Size-Dependent Inhibitory Effects of Antibiotic Drug Nanocarriers against Pseudomonas aeruginosa

Abstract

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.

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

“…We synthesized, puried and characterized three different sized antibiotic nanocarriers with diameters of Ag NPs of (2.4 AE 0.7), (13.0 AE 3.1) and (92.6 AE 4.4) nm and the molar conjugation ratios of antibiotics (Ox) per NP of 8.6 Â 10 2 , 9.4 Â 10 3 , and 6.5 Â 10 5 , respectively, as we reported previously. 46 We rst synthesized, puried and characterized the three different sized Ag NPs, as we reported previously. 28,30,34,37,47,48 We characterized the NP concentrations, the plasmonic optical properties (LSPR images and spectra), sizes and zeta potentials of single NPs using UV-Vis absorption spectroscopy (Hitachi U-2010), dark-eld optical microscopy and spectroscopy (DFOMS), highresolution transmission electron microscopy (HRTEM) (JEOL, JEM-2100F), and dynamic light scattering (DLS) (Nicomp 380ZLS particle sizing system), respectively.…”

“…28,30,34,37,47,48 We used the interaction of thiol groups of MUNH 2 with the NPs to directly attach 11-amino-1-undecanethiol hydrochloride (MUNH 2 ) onto the surface of NPs and create AgMUNH 2 NPs. 46 We removed excess MUNH 2 by thoroughly washing the AgMUNH 2 NPs with DI water three times using centrifugation (Beckman Optima L90k, 4 C). We then covalently conjugated the carboxyl group of Ox with the amine groups of MUNH 2 attached onto the surface of the NPs (AgMUNH 2 NPs) via the peptide bond to prepare antibiotic nanocarriers (AgMUNH-Ox NPs) using a two-step method via EDC and s-NHS as mediators ( Fig.…”

“…We determined the molar concentration of NPs and Ox attached on the NPs for each different sized nanocarrier by measuring the plasmonic absorption spectra of the NPs and the absorbance spectra of Ox at 288 nm, respectively. 46 We then divided the molar concentrations of Ox molecules on the surface of the nanocarriers by the molar concentration of the NPs, to determine the molar conjugation ratios of Ox molecules/ NP for each different sized nanocarrier. We studied the stability of each different sized nanocarrier (AgMUNH-Ox NPs) in the standard (1% tryptone peptone, 0.5% yeast extract, and 0.5% NaCl, pH ¼ 7.2) and modied LB medium (1% tryptone peptone, 0.5% yeast extract, and 0.1% NaCl, pH ¼ 7.2) over 24 h using UV-Vis absorption spectra, DLS and DFOMS.…”

“…49 We imaged the cells in a micro-chamber using dark-eld optical microscopy and epi-uorescence microscopy and counted the green uorescent cells (peak wavelength of uorescence spectra of SYTO9, l max ¼ 520 nm) and the red uorescent cells (peak wavelength of uorescence spectra of propidium iodide, l max ¼ 610 nm) as live and dead cells, respectively. 29,44,46,49 We further characterized the efflux function of the ABC membrane transporter of the cells cultured in the standard and the modied LB medium by measuring the time-course of the uorescence intensity of the dye (Hoechst 33342) accumulated inside the cells in the presence and absence of the ATPase inhibitor (orthovanadate) using uorescence spectroscopy, as described in the following. 29,50,51 We harvested the cultured cells using centrifugation (Beckman Model J2-21 Centrifuge, JA-14 rotor, at 7500 rpm, 23 C, 10 min), thoroughly washed the cells with the PBS buffer (0.5 mM phosphate buffer, 1.5 mM NaCl, pH 7.2) three times, and resuspended the cells in the buffer.…”

“…We characterized sizes and shapes of single Ag NPs using TEM, and LSPR spectra of single Ag, AgMUNH 2 , and AgMUNH-Ox NPs using DFOMS, as we reported previously. 46 We imaged at least 100 NPs for each measurement and repeated each experiment three times for each individual size. Therefore, a minimum of 300 NPs were characterized using TEM and DFOMS.…”