# Zeolite-Y-Loaded Chitosan Nanoparticles as Endodontic Antimicrobial Agent: An In vitro Study

## TL;DR

This study shows that a new chitosan-zeolite nanocomposite effectively kills endodontic bacteria and is safe for cells, even in the presence of tissue inhibitors.

## Contribution

A novel chitosan-zeolite nanocomposite is developed and tested as a biocompatible antimicrobial agent for endodontic applications.

## Key findings

- The nanocomposite showed inhibition zones of 9.57 mm and 7.85 mm against S. mutans and E. faecalis, respectively.
- The nanocomposite completely eradicated both bacteria in the presence of tissue inhibitors.
- The nanocomposite significantly promoted 3T3 fibroblast cell growth, indicating low cytotoxicity.

## Abstract

The objectives were to synthesize a bioactive nanocomposite as an endodontic antimicrobial agent by loading previously synthesized electrosprayed chitosan nanoparticles (Ch-Np) into Zeolite-Y as a carrier and compare its antimicrobial activity against two endodontic pathogens using the agar diffusion test. Additionally, the effect of tissue inhibitors (dentin powder and serum albumin) on the antimicrobial activity of the Ch-Np-Zeolite nanocomposite was studied. Finally, the possible cytotoxicity of the novel nanocomposite against Balb/c 3T3 mouse fibroblast cells was evaluated.

A concentration of 3% (w/v) electrosprayed Ch-Np was mixed with Zeolite-Y in a concentration of 53.3 (w/v) and characterized using high-resolution scanning electron microscopy (HR-SEM) and energy-dispersive X-ray spectroscopy (EDS) analysis. The antimicrobial activity was evaluated against
Streptococcus mutans
and
Enterococcus faecalis
using the agar diffusion test, and the time-kill test was performed using the broth microdilution technique in the presence of tissue inhibitors. The cytotoxicity was evaluated against 3T3 mouse fibroblast cells using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

The difference between the antimicrobial activity of Ch-Np-Zeolite nanocomposite against
S. mutans
and
E. faecalis
was analyzed using the Mann–Whitney's
U
test. The effect of tissue inhibitors on the antimicrobial activity of Ch-Np-Zeolite nanocomposite was analyzed by comparing the mean of log colony-forming unit per milliliter over time. For the cytotoxicity assay, a statistically significant difference between each group and their control was made using a
t
-test with a probability value of
p
≤ 0.05, considered a significant difference.

HR-SEM of the dried paste-like mixture Ch-Np-Zeolite revealed the typical crystal habit of the supporting zeolite, and EDS analysis confirmed that the zeolite parent material retained its elemental composition after loading with Ch-Np. The antimicrobial activity of Ch-Np-Zeolite was demonstrated by the mean diameter inhibition zones of 9.57 and 7.85 mm for
S. mutans
and
E. faecalis
, respectively.
Streptococcus mutans
and
E. faecalis
were completely eradicated in the presence of tissue inhibitors. The Ch-Np-Zeolite nanocomposite significantly promoted the growth of 3T3 fibroblast cells (
p
 < 0.05), supporting its lack of cytotoxicity.

Zeolite-Y-loaded Ch-Np nanocomposite shows promising antimicrobial activity while maintaining its biocompatibility even in the presence of tissue inhibitors.