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Probing of multidrug ABC membrane transporters of single living cells using single plasmonic nanoparticle optical probes

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Abstract

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 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 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.

Probing of efflux functions of single multidrug transporters using single plasmonic nanoparticles

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Acknowledgements

This work is supported in part by NSF (NIRT: BES 0507036) and NIH (R01 GM076440). Ding, Nallathamby, Browning, and Lee are grateful for the support of Dominion Scholar Fellowship, NIH-GRAS (R01 GM076440S1), and NSF-GRAS (CBET 0940923), respectively. We thank J.-M. Jault for providing us with ΔBmrA (ΔYvcC) cells, and CharFac of University of Minnesota (a NNIN site funded by NSF) for their assistance in characterizing Ag nanoparticles using HRTEM.

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  1. Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA, 23529, USA

    Kerry J. Lee, Lauren M. Browning, Tao Huang, Feng Ding, Prakash D. Nallathamby & Xiao-Hong Nancy Xu

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  1. Kerry J. Lee
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  2. Lauren M. Browning
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Correspondence to Xiao-Hong Nancy Xu.

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Lee, K.J., Browning, L.M., Huang, T. et al. Probing of multidrug ABC membrane transporters of single living cells using single plasmonic nanoparticle optical probes. Anal Bioanal Chem 397, 3317–3328 (2010). https://doi.org/10.1007/s00216-010-3864-8

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