Wang Jing, MacEwan Sarah R, Chilkoti Ashutosh
Department of Biomedical Engineering, Duke University , Durham, North Carolina 27708, United States.
Nano Lett. 2017 Feb 8;17(2):1226-1232. doi: 10.1021/acs.nanolett.6b05041. Epub 2017 Jan 3.
Understanding the intracellular distribution and trafficking of nanoparticle drug carriers is necessary to elucidate their mechanisms of drug delivery and is helpful in the rational design of novel nanoparticle drug delivery systems. The traditional immunofluorescence method to study intracellular distribution of nanoparticles using organelle-specific antibodies is laborious and subject to artifacts. As an alternative, we developed a new method that exploits ratiometric fluorescence imaging of a pH-sensitive Lysosensor dye to visualize and quantify the spatial distribution of nanoparticles in the endosomes and lysosomes of live cells. Using this method, we compared the endolysosomal distribution of cell-penetrating peptide (CPP)-functionalized micelles to unfunctionalized micelles and found that CPP-functionalized micelles exhibited faster endosome-to-lysosome trafficking than unfunctionalized micelles. Ratiometric fluorescence imaging of pH-sensitive Lysosensor dye allows rapid quantitative mapping of nanoparticle distribution in endolysosomes in live cells while minimizing artifacts caused by extensive sample manipulation typical of alternative approaches. This new method can thus serve as an alternative to traditional immunofluorescence approaches to study the intracellular distribution and trafficking of nanoparticles within endosomes and lysosomes.
了解纳米颗粒药物载体的细胞内分布和转运对于阐明其药物递送机制是必要的，并且有助于合理设计新型纳米颗粒药物递送系统。使用细胞器特异性抗体研究纳米颗粒细胞内分布的传统免疫荧光方法既费力又容易产生假象。作为一种替代方法，我们开发了一种新方法，该方法利用对pH敏感的溶酶体传感器染料的比率荧光成像来可视化和量化纳米颗粒在活细胞的内体和溶酶体中的空间分布。使用这种方法，我们比较了细胞穿透肽（CPP）功能化胶束与未功能化胶束的内溶酶体分布，发现CPP功能化胶束比未功能化胶束表现出更快的内体到溶酶体的转运。对pH敏感的溶酶体传感器染料的比率荧光成像允许快速定量绘制活细胞内溶酶体中纳米颗粒的分布，同时将替代方法典型的大量样品操作引起的假象降至最低。因此，这种新方法可以作为传统免疫荧光方法的替代方法，用于研究纳米颗粒在内体和溶酶体内的细胞内分布和转运。
