Probing of multidrug ABC membrane transporters of single living cells using single plasmonic nanoparticle optical probes.
Probing of multidrug ABC membrane transporters of single living cells using single plasmonic nanoparticle optical probes.
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DOI：
10.1007/s00216-010-3864-8
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发表时间：
2010-08
影响因子：
4.3
通讯作者：
Xu, Xiao-Hong Nancy
中科院分区：
文献类型：
--
作者：
Lee, Kerry J.;Browning, Lauren M.;Huang, Tao;Ding, Feng;Nallathamby, Prakash D.;Xu, Xiao-Hong Nancy
关键词：
目前，多药 ABC（ATP 结合盒）膜转运蛋白的分子机制仍不清楚。在本研究中，我们合成并表征了纯化的球形银纳米颗粒 (Ag NP)（直径 11.8 ± 2.6 nm），它们在 PBS 缓冲液中和单个活细胞内稳定（非聚集）。我们使用单个 Ag NP 的尺寸依赖性局域表面等离子共振 (LSPR) 光谱来确定其尺寸，并使用暗场光学显微镜和光谱 (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运输动力学。结果表明，较小的纳米颗粒比较大的纳米颗粒在细胞内停留的时间更长，这表明膜转运蛋白的尺寸依赖性流出动力学。值得注意的是，单个活细胞的细胞内 NP 的积累和流出动力学取决于 BmrA 的细胞表达水平、NP 浓度和泵抑制剂（25 µM，原钒酸盐），表明 NP 是 BmrA 转运蛋白的底物，浓度梯度驱动的被动扩散是 NP 进入细胞的主要机制。给定细胞的细胞内 NP 的积累和流出动力学与使用 BmrA 底物（Hoechst 染料）观察到的相似，表明 NP 是实时研究单个活细胞的多药物膜转运蛋白的合适探针。与荧光探针不同，单银纳米粒子表现出尺寸依赖性的局域表面等离子体共振光谱和优异的光稳定性，使它们能够实时探测单个活细胞膜转运蛋白的尺寸依赖性流出动力学，以更好地了解多药耐药性。
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 shows 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 µM, 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 exhibit 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.
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影响因子：
56.9
作者：
Locher, KP;Lee, AT;Rees, DC
通讯作者：
Rees, DC
影响因子：
17.1
作者：
Lee, Kerry J.;Nallathamby, Prakash D.;Xu, Xiao-Hong Nancy
通讯作者：
Xu, Xiao-Hong Nancy
影响因子：
6.7
作者：
Browning LM;Lee KJ;Huang T;Nallathamby PD;Lowman JE;Xu XH
通讯作者：
Xu XH
影响因子：
4.8
作者：
Gandlur, SM;Wei, L;Kaur, P
通讯作者：
Kaur, P
影响因子：
3.5
作者：
Kyriacou, SV;Nowak, ME;Xu, XHN
通讯作者：
Xu, XHN