In this paper, the salt-assisted chitosan nanoparticles (CS NPs) containing metronidazole (MTZ) were prepared using the ionic gelation technique in the presence of NaCl. The effect of different concentrations of NaCl on particle size, zeta potential, polydispersity index (PDI), and entrapment efficiency (EE %) was investigated. Also, the stability of MTZ-loaded CS NPs in the absence/presence of NaCl was evaluated over a 6-month storage period. Furthermore, drug release at pH =7.4 was examined and the corresponding mechanism was explored. Finally, the time-kill assay of free MTZ and salt-assisted MTZ-loaded CS NPs against Bacteroides fragilis was performed by applying the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). It was found that in the presence of 20 mM NaCl, the optimum NPs can be achieved with the particle size of 284 nm, PDI of 0.099, EE% of 57.4 %, and zeta potential of +46.32 mV. More stability of salt-assisted nanoparticles, as well as lower size enhancement versus time, were observed due to higher surface charge density calculated using the Gouy-Chapman theory. The in-vitro drug release profiles demonstrated a more controlled drug release of MTZ from CS NPs compared to free MTZ, because of the shrinkage properties of CS at high pH. The kinetic modeling of drug release approved the Fickian diffusion of drug based on the Korsmeyer- Peppas model. The time-kill plots confirmed the higher antibacterial activity of salt-assisted MZ-loaded CS NPs compared to the free MTZ against B. fragilis bacterium. In conclusion, the salt-assisted MTZ-loaded CS NPs prepared in the presence of a proper concentration of NaCl, can be an acceptable nanoparticle form for designing MTZ drug delivery systems.

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