8 research outputs found

    Study of ABC Membrane Transporters in Single Live Cells

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    The multidrug ATP-binding cassette (ABC) membrane transporters (efflux pumps) are found in both prokaryotes and eukaryotes and they can extrude diverse structurally unrelated substrates, such as antibiotics and chemotherapeutic agents out of the cells. The efflux pumps are responsible for multidrug resistance (MDR) and the failure of numerous treatments in infections and cancers. All ABC membrane transporters share a common modular topology containing two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). The underlying molecular mechanisms regarding how the similar structural ABC membrane transporters could selectively extrude a wide variety of substrates and cause MDR, are not yet fully understood. Radioisotopes and fluorophores have been widely used as probes to study efflux kinetics of multidrug membrane transporters in bulk cells which could have masked interesting rare events from individual cells. Moreover, radioisotopes and fluorophores do not process size-dependent physicochemical properties, making them unsuitable to serve as various sized substrates for the study of efflux function of the ABC transporter. In this dissertation, we focus on the development of three different sized single silver nanoparticles (Ag NPs) to serve as both drug nanocarriers and imaging probes to study size-dependent efflux function of ABC membrane transporters in single live cells (e.g., Escherichia coli) in situ in real time. We synthesized and characterized Ag NPs with diameters of 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm, functionalized them with a monolayer of 11-amino-1-undecanethiol (AUT) to prepare AgMUNH2 NPs (control nanocarriers). We then covalently linked the AgMUNH2 NPs with ofloxacin (Oflx) to prepare AgMUNH-Oflx NPs (antibiotic drug nanocarriers) with conjugation ratios of 8.6x102, 9.4x103, and 6.5x105 Oflx molecules per NP, respectively. We studied inhibitory effects of these antibiotic drug nanocarriers against E. coli and found size-dependent inhibitory effects in which the same amount of Oflx carried by the largest nanocarriers exhibited the highest inhibitory effects, and the smallest nanocarriers exhibited the lowest inhibitory effects. The AgMUNH2 NPs did not show significant inhibitory effects on cell growth. Furthermore, we used Ag NP-based nanocarriers as imaging probes to study efflux function of multidrug ABC membrane transporters in single live E. coli cells, because Ag NPs process distinctive size-dependent photostable plasmonic optical properties. We found that the accumulation rates of nanocarriers highly depended on the NP concentration, the presence of ATPase pump inhibitor, and the types and sizes of nanocarriers. Interestingly, the ABC membrane transporters extrude AgMUNH-Oflx NPs more effectively and rapidly than AgMUNH2 NPs indicating that efflux pumps could be equipped with a sensing machinery to detect, recognize and extrude toxic substrates (e.g., antibiotics). Notably, the cells could extrude the smaller nanocarriers more effectively, leading to the least inhibitory effects. Therefore, the smaller drug nanocarriers could serve as excellent imaging probes to study the efflux function while the larger nanocarriers serve as powerful drug delivery vehicles. This study demonstrates the possibility of developing optimal-sized nanocarriers to achieve the maximum drug efficacy and potentially avoiding MDR

    A simulation model for triclosan concentrations in the North and Middle Rivers, Virginia

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    Incidences of fish kills and intersex phenomena have occurred extensively in the Shenandoah River since 2004. Pharmaceuticals including triclosan have been detected at low concentrations in the Shenandoah River. Scientists hypothesize that triclosan, an antibacterial agent, may be one of the pharmaceuticals that is responsible for fish kills and intersex phenomena. Methyl triclosan (MTS) were found in fish tissues at a significantly higher concentration in the part of the Shenandoah River where fish kills are present compared to a non-impact river. Triclosan is widely used in personal care products, such as soaps, shampoos, and toothpastes and is rinsed down the drain. It enters the aquatic environment via wastewater discharges and can accumulate in the surface waters since waters and wastewater treatment technologies do not completely remove triclosan from treated wastewater. This thesis explores an application of the system dynamics problem solving and modeling methodology to predict triclosan concentration levels in parts of the North and Middle Rivers, main tributaries of the South Fork Shenandoah River. A simulation model calculate triclosan concentrations in the North and Middle River based on numerous factors including watershed characteristics, streamflow, and removal efficiency of triclosan in wastewater treatment plants. The effect of removal efficiency in WWTPs is the most sensitive factor to the triclosan concentration levels regarding simulated results. Concentrations significantly change whether treatment systems are improved or deteriorated. Treatment technology improvement would be a significant approach to reduce triclosan concentrations in the North and Middle Rivers. For further research, the model platform could be applied to predict concentrations of triclosan or other pollutants in other rivers. However, a number of variables are needed to be modified to fulfill this purpose

    Size-Dependent Inhibitory Effects of Antibiotic Nanocarriers on Filamentation of \u3ci\u3eE. coli\u3c/i\u3e

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    Multidrug membrane transporters exist in both prokaryotic and eukaryotic cells and cause multidrug resistance (MDR), which results in an urgent need for new and more effective therapeutic agents. In this study, we used three different sized antibiotic nanocarriers to study their mode of action and their size-dependent inhibitory effects against Escherichia coli (E. coli). Antibiotic nanocarriers (AgMUNH–Oflx NPs) with 8.6 × 102, 9.4 × 103 and 6.5 × 105 Oflx molecules per nanoparticle (NP) were prepared by functionalizing Ag NPs (2.4 ± 0.7, 13.0 ± 3.1 and 92.6 ± 4.4 nm) with a monolayer of 11-amino-1-undecanethiol (MUNH2) and covalently linking ofloxacin (Oflx) with the amine group of AgMUNH2 NPs, respectively. We designed a modified cell culture medium for nanocarriers to be stable (non-aggregated) over 18 h of cell culture, which enabled us to quantitatively study their size and dose dependent inhibitory effects against E. coli. We found that the inhibitory effects of Oflx against E. coli highly depended upon the dose of Oflx and the size of the nanocarriers, showing that an equal amount of Oflx that was delivered by the largest nanocarriers (92.6 ± 4.4 nm) were most potent with the lowest minimum inhibitory concentration (MIC50) and created the longest and highest percentage of filamentous cells, while the smallest nanocarriers (2.4 ± 0.7) were least potent with the highest MIC50 and produced the shortest and lowest percentage of filamentous cells. Interestingly, the same amount of Oflx on 2.4 ± 0.7 nm nanocarriers showed a 2× higher MIC and created 2× shorter filamentous cells than free Oflx, while the Oflx on 13.0 ± 3.1 and 92.6 ± 4.4 nm nanocarriers exhibited 2× and 6× lower MICs, and produced 2× and 3× longer filamentous cells than free Oflx, respectively. Notably, the three different sized AgMUNH2 NPs (absence of Oflx) showed negligible inhibitory effects and did not create filamentous cells. The results show that the filamentation of E. coli highly depends upon the sizes of nanocarriers, which leads to the size-dependent inhibitory effects of nanocarriers against E. coli

    Toxic Effects of Silver Ions on Early Developing Zebrafish Embryos Distinguished From Silver Nanoparticles

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    Currently, effects of nanomaterials and their ions, such as silver nanoparticles (Ag NPs) and silver ions (Ag+), on living organisms are not yet fully understood. One of the vital questions is whether nanomaterials have distinctive effects on living organisms from any other conventional chemicals (e.g., their ions), owing to their unique physicochemical properties. Due to various experimental protocols, studies of this crucial question have been inconclusive, which hinders rational design of effective regulatory guidelines for safely handling NPs. In this study, we chronically exposed early developing zebrafish embryos (cleavage-stage, 2 hours post-fertilization, hpf) to a dilution series of Ag+ (0–1.2 μM) in egg water (1 mM NaCl, solubility of Ag+ = 0.18 μM) until 120 hpf. We systematically investigated effects of Ag+ on developing embryos and compared them with our previous studies of effects of purified Ag NPs on developing embryos. We found the concentration- and time-dependent effects of Ag+ on embryonic development, and only half of the embryos developed normally after being exposed to 0.25 μM (27 μg/L) Ag+ until 120 hpf. As the Ag+ concentration increases, the number of embryos that developed normally decreases, while the number of embryos that became dead increases. The number of abnormally developing embryos increases as the Ag+ concentration increases from 0 to 0.3 μM and then decreases as the concentration increases from 0.3 to 1.2 μM because the number of embryos that became dead increases. The concentration-dependent phenotypes were observed, showing fin fold abnormality, tail and spinal cord flexure, and yolk sac edema at low Ag+ concentrations (≤0.2 μM) and head and eye abnormalities along with fin fold abnormality, tail and spinal cord flexure, and yolk sac edema at high concentrations (≥0.3 μM). Severities of phenotypes and the number of abnormally developing embryos were far less than those observed in Ag NPs. The results also show concentration-dependent effects on heart rates and hatching rates of developing embryos, attributing to the dose-dependent abnormally developing embryos. In summary, the results show that Ag+ and Ag NPs have distinctive toxic effects on early developing embryos, and toxic effects of Ag+ are far less severe than those of Ag NPs, which further demonstrates that the toxicity of Ag NPs toward embryonic development is attributed to the NPs themselves and their unique physicochemical properties but not the release of Ag+ from the Ag NPs

    Size-Dependent Inhibitory Effects of Antibiotic Drug Nanocarriers Against Pseudomonas Aeruginosa

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    Multidrug membrane transporters (efflux pumps) are responsible for multidrug resistance (MDR) and the low efficacy of therapeutic drugs. Noble metal nanoparticles (NPs) possess a high surface-area-to-volume ratio and size-dependent plasmonic optical properties, enabling them to serve both as imaging probes to study sized-dependent MDR and as potential drug carriers to circumvent MDR and enhance therapeutic efficacy. To this end, in this study, we synthesized three different sizes of silver nanoparticles (Ag NPs), 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm, functionalized their surface with a monolayer of 11-amino-1-undecanethiol (AUT), and covalently conjugated them with antibiotics (ofloxacin, Oflx) to prepare antibiotic drug nanocarriers with conjugation ratios of 8.6 × 102, 9.4 × 103, and 6.5 × 105 Oflx molecules per NP, respectively. We purified and characterized the nanocarriers and developed cell culture medium in which the cells grew normally and the nanocarriers were stable (non-aggregated), to quantitatively study the size, dose, and efflux pump (MexAB-OprM) dependent inhibitory effect of the nanocarriers against two strains of Pseudomonas aeruginosa, WT (normal expression of MexAB-OprM) and ΔABM (deletion of MexAB-OprM). We found that the inhibitory effect of these nanocarriers highly depended on the sizes of NPs, the doses of antibiotic, and the expression of MexAB-OprM. The same amount of Oflx on the largest nanocarriers (92.6 ± 4.4 nm) showed the highest inhibitory effect (the lowest minimal inhibitory concentration) against P. aeruginosa. Surprisingly, the smallest nanocarriers (2.4 ± 0.7 nm) exhibited a lower inhibitory effect than free Oflx. The results suggest that size-dependent multivalent effects, the distribution and localization of Oflx (pharmacodynamics), and the efflux of Oflx all play a role in the inhibitory effects. Control experiments using three sizes of AgMUNH2 NPs (absence of Oflx) showed that these NPs do not exhibit any significant inhibitory activity toward both strains. These new findings demonstrate the need for and possibility of designing optimal sized antibiotic nanocarriers to achieve the highest efficacy against P. aeruginosa

    Antibiotic Drug Nanocarriers for Probing of Multidrug ABC Membrane Transporter of \u3ci\u3eBacillus subtilis\u3c/i\u3e

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    Multidrug membrane transporters can extrude a wide range of substrates, which cause multidrug resistance and ineffective treatment of diseases. In this study, we used three different sized antibiotic drug nanocarriers to study their size-dependent inhibitory effects against Bacillus subtilis. We functionalized 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm silver nanoparticles (Ag NPs) with a monolayer of 11-amino-1-undecanethiol and covalently linked them with antibiotics (ofloxacin, Oflx). The labeling ratios of antibiotics with NPs are 8.6 × 102, 9.4 × 103, and 6.5 × 105 Oflx molecules per NP, respectively. We designed cell culture medium in which both BmrA and ΔBmrA cells grew and functioned normally while ensuring the stabilities of nanocarriers (nonaggregation). These approaches allow us to quantitatively study the dependence of their inhibitory effect against two isogenic strains of B. subtilis, WT (normal expression of BmrA) and ΔBmrA (deletion of bmrA), upon the NP size, antibiotic dose, and BmrA expression. Our results show that the inhibitory effects of nanocarriers highly depend on NP size and antibiotic dose. The same amount of Oflx on 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm nanocarriers shows the 3× lower, nearly the same, and 10× higher inhibitory effects than that of free Oflx, against both WT and ΔBmrA, respectively. Control experiments of the respective sized AgMUNH2 NPs (absence of Oflx) show insignificant inhibitory effects toward both strains. Taken together, the results show multiple factors, such as labeling ratios, multivalent effects, and pharmacodynamics (Oflx localization and distribution), which might play the roles in the size-dependent inhibitory effects on the growth of both WT and ΔBmrA strains. Interestingly, the inhibitory effects of nanocarriers are independent of the expression of BmrA, which could be attributed to the higher efflux of nanocarriers by other membrane transporters in both strains

    Synthesis and Characterization of Stable and Purified Silver Nanoparticles (Ag NPs) for Biomedical Applications

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    Silver nanoparticles (Ag NPs) have been used for a wide range of applications because of their distinctive optical properties and unique biological effects. These properties highly depend on their sizes, shapes and their surrounding environments. Furthermore, Ag NPs possess high quantum yield of Rayleigh scattering, which enables them to serve as highly sensitive imaging probes and sensors for live cell imaging, as well as assays for sensitive detection of biomarkers for early disease diagnosis. We have successfully synthesized, purified and characterized Ag NPs with average diameters of 2, 12 and 43 nanometer (nm). We have studied the stability of Ag NPs in aqueous solution and cell culture medium, which shows our Ag NPs are stable (non-aggregated) in aqueous solution and given cell culture medium over a long period of time. We have studied their cytotoxic and therapeutic effects on various biological organisms and explored a wide range of their biomedical applications. The detailed experimental designs and updated results will be presented.https://digitalcommons.odu.edu/sciences_achievement/1019/thumbnail.jp

    Size-Dependent Inhibitory Effects of Antibiotic Drug Nanocarriers against <i>Pseudomonas aeruginosa</i>

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    Multidrug membrane transporters (efflux pumps) are responsible for multidrug resistance (MDR) and the low efficacy of therapeutic drugs. Noble metal nanoparticles (NPs) possess a high surface-area-to-volume ratio and size-dependent plasmonic optical properties, enabling them to serve both as imaging probes to study sized-dependent MDR and as potential drug carriers to circumvent MDR and enhance therapeutic efficacy. To this end, in this study, we synthesized three different sizes of silver nanoparticles (Ag NPs), 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm, functionalized their surface with a monolayer of 11-amino-1-undecanethiol (AUT), and covalently conjugated them with antibiotics (ofloxacin, Oflx) to prepare antibiotic drug nanocarriers with conjugation ratios of 8.6 × 10<sup>2</sup>, 9.4 × 10<sup>3</sup>, and 6.5 × 10<sup>5</sup> Oflx molecules per NP, respectively. We purified and characterized the nanocarriers and developed cell culture medium in which the cells grew normally and the nanocarriers were stable (non-aggregated), to quantitatively study the size, dose, and efflux pump (MexAB-OprM) dependent inhibitory effect of the nanocarriers against two strains of Pseudomonas aeruginosa, WT (normal expression of MexAB-OprM) and ΔABM (deletion of MexAB-OprM). We found that the inhibitory effect of these nanocarriers highly depended on the sizes of NPs, the doses of antibiotic, and the expression of MexAB-OprM. The same amount of Oflx on the largest nanocarriers (92.6 ± 4.4 nm) showed the highest inhibitory effect (the lowest minimal inhibitory concentration) against P. aeruginosa. Surprisingly, the smallest nanocarriers (2.4 ± 0.7 nm) exhibited a lower inhibitory effect than free Oflx. The results suggest that size-dependent multivalent effects, the distribution and localization of Oflx (pharmacodynamics), and the efflux of Oflx all play a role in the inhibitory effects. Control experiments using three sizes of AgMUNH<sub>2</sub> NPs (absence of Oflx) showed that these NPs do not exhibit any significant inhibitory activity toward both strains. These new findings demonstrate the need for and possibility of designing optimal sized antibiotic nanocarriers to achieve the highest efficacy against P. aeruginosa
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