Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA.
Anal Bioanal Chem. 2010 Aug;397(8):3317-28. doi: 10.1007/s00216-010-3864-8. Epub 2010 Jun 11.
Currently, molecular mechanisms of multidrug ABC (ATP-binding cassette) membrane transporters remain elusive. In this study, we synthesized and characterized purified spherically shaped silver nanoparticles (Ag NPs) (11.8 +/- 2.6 nm in diameter), which were stable (non-aggregation) in PBS buffer and inside single living cells. We used the size-dependent localized surface plasmon resonance (LSPR) spectra of single Ag NPs to determine their sizes and to probe the size-dependent transport kinetics of the ABC (BmrA, BmrA-EGFP) transporters in single living cells (Bacillus subtilis) in real time at nanometer resolution using dark-field optical microscopy and spectroscopy (DFOMS). The results show that the smaller NPs stayed longer inside the cells than larger NPs, suggesting size-dependent efflux kinetics of the membrane transporter. Notably, accumulation and efflux kinetics of intracellular NPs for single living cells depended upon the cellular expression level of BmrA, NP concentrations, and a pump inhibitor (25 muM, orthovanadate), suggesting that NPs are substrates of BmrA transporters and that passive diffusion driven by concentration gradients is the primary mechanism by which the NPs enter the cells. The accumulation and efflux kinetics of intracellular NPs for given cells are similar to those observed using a substrate (Hoechst dye) of BmrA, demonstrating that NPs are suitable probes for study of multidrug membrane transporters of single living cells in real-time. Unlike fluorescent probes, single Ag NPs exibit size-dependent LSPR spectra and superior photostability, enabling them to probe the size-dependent efflux kinetics of membrane transporters of single living cells in real-time for better understanding of multidrug resistance.
目前，多药 ABC（ATP 结合盒）膜转运蛋白的分子机制仍不清楚。在这项研究中，我们合成并表征了纯化的球形银纳米粒子（Ag NPs）（直径为 11.8 +/- 2.6nm），它们在 PBS 缓冲液中和单个活细胞内稳定（不聚集）。我们使用单个 Ag NPs 的尺寸依赖的局域表面等离子体共振（LSPR）光谱来确定它们的尺寸，并使用暗场光学显微镜和光谱学（DFOMS）以纳米分辨率实时探测单个活细胞（枯草芽孢杆菌）中 ABC（BmrA、BmrA-EGFP）转运蛋白的尺寸依赖性转运动力学。结果表明，较小的 NPs 在细胞内停留的时间比较大的 NPs 长，这表明膜转运蛋白的外排动力学具有尺寸依赖性。值得注意的是，单个活细胞内的 NP 积累和外排动力学取决于 BmrA 的细胞表达水平、NP 浓度和泵抑制剂（25 μM，正钒酸盐），这表明 NP 是 BmrA 转运蛋白的底物，并且浓度梯度驱动的被动扩散是 NP 进入细胞的主要机制。对于给定的细胞，NP 的积累和外排动力学与观察到的 BmrA 底物（Hoechst 染料）相似，表明 NP 是研究单个活细胞中多药膜转运蛋白的实时研究的合适探针。与荧光探针不同，单个 Ag NPs 表现出尺寸依赖的 LSPR 光谱和优异的光稳定性，使它们能够实时探测单个活细胞中膜转运蛋白的尺寸依赖性外排动力学，从而更好地理解多药耐药性。
