Browning Lauren M, Lee Kerry J, Cherukuri Pavan K, Nallathamby Prakash D, Warren Seth, Jault Jean-Michel, Xu Xiao-Hong Nancy
Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA.
UMR5086 CNRS/UCBLyon I, MMSB-IBCP, 7 Passage du Vercors 69367 Lyon cedex 07, France.
RSC Adv. 2016;6(43):36794-36802. doi: 10.1039/C6RA05895G. Epub 2016 Mar 30.
ATP-binding cassette (ABC) membrane transporters exist in all living organisms and play key roles in a wide range of cellular and physiological functions. The ABC transporters can selectively extrude a wide variety of structurally and functionally unrelated substrates, leading to multidrug resistance. Despite extensive study, their efflux molecular mechanisms remain elusive. In this study, we synthesized and characterized purified silver nanoparticles (Ag NPs) (97 ± 13 nm in diameter), and used them as photostable optical imaging probes to study efflux kinetics of ABC membrane transporters (BmrA) of single live cells ). The NPs with concentrations up to 3.7 pM were stable (non-aggregated) in a PBS buffer and biocompatible with the cells. We found a high dependence of accumulation of the intracellular NPs in single live cells (WT, Ct-BmrA-EGFP, Δ) upon the cellular expression level of BmrA and NP concentration (0.93, 1.85 and 3.7 pM), showing the highest accumulation of intracellular NPs in Δ (deletion of BmrA) and the lowest ones in Ct-BmrA-EGFP (over-expression of BmrA). Interestingly, the accumulation of intracellular NPs in Δ increases nearly proportionally with the NP concentration, while those in WT and Ct-BrmA-EGFP do not. This suggests that the NPs enter the cells via passive diffusion driven by concentration gradients and are extruded out of cells by BmrA transporters, similar to conventional pump substrates (antibiotics). This study shows that such large substrates (84-100 nm NPs) can enter into the live cells and be extruded out of the cells by BmrA, and the NPs can serve as nm-sized optical imaging probes to study the size-dependent efflux kinetics of membrane transporters in single live cells in real time.
ATP结合盒（ABC）膜转运蛋白存在于所有生物中，在广泛的细胞和生理功能中发挥关键作用。ABC转运蛋白可以选择性地排出多种结构和功能无关的底物，导致多药耐药性。尽管进行了广泛的研究，但其外排分子机制仍然难以捉摸。在本研究中，我们合成并表征了纯化的银纳米颗粒（Ag NPs）（直径为97±13 nm），并将其用作光稳定的光学成像探针，以研究单个活细胞的ABC膜转运蛋白（BmrA）的外排动力学。浓度高达3.7 pM的纳米颗粒在PBS缓冲液中稳定（未聚集），并且与细胞具有生物相容性。我们发现单个活细胞（野生型、Ct-BmrA-EGFP、缺失型）中细胞内纳米颗粒的积累高度依赖于BmrA的细胞表达水平和纳米颗粒浓度（0.93、1.85和3.7 pM），显示出在缺失型（BmrA缺失）中细胞内纳米颗粒的积累最高，而在Ct-BmrA-EGFP（BmrA过表达）中最低。有趣的是，缺失型中细胞内纳米颗粒的积累几乎与纳米颗粒浓度成比例增加，而野生型和Ct-BrmA-EGFP中的积累则不然。这表明纳米颗粒通过浓度梯度驱动的被动扩散进入细胞，并被BmrA转运蛋白排出细胞，类似于传统的泵底物（抗生素）。这项研究表明，如此大的底物（84-100 nm纳米颗粒）可以进入活细胞并被BmrA排出细胞，并且纳米颗粒可以作为纳米尺寸的光学成像探针，实时研究单个活细胞中膜转运蛋白的尺寸依赖性外排动力学。
