Single gold nanoparticle plasmonic spectroscopy for study of chemical-dependent efflux function of single ABC transporters of single live Bacillus subtilis cells

Abstract

ATP-binding cassette (ABC) membrane transporters serve as self-defense transport apparatus in many living organisms and they can selectively extrude a wide variety of substrates, leading to multidrug resistance (MDR). The detailed molecular mechanisms remain elusive. Single nanoparticle plasmonic spectroscopy highly depends upon their sizes, shapes, chemical and surface properties. In our previous studies, we have used the size-dependent plasmonic spectra of single silver nanoparticles (Ag NPs) to study the real-time efflux kinetics of the ABC (BmrA) transporter and MexAB-OprM transporter in single live cells (Gram-positive and Gram-negative bacterium), respectively. In this study, we prepared and used purified, biocompatible and stable (non-aggregated) gold nanoparticles (Au NPs) (12.4 ± 0.9 nm) to study the efflux kinetics of single BmrA membrane transporters of single live Bacillus subtillis cells, aiming to probe chemical dependent efflux functions of BmrA transporters and their potential chemical sensing capability. Similar to those observed using Ag NPs, accumulation of the intracellular Au NPs in single live cells (WT and ΔBmrA) highly depends upon the cellular expression of BmrA and the NP concentration (0.7 and 1.4 nM). The lower accumulation of intracellular Au NPs in WT (normal expression of BmrA) than ΔBmrA (deletion of bmrA) indicates that BmrA extrudes the Au NPs out of the WT cells. The accumulation of Au NPs in the cells increases with NP concentration, suggesting that the Au NPs most likely passively diffuse into the cells, similar to antibiotics. The result demonstrates that such small Au NPs can serve as imaging probes to study the efflux function of the BmrA membrane transporter in single live cells. Furthermore, the dependence of the accumulation rate of intracellular Au NPs in single live cells upon the expression of BmrA and the concentration of the NPs is about twice higher than that of the same sized Ag NPs. This interesting finding suggests the chemical-dependent efflux kinetics of BmrA and that BmrA could distinguish nearly identical sized Au NPs from Ag NPs and might possess chemical sensing machinery.

Gold Nanorods Functionalized with Cathepsin B Targeting Peptide and Doxorubicin for Combinatorial Therapy against Multidrug Resistance

“…It was found that the larger nanoparticles were more favorable to overcome the MDR, due to the size-exclusion effect, i.e., too-big-to-be-pumped-out. The P-gp is ∼16 nm long and 4.5–6.5 nm wide, with the active site shaped funnel 0.9–2.5 nm in the middle. − The AuNRs used in the present work are 48.8 × 14.4 nm, much larger than the active site of P-gp; therefore, AuNR–LAX could not match well with P-gp, and thus it could effectively avoid efflux by the P-gp pump and increase the accumulation of nanomedicine inside the cells. It is also favorable for AuNR–LAX to be applied in photothermal therapy of MDR phenotype cancer.…”

“…After AuNR–LAX entered into cancer cells, GFLG was cleaved by cathepsin B, and DOX was thus released; therefore, the fluorescence of DOX was recovered from AuNR nanosurface quenching, making the DOX release visible and trackable inside the cancer cells. − Because the AuNRs show a property of local surface plasmon resonance (LSPR) that can shift from ∼600 nm to the near-infrared (NIR) region with large AuNR aspect ratio, the AuNRs can perform as a nanoscale transducer to convert NIR light into heat, providing a platform for cancer treatment via a photothermal effect. − Moreover, the size of the nanoparticles affects the drug bypassing P-glycoprotein (P-gp), a major drug transporter in MDR phenotype cancer cells. The larger nanoparticles hold high potential in overcoming MDR because their size is much larger than the active P-gp binding site. − In the present work, AuNRs with aspect ratio of 3.4 (48.8 × 14.4 nm, Figure ) were prepared and used as a nanocarrier to construct an enzyme-responsive DDS AuNR–LAX. After AuNR–LAX was internalized by the cancer cells, the peptide linker GFLG could be cleaved by cathepsin B, and then DOX was released in a controlled fashion to exert the anticancer activity and, combined with the NIR photothermal effect, to improve the therapeutic index toward both parental and DOX-resistant cancer cells and to alleviate the adverse effect of DOX on the healthy cells.…”

Study of silver nanoparticle effects on some molecular responses and metabolic pathways of Phytophthora parasitica

“…transporter is also indicative of self-defense. ABC transporters exude a variety of substrates, including extracellular toxins from the cell [55,56]. However, research has indicated that AgNPs induce cell death and inhibit the effl ux mechanism of the emergence of multidrug resistant (MDR) cancer cells [57].…”

“…ABC transporters have also been reported to be involved in toxicant effl ux and virulence [58][59][60]. These transporters serve as an apparatus for cell defense in many organisms, extruding various substrates [55]. The high expression in treated samples in response to AgNPs would indicate a stress response.…”

Size-dependent inhibitory effects of antibiotic nanocarriers on filamentation ofE. coli

“…We have demonstrated that we can image and characterize the sizes of single Ag NPs at nanometer (nm) resolution in single live cells in situ in real time using size-dependent localized surface plasmon resonance (LSPR) spectra of single Ag NPs by dark-eld optical microscopy and spectroscopy (DFOMS). [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] We have used the size-dependent LSPR spectra of single Ag NPs to track the transport of single NPs in and out of single live cells and to determine the pore sizes of membrane transporters. 29,30 In our previous studies, we found dose and size dependent inhibitory effects of antibiotic nanocarriers (AgMUNH-Ox NPs) against Pseudomonas aeruginosa (Gram-negative bacterium) and Bacillus subtilis (Gram-positive bacterium).…”