221 ISSN 1229-9197 (print version) ISSN 1875-0052 (electronic version) Fibers and Polymers 2017, Vol.18, No.2, 221-230 Antimicrobial Properties of Chitosan Nanoparticles: Mode of Action and Factors Affecting Activity K. Divya, Smitha Vijayan, Tijith K. George, and M. S. Jisha* School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala-685560, India (Received July 14, 2016; Revised November 16, 2016; Accepted November 18, 2016) Abstract: The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on chitosan for biomedical applications. The presence of primary amine groups in repeating units of chitosan grants it several properties like antibacterial activity, antitumor activity and so on. Chitosan forms nanoparticles spontaneously on the addition of polyanion tripolyphosphate which has greater antimicrobial activity than parent chitosan. In the present study, chitosan nanoparticles (ChNP) were prepared by the ionic gelation method. The physiochemical characteristics of nanoparticles were analyzed using XRD, SEM, FTIR. The antibacterial activity of chitosan nanoparticles against medical pathogens Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa was evaluated by calculation of minimum inhibitory concentration (MIC) and compared with chitosan and chitin activity. The mode of action and factors affecting antibacterial activity were also analyzed. ChNP compounds exhibited superior antimicrobial activity against all microorganisms in comparison with chitosan and chitin. The antibiofilm activity was studied using crystal violet assay and growth on congo red agar. The study is thus a good demonstration of the applicability of chitosan nanoparticles as an effective antimicrobial agent with antibiofilm activity as well. Keywords: Antimicrobial property, Antibiofilm activity, Chitosan nanoparticles, Chitosan, TTC test Introduction The evolution of antibiotic resistant microorganisms has attracted much attention recently. It has been reported that there is no single antimicrobial agent that has not exhibited resistance by microorganisms [1]. This new development has forced scientists to formulate novel antimicrobial agents that show an effect against the continually increasing antibiotic resistant pathogenic microorganisms [2]. Chitosan has a new found popularity in this regard as bacteria are not reported to develop resistance to chitosan [3]. Chitosan is the second most abundant polysaccharide in nature. It is a linear polysaccharide of (1→4)-linked 2- acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc) units and 2-amino-2-deoxy-beta-D-gulcopyranose (GlcN) units [4]. It is acclaimed for its nontoxic nature, biodegradability, polycationic, antimicrobial activity, antitumor activity, antioxidative activity, anticholesterolemic, hemostatic and analgesic effect [5-12]. Chitosan nanoparticle (ChNP) can be prepared easily by the incorporation of polyanion like tripolyphosphate (TPP) in chitosan solution under continuous stirring. These nanoparticles are found to have increased activity compared to the parent chitosan [13]. Chitosan exhibits a wide range of antimicrobial activity against bacteria, filamentous fungi, yeast and even virus. Studies have reported antibacterial activity of ChNP against Escherichia coli, Staphylococcus aureus, Streptococcus mutans, Salmonella, typhimurium, Salmonella choleraesuis, Pseudomonas aeruginosa [14-17]. Antifungal activity of CHNP has also been reported against Aspergillus niger, Fusarium solani, Rhizoctonia solani, Collectotrichum gloeosporioides, Candida albicans [13,18]. Antiviral activity of ChNP against Human cytomegalovirus (HMCV) strain AD- 169, H1N1 Influenza A virus has also been reported recently [19,20]. Several studies have shown chitosan to exhibit higher antibacterial activity against gram positive bacteria than gram negative bacteria, while some other studies have shown that gram negative bacteria as more susceptible than gram positive [21,22]. Still many works have demonstrated that there is no significant difference between the antibacterial activity and bacterial species [23]. The exact mechanism of antibacterial activity is yet to be fully understood. The most prevalent proposed antibacterial activity of chitosan is by binding to the negatively charged bacterial cell wall causing disruption of the cell thus altering the membrane permeability, followed by attachment to DNA causing inhibition of DNA replication and subsequently cell death [24]. Another possible mechanism is that chitosan acts as a chelating agent that selectively binds to trace metal elements causing toxin production and inhibiting microbial growth [25]. The antibacterial activity of chitosan is also dependent on various factors both intrinsic and extrinsic. Intrinsic factors include the molecular weight and degree of deacetylation of parent chitosan, size and concentration of nanoparticle etc. The extrinsic factors include pH, temperature, reactive time and so on [26]. In the present study, we have synthesized and characterized ChNP by ionic gelation method. The antibacterial activity of ChNP against Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, P. aeruginosa was evaluated. The *Corresponding author: jishams@mgu.ac.in DOI 10.1007/s12221-017-6690-1