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

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.

Single Nanoparticle Plasmonic Spectroscopy for Study of the Efflux Function of Multidrug ABC Membrane Transporters of Single Live Cells.

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 (B. subtillis). 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, ΔbmrA) 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 ΔbmrA (deletion of BmrA) and the lowest ones in Ct-BmrA-EGFP (over-expression of BmrA). Interestingly, the accumulation of intracellular NPs in ΔbmrA 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.

Single Nanoparticle Plasmonic Spectroscopy for Study of Charge-Dependent Efflux Function of Multidrug ABC Transporters of Single Live Bacillus subtilis Cells.

Multidrug membrane transporters can selectively extrude a wide variety of structurally and functionally unrelated substrates, and they are responsible for ineffective treatment of a wide range of diseases (e.g., infection and cancer). Their underlying molecular mechanisms remain elusive. In this study, we functionalized Ag NPs (11 nm in diameter) with two biocompatible peptides (CALNNK, CALNNE) to prepare positively and negatively charged Ag-peptide NPs (Ag-CALNNK NPs+ζ, Ag-CALNNE NPs-4ζ), respectively. We used them as photostable plasmonic imaging probes to study charge-dependent efflux kinetics of BmrA (ABC) membrane transporter of single live Bacillus (B.) subtilis cells. Two strains of the cells, normal expression of BmrA (WT) or devoid of BmrA (ΔBmrA), were used to study the charge-dependent efflux kinetics of single NPs upon the expression of BmrA. The NPs (1.4 nM) were stable (non-aggregated) in a PBS buffer and biocompatible to the cells. We found the high dependent accumulation of the intracellular NPs in both WT and ΔBmrA upon the charge and concentration of NPs. Notably, the accumulation rates of the positively charged NPs in single live WT cells are nearly identical to those in ΔBmrA cells, showing independence upon the expression of BmrA. In contrast, the accumulation rates of the negatively charged NPs in WT are much lower than in ΔBmrA, showing high dependence upon the expression of BmrA and suggesting that BmrA extrude the negatively charged NPs, but not positively charged NPs, out of the WT. The accumulation of positively charged NPs in both WT and ΔBmrA increases nearly proportionally to the NP concentration. The accumulation of negatively charged NPs in ΔBmrA, but not in WT, also increases nearly proportionally to the NP concentration. These results suggest that both negatively and positively charged NPs enter the cells via passive diffusion driven by concentration gradients across the cellular membrane, and BmrA can only extrude the negatively charged NPs out of the WT. This study shows that single NP plasmon spectroscopy can serve as a powerful tool to identify single plasmonic NPs and to probe the charge-dependent efflux kinetics and function of single membrane transporters in single live cells in real time.

Single nanoparticle spectroscopy for real-time in vivo quantitative analysis of transport and toxicity of single nanoparticles in single embryos.

Nanomaterials exhibit distinctive physicochemical properties and promise a wide range of applications from nanotechnology to nanomedicine, which raise serious concerns about their potential environmental impacts on ecosystems. Unlike any conventional chemicals, nanomaterials are highly heterogeneous, and their properties can alter over time. These unique characteristics underscore the importance of study of their properties and effects on living organisms in real time at single nanoparticle (NP) resolution. Here we report the development of single-NP plasmonic microscopy and spectroscopy (dark-field optical microscopy and spectroscopy, DFOMS) and ultrasensitive in vivo assay (cleavage-stage zebrafish embryos, critical aquatic species) to study transport and toxicity of single silver nanoparticles (Ag NPs, 95.4 ± 16.0 nm) on embryonic developments. We synthesized and characterized purified and stable (non-aggregation) Ag NPs, determined their sizes and doses (number), and their transport mechanisms and effects on embryonic development in vivo in real time at single-NP resolution. We found that single Ag NPs passively entered the embryos through their chorionic pores via random Brownian diffusion and stayed inside the embryos throughout their entire development (120 h), suggesting that the embryos can bio-concentrate trace NPs from their environment. Our studies show that higher doses and larger sizes of Ag NPs cause higher toxic effects on embryonic development, demonstrating that the embryos can serve as ultrasensitive in vivo assays to screen biocompatibility and toxicity of the NPs and monitor their potential release into aquatic ecosystems.

In vivo quantitative study of sized-dependent transport and toxicity of single silver nanoparticles using zebrafish embryos.

Nanomaterials possess distinctive physicochemical properties (e.g., small sizes and high surface area-to-volume ratios) and promise a wide variety of applications, ranging from the design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advances in nanotechnology. In this study, we have synthesized and characterized purified and stable (nonaggregation) silver nanoparticles (Ag NPs, 41.6 ± 9.1 nm in average diameter) and utilized early developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe the diffusion and toxicity of Ag NPs. We found that single Ag NPs (30-72 nm diameters) passively diffused into the embryos through chorionic pores via random Brownian motion and stayed inside the embryos throughout their entire development (120 hours-post-fertilization, hpf). Dose- and size-dependent toxic effects of the NPs on embryonic development were observed, showing the possibility of tuning biocompatibility and toxicity of the NPs. At lower concentrations of the NPs (≤0.02 nM), 75-91% of embryos developed into normal zebrafish. At the higher concentrations of NPs (≥0.20 nM), 100% of embryos became dead. At the concentrations in between (0.02-0.2 nM), embryos developed into various deformed zebrafish. Number and sizes of individual Ag NPs embedded in tissues of normal and deformed zebrafish at 120 hpf were quantitatively analyzed, showing deformed zebrafish with higher number of larger NPs than normal zebrafish and size-dependent nanotoxicity. By comparing with our previous studies of smaller Ag NPs (11.6 ± 3.5 nm), we found striking size-dependent nanotoxicity that, at the same molar concentration, the larger Ag NPs (41.6 ± 9.1 nm) are more toxic than the smaller Ag NPs (11.6 ± 3.5 nm).