As a typical plant virus which has biocompatibility and high transfection efficiency, tobacco mosaic virus (TMV) has shown broad application potential in drug or gene delivery field. Elucidating its intracellular trafficking is of great importance in investigation of its cytotoxicity, targeting site, and delivery efficiency, and is advantageous to designing new TMV-based drug delivery systems with different targets. By taking advantage of the regulated pH value of different organelles in a mammalian cell, we exploit a pH detection strategy to investigate the intracellular trafficking pathway of TMV. Here, we report a single-wavelength excited ratiometric fluorescent pH probe. This probe is constructed by simultaneously coupling pH-sensitive fluorescein isothiocyanate (FITC) and pH-insensitive rhodamine B isothiocyanate (RBIRC) onto the inner surface of TMV. The fluorescence intensity ratio of FITC to RBITC excited at 488 nm responds specifically towards pH value over other interferential agents. By taking use of this single-wavelength excited ratiometric pH probe and confocal laser scanning microscopy, it is shown that the endocytosed TMV is located in a pH decreasing microenvironment and eventually enters lysosomes. This work may provide important guidance on construction of TMV-based nano carriers.

作为具有生物相容性和高转染效率的典型植物病毒，烟草花叶病毒（TMV）在药物或基因传递领域具有广泛的应用潜力。阐明其细胞内运输在研究其细胞毒性，靶向部位和递送效率方面非常重要，并且有利于设计具有不同靶点的新型基于TMV的药物递送系统。通过利用哺乳动物细胞中不同细胞器调节的pH值，我们利用pH检测策略来研究TMV的细胞内运输途径。在这里，我们报告一个单波长激发比例荧光pH探针。通过将pH敏感的异硫氰酸荧光素（FITC）和pH敏感的若丹明B异硫氰酸酯（RBIRC）偶联到TMV的内表面上来构造此探针。在488 nm处激发的FITC与RBITC的荧光强度比对pH值的反应特别强于其他干扰剂。通过使用这种单波长激发比例式pH探针和共聚焦激光扫描显微镜，可以证明内吞的TMV位于pH降低的微环境中并最终进入溶酶体。这项工作可能为基于TMV的纳米载体的构建提供重要指导。通过使用这种单波长激发比例式pH探针和共聚焦激光扫描显微镜，可以证明内吞的TMV位于pH降低的微环境中并最终进入溶酶体。这项工作可能为基于TMV的纳米载体的构建提供重要指导。通过使用这种单波长激发比例式pH探针和共聚焦激光扫描显微镜，可以证明内吞的TMV位于pH降低的微环境中并最终进入溶酶体。这项工作可能为基于TMV的纳米载体的构建提供重要指导。