N 端与已知融合肽之间的一些结构相似性，我们假设除了特异性靶向外，噬菌体蛋白可能具有内体逃逸潜力，并且载药噬菌体蛋白靶向脂质体的细胞毒性增加可以通过两者的有利结合来解释，即细胞靶向和载药纳米载体的内体逃逸。荧光共振能量转移（FRET）技术的使用使我们能够清楚地证明噬菌体蛋白的 pH 依赖性膜融合活性。用荧光显微镜观察了噬菌体脂质体的内体逃逸和细胞质递送。通过巴弗洛霉素 A1 抑制内体酸化导致噬菌体-Doxil 的细胞毒性降低，而氯喹对内体的破坏对噬菌体-Doxil 的疗效几乎没有影响，证实了其具有内体逃逸能力。我们的结果证明了噬菌体蛋白的内体逃逸特性，并深入了解了噬菌体-Doxil 增强细胞毒性的机制。
