6 research outputs found
Design of Drug Nano-Carriers for Study of Multidrug Resistance in Single Live Cells
Multidrug resistance (MDR) exists in both prokaryotic and eukaryotic cells. MDR is responsible for ineffective treatment of a wide range of diseases, such as infections and cancer. The ATP-binding cassette (ABC) membrane transporters (efflux pumps) are one of the largest and most diverse super-families of membrane proteins found in all living organisms, ranging from bacteria to humans. All ABC transporters share a common structure of four core domains; two transmembrane domains (TMD) with variable sequence and topology and two nucleotide-binding domains (NBD) with conserved sequences. Conventional methods for the study of the efflux functions are radioactively labeled substrates and fluorescent dyes, which provide the sum of accumulation kinetics of a population of cells. Notably, individual cells act independently of efflux and accumulation kinetics and therefore, the accumulation kinetics of single cells is lost in such bulk measurements. Furthermore, single fluorophores or radioisotopes themselves do not possess distinctive size-dependent physicochemical properties that would allow their sizes to be measured in situ in real time. Therefore, they cannot serve as various sized pump substrates for the study of size-dependent efflux function of single membrane transporters in single live cells. This dissertation focuses on development of single nanoparticle (NP) plasmonic spectroscopy and design of three different sized drug nano-carriers to study the size-dependent efflux function of ABC membrane transporters (e.g., MsbA and BmrA) in single live cells, two model organisms such as E.coli (gram-negative) and B.subtilis (gram-positive) bacterial cells., aiming to understand and overcome MDR. We designed, synthesized, and purified silver (Ag) nanoparticles (NPs) with diameters of 2.4 ± 0.7 nm, 13.0 ± 3.1, and 92.6 ± 4.4 nm, functionalized them with monolayer of 11-amino-1-undecanethiol hydrochloride (MUNH2), and linked them with antibiotics ofloxacin (Oflx) via two-step reaction process. We determined the conjugation ratio (the number of Oflx molecules attached to a single NP) of 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm as 8.6x102, 9.4x103, and 6.5x105 respectively. We characterized single nano-carriers using dark-field optical microscopy and spectroscopy (DFOMS). These drug nano-carriers exhibit photostability, which enabled us to study the size-dependent efflux functions of single membrane transporters in single live cells in real-time for a desired period of time. Using these three different sized nano-carriers, we found the size dependent inhibitory effects of the drug nano-carriers in single live cells, two strains of B.subtilis, WT (normal expression of BmrA) and ∆BmrA (deletion of BmrA). We have demonstrated that single plasmonic NPs can serve as unique size-dependent molecular imaging probes to study efflux functions of single membrane transporters in single live cells in real-time. Furthermore, we found that the smaller drug nano-carriers are much more biocompatible than the larger drug nano-carriers. In other words, the larger drug nano-carriers show higher inhibitory effects against the bacterial cells. Thus, the smallest nano-carriers (2.4 ± 0.7 nm) could be used to study the efflux function of ABC (BmrA) membrane transporters in single live cells, while the large nano-carriers could be used to effectively deliver antibiotics that could overcome MDR and enhance antibiotics efficacy
Size-Dependent Inhibitory Effects of Antibiotic Nanocarriers on Filamentation of \u3ci\u3eE. coli\u3c/i\u3e
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
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
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
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
Size-Dependent Inhibitory Effects of Antibiotic Drug Nanocarriers against <i>Pseudomonas aeruginosa</i>
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