Bacteria are responsible for numerous diseases in humans. Ever increasing bacterial infection and the emergence of antibiotic resistant bacterial strains is one of the major concerns. To address this challenge, new biocompatible inexpensive molecules having antibacterial properties need to be investigated in combination with nanoscience and natural polymers.
In recent years, chitosan-based nanoparticles have become important molecules in medical research due to their high specificity, sensitivity and reduced toxic effect on mammalian cells (Sharifi-Rad et al., 2021). Chitosan is a naturally occurring cationic, biocompatible polysaccharide approved by the US FDA for tissue engineering and drug delivery in the biomedical field (Gagliardi et al., 2021). Indeed, the amino group of biopolymers plays a vital role in enhancing biomedical properties such as cell-to-cell adhesion, low immunogenicity and bio-tolerability under different conditions (Bravo-Osuna et al., 2007; Ali and Ahmed, 2018). Studies have observed that chitosan-based NPs are also particularly suitable for local delivery at the dermis and mucosa because of their cell adhesion and antibacterial properties (Sharifi-Rad et al., 2021).
In the current study, we synthesized chitosan-coupled silver nanoparticles using hydrazine at a minimum quantity as a reducing agent. During the synthesis process, the addition of silver to the chitosan solution led to the formation of a silver core shell surrounded by hydroxyl and amine groups of the chitosan biopolymer by electrostatic attraction, which was responsible for the capping and stability of NPs (Sharifi-Rad et al., 2021). Reducing agent hydrazine hydrate (N2H4·H2O) at lower concentrations (0.1–0.4%) aids in Ch-AgNP formation by eliminating nitrogen gas and also helps to decrease the NPs size (Shah et al., 2015). Moreover, reaction conditions also play a crucial factor in determining the size, shape and yield of nanoparticles (Usman et al., 2012). The peak observed at 455 nm in a spectrophotometer is related to surface plasmon resonance (SPR) due to the size and shape of the NPs, along with precursors and the dielectric environment of the solution (Singha et al., 2017). Furthermore, the observed SPR is due to the interaction between free photons and electrons in the conduction band (Hammond et al., 2014). A previous finding suggests that the peak observed at this range might be due to the interaction of amino and hydroxyl groups of the chitosan polymer with silver ions (Sanpui et al., 2008). However, a study carried out by Dara et al. (2020) showed that the chitosan-silver NPs showed an SPR band at 419 nm, which could be due to the change in conditions of the reaction carried out and the reducing agent used (Yadav et al., 2017). Furthermore, NPs synthesized in the current study have a positive surface charge that is directly related to the higher electrostatic repulsion between the NPs. The functional group involved in the formation of Ag-Ch NPs was confirmed by FT-IR and the obtained results are in agreement with the observed results of previous studies (An et al., 2011). XRD patterns revealed a broadening of Bragg’s peaks, indicating the successful formation of AgNPs. TEM images confirmed the particle size, which correlates to SEM results. In this study, the cytotoxicity assay also revealed that chitosan-based silver nanoparticles were less toxic at a 5.53 µg/mL concentration to HEK-293 cells (IC50). Despite silver’s toxicity to the cells at higher concentrations, AgNPs showed promising results in wound healing activity, which could be due the nature of the nanoparticles that helps to keep beneficial enzymes intact in the host during the wound healing process and are also less prone to bacterial resistance (An et al., 2011).
Silver is one of the most extensively used metals for the synthesis of nanoparticles due to its wide spectrum of antibacterial activity. We reported in our previous study that silver in nano form is known to enter the microbial cell membranes easily and damage the sub-cellular structures (Vanti et al., 2020). In the present study, synthesized NPs showed promising antibacterial activity against S. aures and E. coli that are in corroboration with earlier reports. A previous study showed 100% inhibition of S. aureus and E. coli at > 0.3 mg/cm2 when tested with silver nanoparticle-loaded collagen-chitosan dressing material, whereas collagen-chitosan alone dressing material showed 100% inhibition of the same pathogens (Li et al., 2020). In the current study, Ch-AgNPs showed better antibacterial activity when compared to chitosan alone). Similarly, Ch-AgNPs showed better antibacterial activity against E. coli in the preservation of minced meat when compared to that of chitosan alone (Badawy et al., 2019). Potara and co-workers reported that antibacterial activity against S. aureus was a synergistic effect due to the combination of chitosan and AgNPs (Potara et al., 2011; Shinde et al., 2021).
Chitosan-based nanomaterials were studied extensively in recent years in a wound healing assay due to their antibacterial and biocompatible nature. Several researchers adopted a combination of chitosan and metal nanoparticles in the synthesis of chitosan-based nanoparticles in order to enhance antimicrobial activity and biological safety for clinical application. Many articles reported on the preparation of chitosan-based metal nanoparticles in different forms such as hydrogels, fibers, sponges, films, membranes and scaffolds for studying wound healing assays (Hajji et al., 2019; Kharaghani, et al., 2018; Ahmed, et al., 2018; Ran et al., 2019; Wichai et al., 2019; Shah et al., 2019; Cheng and Yu, 2019). However, limited knowledge is currently available on the powdered form of chitosan-based silver nanoparticles and their application in wound treatment (Shehabeldine et al., 2022).
In the present study, a wound closure assay on L292 fibroblast cells revealed the ability of Ch-AgNPs to heal topical wounds at a 0.5 µg/mL concentration (10 times less than that of IC50). A study carried out by An et al. (2011) showed that a combination of silver and a biopolymer could increase the antimicrobial and wound healing ability. In our study, enhanced wound healing ability could be achieved due to the chitosan biopolymer, which accelerates the wound healing process by triggering inflammatory cells, macrophages and fibroblast cells that help in proliferating cells during the wound healing process (Liu et al., 2018; Matica et al., 2019; Shivakumar et al., 2021).
Despite the use of advanced antiseptics, wound infections remain the major concern to clinicians due to the emergence of drug-resistant pathogens. Therefore, a reliable and nontoxic antimicrobial agent that also helps in the wound healing process of host cells is necessary. To this end, our study suggests that chitosan-based silver nanoparticles could be used as an antibacterial agent that not only inhibits the growth of infectious pathogens but also promotes the proliferation and migration of host cells to the wound area. The overall schematic representation of chitosan-based silver nanoparticles synthesis and its application is presented in Supplementary Fig. 1. However, comprehensive studies are required to better understand the molecular mechanism of NPs involved in the antibacterial and wound healing ability.