Phyto-assisted synthesis of zinc oxide nanoparticles for developing antibiofilm surface coatings on central venous catheters

Abstract

Medical devices such as Central Venous Catheters (CVCs), are routinely used in intensive and critical care settings. In the present scenario, incidences of Catheter-Related Blood Stream Infections (CRBSIs) pose a serious challenge. Despite considerable advancements in the antimicrobial therapy and material design of CVCs, clinicians continue to struggle with infection-related complications. These complications are often due colonization of bacteria on the surface of the medical devices, termed as biofilms, leading to infections. Biofilm formation is recognized as a critical virulence trait rendering infections chronic and difficult to treat even with 1,000x, the minimum inhibitory concentration (MIC) of antibiotics. Therefore, non-antibiotic-based solutions that prevent bacterial adhesion on medical devices are warranted. In our study, we report a novel and simple method to synthesize zinc oxide (ZnO) nanoparticles using ethanolic plant extracts of Eupatorium odoratum. We investigated its physio-chemical characteristics using Field Emission- Scanning Electron Microscopy and Energy dispersive X-Ray analysis, X-Ray Diffraction (XRD), Photoluminescence Spectroscopy, UV-Visible and Diffuse Reflectance spectroscopy, and Dynamic Light Scattering characterization methods. Hexagonal phase with wurtzite structure was confirmed using XRD with particle size of ∼50 nm. ZnO nanoparticles showed a band gap 3.25 eV. Photoluminescence spectra showed prominent peak corresponding to defects formed in the synthesized ZnO nanoparticles. Clinically relevant bacterial strains, viz., Proteus aeruginosa PAO1, Escherichia coli MTCC 119 and Staphylococcus aureus MTCC 7443 were treated with different concentrations of ZnO NPs. A concentration dependent increase in killing efficacy was observed with 99.99% killing at 500 μg/mL. Further, we coated the commercial CVCs using green synthesized ZnO NPs and evaluated it is in vitro antibiofilm efficacy using previously optimized in situ continuous flow model. The hydrophilic functionalized interface of CVC prevents biofilm formation by P. aeruginosa, E. coli and S. aureus. Based on our findings, we propose ZnO nanoparticles as a promising non-antibiotic-based preventive solutions to reduce the risk of central venous catheter-associated infections.

Keywords: anti-biofilm coatings; anti-microbial resistance; device associated infections; green synthesis; medical devices; nanoparticle coatings; plant mediated synthesis; zinc oxide nanoparticles (ZnO NPs).

FIGURE 1

XRD pattern for the synthesized ZnO NPs.

FIGURE 2

Williamson-Hall plot for the ZnO NPs, particle size was determined from the intercept.

FIGURE 3

SEM and EDAX Analysis. Field Emission-Scanning Electron Microscopy Images of Zinc Oxide Nanoparticles synthesized using green synthesis showing spherical, hexagonal structures of Zinc Oxide. EDAX spectra reveals presence of Zn and O confirming pure synthesis of Zinc Oxide nanoparticles.

FIGURE 4

Kubelka- Munk function (KM) versus photon energy for ZnO NPs. Inset- Absorption spectra for synthesized ZnO NPs.

FIGURE 5

(A) Photoluminescence spectra measured at room temperature for green synthesized ZnO NPs. Dotted curve shows the fitted Gaussian peaks to obtain defect states in the ZnO NPs. (B) Energy level diagram of synthesized ZnO NPs showing defect states (Vempati et al., 2012) and the possible transition corresponding to observed defect states in PL spectra.

FIGURE 6

Dynamic Light Scattering Characterization of ZnO synthesized using Green Synthesis. (A) Particle Size Distribution, D₁₀ = 32.19 nm, Median Diameter, D₅₀ = 48.36 nm, D₉₀ = 69.95 nm. (B) Correlogram. (C) Intensity Fluctuation Plot. (D) Zeta Potential distribution upon nanoparticles.

FIGURE 7

SEM and EDAX Analysis of surface morphology of coated and uncoated catheters. (A) Coated Surface of CVC. (B) Uncoated Surface of CVC.

FIGURE 8

Antibacterial Efficacy of Eupatorium odoratum mediated ZnO NPs. Inhibition of: (A) Staphylococcus aureus, (B) Escherichia coli, and (C) Proteus aeruginosa growth in the presence of different concentrations of ZnO NPs; Percentage killing of: (D) Staphylococcus aureus, (E) Escherichia coli, and (F) Proteus aeruginosa, after 24 h of exposure to different concentrations of ZnO NPs.

FIGURE 9

Antibiofilm Efficacy of ZnO NP coated CVCs. (A) Biofilm formation of Staphylococcus aureus is inhibited by >97%. (B) Biofilm formation of Escherichia coli is inhibited nearly by 90%. (C) Biofilm formation of Proteus aeruginosa is inhibited by >90%. ZnO-NPs Coated: ZnO NPs-HPMC composite coating. Statistical analysis was done by 1-way analysis of variance (unpaired t-test with Welch’s correction) using GraphPad Prism software (version 8.0.1). Differences were considered significant at p < 0.05*, p < .0005 ***; p < 0.0001 ****.