In situ synthesis of bio-green silver nanoparticles incorporated zeolite a using orthosiphon aristatus leaves extract for antibacterial wound healing

Abstract

Silver in its various forms is well known to have a potent antibacterial property. Despite the high antibacterial activity and efficacy of silver nanoparticles (AgNP), its frequent use could lead to bacterial resistance. Without a proper release mechanism, the efficacy of AgNP is often questioned. Additionally, chemical and physical methods to synthesize AgNP pose threats to the environment and health. Thus, alternative approach using biological resources are desired. However, AgNP produced through this method still needs preclinical evaluation on toxicity and biocompatibility. Thus, a novel in situ biosynthesis of AgNP-incorporated synthesized zeolite A (AgNP-SZ) was developed. The AgNP-SZ was then assessed for their antibacterial activity, in vitro cytotoxicity and wound healing potency. Zeolite A (SZ) was synthesized from kaolinite through hydrothermal method whereas AgNP was produced from AgNO3 using Orthosiphon aristatus leaves extract as the green reducing and capping agent. The AgNP-SZ was synthesized using 0.4 mL 5 % O. aristatus leaf extract solution and mixed physically with Ag-SZ. The synthesized materials SZ, Ag-SZ and AgNP-SZ were characterized for their morphological and physicochemical properties. In the present study, the characterization results validated that the synthesized product was zeolite A. Characterization by Transmission Electron Microscope (TEM) showed AgNP with particle size of 20.01 nm in diameter and area of 381.61 nm2 was incorporated in the zeolite A. TEM analysis, surface and pore analysis (BET/BJH), thermogravimetric and differential temperature analysis (TGA-DTA), and inductively coupled plasma-optical emission spectrometry (ICP OES) were used to assess the synthesized products. These characterizations validated the O. aristatus leaves extract acted as natural reducing and capping agents with a timely release mechanism of AgNP from zeolite A. SZ, Ag-SZ and AgNP-SZ were assessed for antibacterial activity against E. coli and S. aureus using disc diffusion technique (DDT) and minimum inhibitory/bactericidal concentration (MIC/MBC), biofilm inhibition against P. aeruginosa, in vitro cytotoxicity against human skin fibroblast (HSF 1184) cells and wound healing potency through in vitro scratch assay. The powder form of the samples was pressed into pellets for DDT, whereas MIC/MBC and biofilm study utilized the powder form in both water and saline solution. Inhibition zones and bacterial growth inhibition were observed. The DDT showed clear zone of inhibitions for Ag-loaded materials on both bacteria, with E. coli was more susceptible than S. aureus in both water and saline solutions based on the MIC/MBC values. The AgNP-SZ also showed potential biofilm inhibition action against S. aureus compared to P. aeruginosa. SZ, Ag-SZ and AgNP-SZ at 0.5, 1.0, 1.5, and 2.0 mg/mL were tested for cytotoxicity. In vitro scratch assay determined the HSF 1184 cell migration rate after treatment with the synthesized products. The absence of cytotoxicity in all concentrations of AgNP-SZ proved that the material is biocompatible. Although cell migration rate by AgNP-SZ was slower compared to the SZ and control in in vitro scratch assay, the material did not hinder cell migration and proliferation. These findings show the potential of green synthesized AgNP-incorporated zeolite A using plant extract to substitute conventional methods, with good antibacterial application and sustainable production.