Antimicrobial activity of chitosan nanoparticles

ABSTRACT Chitosan is a deacetylated chitin which is found naturally, particularly in fungal cell walls and crustacean shells. Chitosan is biocompatible and fully biodegradable and is extensively analyzed for antimicrobial property. Chitosan has also been explored as a drug carrier due to its biocompatible properties. Some studies have demonstrated that use of chitosan to coat nanoparticles made of other materials would help in reducing their impact on the body and also increase their bioavailability. The molecular weight of chitosan and the degree of deacetylation can be modified to derive different physicomechanical properties. Chitosan exhibits potent antifungal activity against several fungal strains including Rhizopus oryzae, Aspergillus niger and Alternaria alternata. Various factors such as molecular weight, dose and functional groups attached to the chitosan have been shown to modulate the antifungal activity of chitosan. Chitosan is known to exert antifungal activity without the need for any chemical modification, however, new derivatives of chitosan can be created to target specific microbial pathogens. The development of novel and ecofriendly methods of chitosan nanoparticles (CSNPs) preparation is in progress for developing chitosan as an efficient antimicrobial agent and in drug delivery system. This review is to focus on recent application of CSNPs as antibacterial and antifungal agent and to highlight the effectiveness of employing chitosan with silver and other metal nanoparticles.


Introduction
Chitosan, α (1-4) 2-amino-2-deoxyβ-D glucan, obtained by chitin deacetylation, is found in the exoskeleton of crustacean and several other organisms including insects and fungi [1]. Chitosan has favorable properties which have attracted the interest of researchers over the past two decades. For instance, it is non-toxic, biocompatible, edible and also possess antimicrobial properties [2][3][4][5]. For years, chitosan has been profoundly inspected as a carrier for drug delivery and biomedical applications [6][7][8]. Much of the research up to now has been focused on studying the antimicrobial activity of chitosan in form of solutions, gels, fibers and films [9][10][11][12][13][14][15]. The interest in employing chitosan nanoparticles (CSNPs) arises mainly due to its well-founded polymeric and cationic properties [16]. Various techniques for chitosan nanoparticles synthesis have been investigated by several research groups such as preparation of CSNPs by: ionic gelation method [17], emulsion crosslinking [18], spray drying [19], emulsion-droplet coalescence [20], reverse micellar method [21], nanoprecipitation [22], desolvation [23], modified ionic gelation with radical polymerisation [24] and emulsion solvent diffusion method [25]. CSNPs is considered as a highly promising candidate for utilisation as biomaterial in food related applications due to its accessibility, lack of toxicity and antimicrobial properties. Due to their small size and large surface to weight ratio, employing CSNPs in biomedical field has been examined extensively. Chitosan is one of the bio-polymers that is utilised as a reducing agent and a protecting polymer in the formation of metallic nanoparticles.

Chitosan nanoparticles as antibacterial agents
In the last 25 years, bacterial resistance has emerged as a major threat to humanity which could lead to a situation in the near future, where even trivial infections could become life threatening. Due to the emergence of multidrug-resistant microbes and lack of new antimicrobial drugs in market, there is an urgent need to discover and develop novel and more potent antimicrobial compounds. Chitosan-based nanoparticles have shown a tremendous potential as an antibacterial agent. In a recent study, CSNPs were prepared using varying concentration of chitosan and tripolyphosphate (TPP) by using ionic gelation method. The CSNPs prepared by employing 0.25% chitosan and 0.1% TPP exhibited an efficient antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa [26]. TPP, a non-toxic polyanion, is very safe and have been used for food industry applications. The protonated-Nh 2 groups in chitosan interact with the negatively charged counterion of TPP via an electrostatic interaction, and thus, ionic cross-linked networks are formed [27,28]. A similar study investigated the antibacterial potency of CSNPs against Staphylococcus aureus and Escherichia coli. CSNPs in the presence of TPP exhibited potent inhibition of both E. coli and S. aureus, compared to the CSNP without TPP which showed less activity [29]. Chitosan derivatives exhibit a pronounced antibacterial potency toward various bacterial strains. In a recent study, CSNPs obtained from a chitosan derivative, i.e. betaine, was tested for antibacterial activity. Chitosan derivatives with medium molecular weight and high degree of substitution showed greater antibacterial activity than commercial antibiotics. It was also revealed that increasing the degree of substitution led to an increase in antibacterial activity of chitosan with different molecular weight [30]. CSNPs obtained from the most effective chitosan heterocyclic derivative with moderate molecular mass, enhanced the antibacterial activity approximately 3-folds than the pure chitosan [31]. CSNPs at 10% and 20% inhibited the growth of Streptococcus mutans, Pseudomonas aeruginosa and Enterococcus faecalis completely [32].
CSNPs were loaded with antibiotics including tetracycline, gentamycin or ciprofloxacin to enhance antibacterial properties after its application on cotton fabrics. Fabric samples of cotton and cotton/polyester fabrics were treated with different concentrations of antibiotic-CSNPs. Cotton fabrics treated with CSNPs and with antibiotic-CSNPs inhibited the growth of S. aureus and E. coli with zone of inhibition of 10-26 mm and from 8.5 to 24 mm, respectively. This study also revealed that gentamycin-CSNPs exhibited higher antibacterial activity as compared to ciprofloxacin-CSNPs and tetracycline-CSNPs at the same concentration and the zone of inhibition (ZoI) increased with the increase of antibiotic concentration. It was also observed that, the fiber structure of cotton fabrics makes them more effective than polyester fabrics due to the number of functional groups of cotton reacting with a sufficient amount of antibiotic-CSNPs and thus enhancing antibacterial activity [33].
CSNPs additionally show synergistic activity to improve antimicrobial activity. however, using high concentration of CSNPs suspensions in industrial application might not be cost-efficient and might make visual or textural changes to commodities being targeted. To overcome this issue, the blend of an efficient antimicrobial agents with CSNPs would have the option to decrease the effective concentration of CSNPs.
Recent study reported an innovative strategy for CSNPs production using chemical cross-linking with cinnamaldehyde and replacing the traditional method of using TPP as ionic cross linker. Synergistic antibacterial activity was observed and the inhibitory effect of chitosan was significantly increased from 65% to 98% against S. aureus and from 62% to 96% against E. coli [34]. Another study examined the synergistic action of CSNPs with the antimicrobial alkaloid berberine against B. subtilis and S. aureus. The evaluation of synergistic effect of CSNPs and berberine was performed by using the broth dilution and the agar diffusion methods. Both CSNPs and berberine showed remarkable inhibitory effect against bacterial pathogens which was improved by their synergistic action [35].
Silver nanoparticles (AgNPs) have garnered the great interest from research community due to their superior properties, as well as conductivity, chemical stability, catalytic properties and antimicrobial activity. The modified CS in AgNPs-chitosan/montmorillonite nanocomposite films may control the morphological characteristics of AgNPs besides the significant inhibition of E. coli and B. subtilis [36].
Chitosan/tea polyphenols-silver nanocomposite film (CS/TP-AgNPs) was tested for antibacterial potency. Tripolyphosphate was added to the chitosan film as a crosslinker and a reducing agent of the silver nanoparticles agent. CS/TP-AgNPs film inhibited S. aureus and E. coli more efficiently compared to the effect of chitosan films alone [37]. The bactericidal action of silver nanoparticle-loaded microspheres (ChM-Ag) was very efficient in inhibiting E. coli and S. aureus [38]. The antibacterial activities of chitosan nanoparticles and copper-loaded nanoparticles against E coli, Salmonella choleraesuis, Salmonella typhimurium and S. aureus have been reported [39]. An eco-friendly CS-AgNPs hybrid was developed from AgNPs biologically prepared using T. portulacifolium leaf extract as a reducing agent and the inhibitory effects of these hybrid NPs were tested against two bacterial strains: E. coli and Serratia marcescens. These hybrid CS-AgNPs inhibited the growth of E. coli and S. marcescens. The antibacterial activity of CS-AgNPs increased with the increase in the concentration of CS-AgNPs [40].
Fungal chitosan (FCS) from Cunninghamella elegans, encapsulated with green silver nanoparticles from Gynura procumbens (GP-AgNPs) were developed to improve the antibacterial activity. The CS-AgNPs inhibited Bacillus cereus, S. aureus, L. monocytogenes, E. coli and Salmonella enterica [41]. Green AgNPs from Prosopisjuli flora (PJ) leaf extract encapsulated with chitosan showed a significant inhibition zone against E. coli compared to streptomycin [42]. Similar study investigated green AgNPs from Sygyzium aromaticum bud extract encapsulated with chitosan for antibacterial activity. CS-AgNPs inhibited vancomycin resistance Staphylococcus aureus with a zone of inhibition of 23.2 ± 0.51 mm and methicillin resistant Staphylococcus aureus with a zone of inhibition of 25.8 ± 0.32 mm.The results revealed their more prominent antibacterial potential compared to other antibacterial agents [43]. CS-AgNPs were synthesised via green method using chitosan, glucose and ethylene glycol. The antibacterial tests implied that CS-AgNPs prepared using glucose (G-AgNPs-CS) inhibited the growth of E. coli and S. aureus. 100 μg/ml of G-AgNPs-CS inhibited the growth of E. coli completely, while a much higher concentration i.e. 200 μg/ml of chitosan C-AgNPs-CS and ethylene glycol e-AgNPs-CS were required for complete inhibition of E. coli growth. S. aureus was inactivated by 300 μg/ml of all nanocomposites. The results revealed that the inhibitory effects against E. coli and S. aureus were enhanced with increasing concentration of AgNPs [44]. The antibacterial efficiency of green chitosan-PVA-silver nanoparticles (chitosan and polyvinyl alcohol polymers, used as stabilising agent) was evaluated against B. cereus, E. coli, E. faecalis, Micrococcus luteus, P. aeruginosa, S. enterica, Salmonella typhi and S. aureus. This study revealed that the increase of AgNo 3 used the in AgNPs synthesis enhances the antibacterial activity of CS-AgNPs [45].
Recently, the anti-biofilm activity of silver nanoparticles capped with chitosan (CS-AgNPs) was tested against pathogenic bacteria including S. aureus and P. aeruginosa. CS-AgNPs inhibited the growth of S. aureus (85%) and P. aeruginosa (95%) at 100 μg/ml which was confirmed using confocal-laser scanning microscopy. The anti-biofilm potency of CS-AgNPs enhanced with the increase in concentration from 25 μg/ml to 100 μg/ ml [46]. layer by layer (l-B-l) nanocoating process was used for deposition of polystyrene sulfonate (PSS) and CS-Ag and the formation of body layers of PSS/CS-Ag on fabric. The antibacterial activity of coated fabrics was evaluated against S. aureus, B. subtilis and E. coli. The coated fabrics with 0.1% of PSS/CS and 0.1% (PSS/ CS-Ag) showed 100% bactericidal activity against S. aureus and E. coli, whereas PSS/CS coated fabric led to inhibition of 72% and 68% against S. aureus and E. coli, respectively. Additionally, PSS/CS-Ag showed zones of inhibition of 4.8 mm, 3.2 mm and 2.5 mm against B. subtilis S. aureus and E. coli, respectively [47].
Gold nanoparticles (AuNPs) capped with chitosan (CS-AuNPs), glycol-chitosan (GC-AuNPs) and poly-γglutamic acid (PA-AuNPs) were applied on fabrics and evaluated for antibacterial activity. PA-AuNPs showed a higher antibacterial activity against S. enterica and E. coli-o157:h7 compared to gentamycin. While CS-AuNPs and GC-AuNPs exhibited maximum antibacterial activity against Listeria monocytogenes, followed by S. enterica, E. coli-o157:h7, MRSA and S. aureus. Transmission electron microscope images revealed that glycol-chitosan altered MRSA cell permeability by attaching on the surface of the cell and blocked nutrient flow and disrupt cell membrane, while PA-AuNPs entered S. enterica to cause pore formation, plasmolysis and dissolution [48].

Chitosan nanoparticles as an antifungal agent
CSNPs prepared using low molecular weight (lMW) and high molecular weight (hMW) of chitosan have been evaluated for their antifungal activity against Candida albicans, Fusarium solani and Aspergillus niger. The nanoparticles prepared with different concentrations of chitosan showed an inhibitory effect against the three fungal species. While A. niger exhibited a strong resistance to CNPs which was fabricated with low concentrations of chitosan [49]. The CSNPs were also evaluated as a controlling agent for various plant diseases caused by Rhizoctonia solani, Fusarium oxysporum, Collectotrichum acutatum and Phytophthora infestans. Moreover, CNP shave been shown as an ideal coating agent for the coated vegetables by improving the shelf life of tomato, chilly and brinjal. CSNPs exhibited significant antifungal activity against all fungal species. Vegetables samples treated with different concentrations that ranged from 1% to 5% of CSNPs prevented the weight loss compared to uncoated vegetables samples [50].
Application of CSNPs prepared with TPP as a crosslinker had significantly reduced the loss of wood mass, when the samples were exposed to the white rot fungus (Trametes versicolor) and brown rot fungus (Gloeophyllum trabeum). Their study clearly suggested that CSNP-TPP could provide the treated wood a resistance to fungal decay [51].
The addition of guar gum to chitosan nanoparticle (CGNP) preparation was used to enhance the effect of CSNPs as a protective agent against microorganisms. This study examined the inhibitory effect of CGNP against the rice blast fungus Pyricularia grisea using the disc diffusion method. CGNPs exhibited antifungal effect (71% inhibition) at 0.01 mg/ml concentration by inhibiting the mycelial growth. In-vitro application of 0.01% CGNP to healthy rice leaves followed by inoculation with P. grisea spores after 48 h, showed no symptom of rice blast compared to rice leaves, sprayed with distilled water as controls, which exhibited the blast symptoms. Moreover, the CGNPs enhanced seed growth and seed germination [52]. Tissue conditioner containing CSNPs at 5% and 2.5% concentration was found to completely inhibit the growth of a C. albicans strain [32]. In a recent study, CSNP-hydrogels with crosslinker TPP were prepared to improve the antimicrobial properties. A modified chitosan hydrogel showed higher antifungal activity with an inhibition zone of 25 mm for Candida albicans and 26 mm for A. niger, while chitosan and CSNPs showed no inhibition against the selected species [53].
Antifungal activity of different chitosan derivatives were analysed against Aspergillus fumigatus and Geotrichum candidum. Chitosan, highly substituted with moderate molecular weight molecules, exhibited the highest zone of inhibition (15.3 mm± 0.5) for G. candidum while chitosan, highly substituted with high molecular weight molecules showed the highest zone of inhibition (17.9 mm ± 0.3) for A. fumigatus [54]. A recent study evaluated the use of plant extracts or plant essential oils to enhance the inhibitory effects of CSNPs against fungi and especially, pathogenic fungi. It had been observed that P. dactylifera plant extract loaded CSNPs exhibited significant inhibitory effect against C. albicans [55]. Chitosan-based nanocomposite films loaded with plant essential oil mixtures (i.e. thyme-oregano, thyme-tea tree and thyme-peppermint) exhibited significant antifungal activities towards A. niger, A. flavus, A. parasiticus and Penicillium chrysogenum [56]. The encapsulated clove essential oil with CSNPs showed a potent antifungal activity against A. niger compared to CSNPs and clove essential oil, used alone [57].
Another study evaluated the inhibitory effect of encapsulated anethole-chitosan nanoemulsion on the fungal contamination and aflatoxinB1 production. Ten moulds, i.e. Aspergillus flavus strain, Aspergillus fumigatus, A. niger, Aspergillus luchuensis, Aspergillus repens, Penicillium italicum, P. chrysogenum, Fusarium oxysporum, Alternaria alternata and Cladosporium cladosporioides were selected for evaluating the efficacy of encapsulated anethole-based chitosan nanoemulsion (Ant-eCsNe). The result indicated the efficacy of Ant-eCsNe against the chosen molds by inhibiting the fungal growth at 0.8 μl/ml dose and inhibiting aflatoxin B1 biosynthesis at dose of 0.4 μl/ml. In-vivo field trial data revealed that Ant-eCsNe has a great potential in preserving the stored maize samples from fungal infestation and aflatoxin B1 contamination which make it a good food preservative [58].
AgNPs, produced using the extract of plant pathogenic fungus Colletotrichum gloeosporioides and conjugated with chitosan, showed an antibiofilm activity and a potential to inhibit Candida species at the dose of 50 μg/ml [59]. In a recent study the antifungal properties of Au-chitosan nanoparticles (CS-AuNPs) with different concentrations have been evaluated against two strains of Fusarium oxysporum. CS-AuNPs exhibited antifungal activity against both strains of F. oxysporum by reducing the colony diameter [60]. The CS-AuNPs exhibited the most effective fungicidal activity against C. albicans. Chitosan strongly improved antifungal properties of AuNPs [61].
In addition, nano-encapsulated chitosan in combination with the essential oils of Ocimum sanctum, Ocimum basilicum, and Ocimum canum exhibited a significant inhibitory activity against the pathogenic fungus Aspergillus flavus and aflatoxin B1. This chitosan nano-matrix enhanced the antifungal and aflatoxin B1 inhibition, compared to the free essential oils, and protected seeds samples from fungal contamination and mycotoxin production [62].
Recently, in drug delivery field considerable research has been carried out using chitosan and its derivatives. It has been observed that antifungal activity of CS-AgNPs composites against A. niger, Cryptococcus neoformans and Candida tropicalis is due to the synergistic effect between AgNPs and a chitosan derivative. These derivatives-AgNP composites have superior antimicrobial activities to pure chitosan and analogues and are also superior to antibiotic Amphotericin B [63]. Metal nanoparticles showed an improvement in antimicrobial activity of chitosan, which were prepared by doping chitosan film with silver nanoparticles, i.e. CS-AgNP. Chitosan thin film were doped with (100 − 400 μg) of biogenic AgNPs to enhance its antimicrobial potency. CS-AgNPs (300 and 400 μg) showed superior inhibition of S. aureus growth over non-doped chitosan films. CS-AgNPs at 300 μg concentration exhibited significant antifungal activity against C. albicans [64].

Conclusions
In conclusion, chitosan and its derivatives are safe and effective antimicrobial agents. lately, the focus has been on the capability of chitosan in reducing and capping metal nanoparticles to develop novel and more potent anti-microbial compounds. New techniques are being developed for synthesizing nanoparticles from natural resources such as bacterial and plant extracts. These studies of combination of metal nanoparticles stabilised with chitosan may provide a synergistic antimicrobial activity which may facilitate the development of a new class of antimicrobial agents.

Data availability statement
The data that support the finding of this study are openly available in public domain.

Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding
The authors received no financial support for the research, authorship and/or publication of this article.

Funding
The author(s) reported there is no funding associated with the work featured in this article.