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 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.