Synthesis, Characterization and Antibacterial Activity of ...jairjp.com/OCTOBER 2016/01...

Journal of Academia and Industrial Research (JAIR) Volume 5, Issue 5 October 2016 65

©Youth Education and Research Trust (YERT) jairjp.com Hipalaswins et al., 2016

ISSN: 2278-5213

Synthesis, Characterization and Antibacterial Activity of Chitosan Nanoparticles and its Impact on Seed Germination

W. Murphin Hipalaswins1, M.D. Balakumaran2 and S. Jagadeeswari1*

1Dept. of Microbiology; 2Dept. of Biotechnology; D.G. Vaishnav College, Chennai–600106, Tamil Nadu, India [email protected]*; +91 9840777165

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Abstract

This study focuses on the synthesis of chitosan nanoparticles by ionic gelation technique using sodium tripolyphosphate. The spherical morphology of the synthesized nanoparticles was confirmed by transmission electron microscopy. The nature of functional groups present in chitosan nanoparticles was determined by FT-IR analysis. The antibacterial activity of the chitosan nanoparticles was tested and found more efficient against Gram negative bacteria compared to Gram positive bacteria. The MIC of chitosan nanoparticles was determined for the pathogens and found Enterobacter aerogenes and Escherichia coli possess minimum MIC of 0.312 µg/mL whereas, maximum MIC was observed with Staphylococcus aureus (1.25 µg/mL). The alteration in cell membrane morphology was evaluated by transmission electron microscopy which revealed the aggregation of nanoparticles on the membrane of the cells. The possible mechanism of antibacterial activity was elucidated by membrane leakage studies. The antibacterial activity of the chitosan nanoparticles was enhanced by embedding them into cotton fabrics. The application of chitosan nanoparticles in the seed viability was determined by wet paper towel germination method.

Keywords: Chitosan nanoparticles, antibacterial activity, membrane leakage, cotton fabrics, seed viability.

Introduction The smaller size of nanoparticles has manifested a significant change in its physical properties from its original counterpart. The relationship between size and properties of nanoparticles has augmented its application in various fields (Balakumaran et al., 2016a). Nanoparticles are more efficient, easy to process and more sustainable than the macromolecules (He et al., 2012). Nanoparticles find their application in various fields including medicine, cosmetic, textile, drug delivery and electronic industries (Ahmad et al., 2012). Chitosan is the natural biopolymer obtained by the deacetylation of chitin. In recent years, the biopolymer, chitosan have gained considerable interest due to their biodegradability, biocompatibility and antimicrobial properties (Qi et al., 2004). The antimicrobial property of the chitosan was influenced by various factors namely the source and type of chitosan, degree of deacetylation and other physico-chemical characteristics. In the field of medicine, the textile products have gained considerable interest due to their role as bandages, sutures, wound dressings, scaffolds, surgical gowns, etc. At present, chemical antimicrobial agents such as triclosan, organometallics, phenols, organosilicons, etc. are used in the textile industries to prevent odours resulting from the microbial growth in textiles (Purwar and Joshi, 2004). The chemical antimicrobial agents often results in the undesirable toxic as well as by products which concern the researchers to search for a safer alternative in terms of safety, durability and effectiveness.

In recent years, nanotechnology has gained great interest in the medical field due to their excellent properties. Various reports are available in last decade focusing on the application of chitosan nanoparticles mediated drug delivery (Janes et al., 2001; Xu et al., 2003). Although reports are available on antibacterial activity of chitosan, very few reports are available regarding the application of chitosan nanoparticles on cotton fabrics for the enhanced antibacterial activity (Joshi et al., 2003). Recently Yang et al. (2010) fabricated the wool fabric with the nanochitosan to enhance the antibacterial properties of the fabric. The aim of the present study is to synthesize chitosan nanoparticles by ionic gelation technique and to evaluate the antibacterial property of the synthesized chitosan nanoparticles against selected pathogenic bacterial strains. The possible mechanism behind the antibacterial activity was also elucidated by SEM and membrane leakage studies. The functionalization of cotton fabrics loaded with chitosan nanoparticles for enhanced antibacterial activity was also carried out. Materials and methods Preparation of chitosan nanoparticles: The chitosan nanoparticles were synthesized by drop wise addition of sodium tripolyphosphate (TPP) solution to the chitosan solution dissolved in 1% acetic acid (Calvo et al., 1997). It was carried out under magnetic stirring and the reaction was stopped after the formation of milky white suspension.

RESEARCH ARTICLE



The milky suspension obtained was centrifuged at 900×g for 30 min and the pellet containing chitosan nanoparticles was collected, rinsed several times with distilled water and freeze-dried for further investigation. Characterization: The external and internal morphology of the synthesized chitosan nanoparticles was determined using transmission electron microscopy (IIT Madras, Chennai). The transmission electron microscope image of the chitosan nanoparticles was obtained by passing the beam of electrons on the surface of the specimen. The chemical and functional groups and their interactions while synthesis of chitosan nanoparticles were determined by assessing the FT-IR spectrum of the sample. The chitosan nanoparticles was dissolved in KOH solution and subjected to FT-IR spectroscopy analysis (IIT SAIF, Madras, Chennai). Antibacterial activity: The antibacterial activity of the synthesized chitosan nanoparticles against the selected clinically pathogenic bacterial strains namely Pseudomonas fluorescens MTCC 1748, Proteus mirabilis MTCC 1429, Staphylococcus aureus MTCC 7443, Klebsiella pneumoniae MTTC 109, Escherichia coli MTTC 1687 and Enterobacter aerogenes MTCC 111 were studied. The standard agar well diffusion assay was used for determining the antibacterial activity of the synthesized chitosan nanoparticles (Soleimani et al., 2015). The selected bacterial strains were separately inoculated into 10 mL LB broth and allowed to incubate for 24 h. The overnight grown bacterial cultures were swabbed over the petri dishes pre-seeded with Mueller Hinton agar (MHA) medium as uniform layer. With the help of sterile cork borer, four wells of 0.7 mm dia were created in all the MHA plates. The wells were labeled (a, b, c, d and e) and loaded each with 50 µL of chitosan nanoparticles, chitosan, saline (negative control) and ampicillin solution (positive control), respectively. The plates were incubated at 37°C for 24 h and observed for the clear zone formation around the wells and were measured. MIC determination: Micro broth dilution method was employed for the determination of minimum inhibitory concentration of chitosan nanoparticles against the tested bacterial strains (Aliasghari et al., 2016). About 100 µL of Mueller Hinton broth was added to each wells of ELISA microtitre plate. To the first well, 100 µL of the chitosan nanoparticles prepared at 1:2 dilutions was added and mixed well. From the first well, 100 µL was transferred to second well and the process was repeated. To each well, 10 µL of the bacterial suspension was added. The wells containing MH broth and bacterial suspension served as negative control. After 24 h incubation, the turbidity measurements were done by microplate reader at 630 nm. The lowest concentration of the drug required for the inhibition of visible growth of the bacteria was considered as minimum inhibition concentration (MIC).

Scanning electron microscopy: The alterations in the membrane morphology of the bacterial cells before and after treated with chitosan nanoparticles was assessed using scanning electron microscopy (NCNSNT, University of Madras, Chennai) (Kim et al., 2011). For SEM analysis, the bacterial strain Staphylococcus aureus was selected based on its susceptibility against the chitosan nanoparticles. About 100 µL of chitosan nanoparticles was added to the bacterial cells with the confluence of 106 CFU/mL and allowed to stand for 3 h. The reaction mixture was centrifuged at 300×g for 30 min and the pellet was washed thrice with phosphate buffered saline (PBS). The cells were then fixed with 2.5% glutaraldehyde for 30 min and dehydrated with increased concentration of ethanol. The fixed cells were dried and observed under scanning electron microscope. Protein leakage determination: The protein leakage study was performed to evidence chitosan nanoparticles induced membrane damage which results in the leakage of bacterial cellular proteins (Kim et al., 2011). The bacterial strains were inoculated in nutrient broth and incubated for 24 h. To 5 mL of the overnight grown bacterial cultures, chitosan nanoparticles was added at the respective MIC concentration and allowed to react for 6 h at 37°C. After incubation, 1 mL of the medium was withdrawn, centrifuged (600×g for 30 min) and the supernatant was assayed for the protein concentration by Bradford method (1976). Nucleic acid leakage determination: Upon disruption of membrane integrity, in addition to protein components, the nucleic acids including purine and pyrimidine are also released into the external medium. The bacterial suspension treated with chitosan nanoparticles was assayed for such nucleic acid leakage (Ghosh and Ramamoorthy, 2010). The bacterial strains were inoculated to nutrient broth and incubated for 24 h. To 5 mL of the overnight grown bacterial cultures, chitosan nanoparticles was added at the respective MIC concentration and allowed to react for 6 h at 37°C. The nucleic acid leakage was determined by measuring the OD at 260 nm using UV- visible spectrophotometer. Functionalization of cotton fabrics with chitosan nanoparticles: The cotton fabrics were cut into 1 cm × 1 cm size, washed with distilled water and sterilized. The sterilized cotton fabrics were dried and allowed to embed with synthesized chitosan nanoparticles by immersing them in Erlenmeyer flask containing chitosan nanoparticles (1000 ppm) under constant stirring for 24 h at 120 rpm. After 24 h, the chitosan nanoparticles treated cotton fabrics were allowed to dry at 70°C for 3 min in hot air oven. The well dispersed cotton fabrics were dried and stored at room temperature (El-Rafie et al., 2010). The antibacterial activity of the chitosan nanoparicles treated cotton fabrics was assayed using agar based diffusion assay (Rajendran et al., 2011).



The overnight grown bacterial suspensions were spread over the sterile Mueller-Hinton agar plates using a sterile cotton swab. The nanoparticles treated fabrics and untreated fabrics were placed on pre-seeded MH agar plates and the plates were incubated for 24 h at 37°C. The zone of inhibition around the fabrics was measured after incubation. Chitosan nanoparticles for the enhanced viability of seeds: The viability and purity of the long time stored seeds can be tested by wet paper towel germination assay. The seeds tested in the study namely cow pea (Vigna unguiculata L.), black gram (Phaseolus mungo L.), fenugreek (Trigonella foenum graecum L.) were washed thrice with distilled water and dried using filter paper. The dried seeds were allowed to immerse separately in chitosan and chitosan nanoparticles (1000 ppm) suspension for 10 min and dried using filter paper. The untreated seeds and treated seeds with chitosan and chitosan nanoparticles were placed on sterile filter paper moistened with water. The apparatus was closed and kept in temperature stable area away from direct sun light. After 7 d, the seedlings were unwrapped and observed for the number of seedlings and the length of the shoots was measured. Results and discussion In the present investigation, an ecofriendly method was employed for the synthesis of biodegradable chitosan nanoparticles using sodium-tripolyphosphate as cross linking agent. The present method holds advantages over the available methods by restricting the use of toxic solvents which hinders the efficacy of the nanoparticles. Chitosan nanoparticles are synthesized by the interaction between the positively charged chitosan polymer and the negatively charged cross linking agent, tripolyphosphate (Qi et al., 2004). Due to the positive charge of the chitosan polymer, it can be efficiently used as carrier for the positively charged macromolecules (Suganeswari et al., 2011). The chitosan to tripolyphosphate weight ratio, pH of the medium are the two main factors which determines the particle size of the synthesized nanoparticles (Katas et al., 2006). The morphology of the synthesized chitosan nanoparticles was assessed using TEM. The TEM of chitosan nanoparticles can be used to assess the shape, size and uniformity of the nanoparticles (Paul and Yadav, 2015). The TEM image revealed the spherical structure and smooth shape of the chitosan nanoparticles (Fig. 1). TEM results found in accordance with the results obtained by Mohammadpour et al. (2012) who synthesized chitosan nanoparticles loaded with venom of Mesobuthus eupeus scorpion for the antigen delivery application. Reports were also available for the production of crystalline (Marambio-Jones and Hoek, 2010) and triangular (Chen and Carroll, 2002) shaped chitosan nanoparticles. FT-IR study of chitosan nanoparticles was performed to elucidate the functional groups present in the synthesized nanoparticles.

Fig. 1. SEM of chitosan nanoparticles.

Fig. 2. FT-IR spectrum of chitosan nanoparticles.

A band at 3436 cm-1 corresponds to the combined peaks of the NH2 and OH group stretching vibration in chitosan nanoparticles (Fig. 2). The band at 1713 cm-1 corresponds to the CONH2 group. The intensity of (NH2) band at 1568 cm-1 can be observed clearly in chitosan nanoparticles. The occurrence of band at 1418 cm-1 indicated that the ammonium groups are cross-linked with tripolyphosphate (Xu et al., 2005). The cross-linked chitosan showed a P=O peak at 1084 cm–1. The strong and sharp peak of phosphate at 1084 cm-1 in chitosan nanoparticles confirmed the involvement of TPP during the synthesis of nanoparticles. Based on the FT-IR results obtained by Kucherov et al. (2003), it is postulated that ammonium groups of the chitosan reacts with the phosphoric groups of tripolyphosphate which facilitated the intermolecular as well as intra molecular interactions to form chitosan nanoparticles.

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There is an emergence in the development of antibiotic resistance among bacteria which necessitates several researches to discover novel antimicrobial agents with more potential (Zu et al., 2014). Chitosan nanoparticles exhibited excellent antibacterial activity which can be further augmented for various applications. Agar well diffusion method was used to evaluate the antibacterial activity of chitosan nanoparticles against the selected bacterial pathogens by measuring the zone of inhibition. The chitosan nanoparticles showed greater inhibitory activity against all the bacterial strains (Table 1). Enterobacter aerogenes was found to be more susceptible to the synthesized chitosan nanoparticles followed by Escherichia coli, Klebsiella pneumoniae, Pseudomonas fluorescens and Proteus mirabilis in a significant manner. The nanoparticles were found to be less toxic towards Staphylococcus aureus. The results of the antibacterial activity studies revealed that the chitosan nanoparticles inhibited Gram negative bacteria more efficiently when compared to Gram positive bacteria. It might be attributed due to the presence of single peptidoglycan layer in Gram negative bacteria and multiple peptidoglycan layers in Gram positive bacteria (Anitha et al., 2009). The viability of bacterial strains when exposed to varying concentrations of chitosan nanoparticles was determined using micro broth dilution assay. The MIC pattern of the chitosan nanoparticles are presented in Table 2. The MIC of chitosan nanoparticles for S. aureus was found to be 1.25 mg/mL, whereas 0.625 mg/mL for K. pneumoniae, P. fluorescens and P. mirabilis. MIC of chitosan nanoparticles for E. aerogenes and E. coli was found as 0.312 mg/mL. The MIC values of Gram positive bacteria were greater than the Gram negative bacteria. The results of the MIC studies also revealed that the chitosan nanoparticles were efficient against Gram negative and Gram positive bacteria. Costa et al. (2011) and Aliasghari et al. (2016) showed that chitosan nanoparticles effectively reduced viable counts of Streptococcus sp. and Enterococcus sp.

Fig. 3. SEM analysis of untreated bacterial cells.

Fig. 4. SEM analysis of bacterial cells treated with chitosan nanoparticles.

The SEM analysis of bacterial cells treated with chitosan nanoparticles was done to elucidate the possible mechanism behind the antibacterial activity of chitosan nanoparticles. Accumulation of chitosan nanoparticles over the bacterial cells suggests that the nanoparticles disrupted the membrane integrity of bacterial cell wall (Fig. 3 and 4). The cell wall breakage also observed in SEM microscopy evidenced the entry of nanoparticles through the pores.

Table 1. Antibacterial activity of chitosan nanoparticles.

Bacterial strains Antibacterial activity (Zone of inhibition in mm) Chitosan nanoparticles Chitosan Saline Ampicillin

Staphylococcus aureus 0.7 0.3 - 1.4 Proteus mirabilis 1.7 0.6 - 1.5 Escherichia coli 2.2 0.9 - 1.8 Pseudomonas fluorescens 1.9 0.8 - 1.7 Klebsiella pneumoniae 2.0 1.0 - 1.6 Enterobacter aerogenes 2.4 1.1 - 1.9

Table 2. MIC of chitosan nanoparticles against bacterial strains. Bacterial strains Minimum inhibitory concentration (µg/mL) Staphylococcus aureus 1.25 Proteus mirabilis 0.625 Escherichia coli 0.312 Pseudomonas fluorescens 0.625 Klebsiella pneumoniae 0.625 Enterobacter aerogenes 0.312



From the results, it is clear that chitosan nanoparticles killed the bacterial strains by damaging their cell membrane. Reports are available evidencing the concentration dependent antibacterial activity of chitosan nanoparticles against bacteria which creates pits in their cell membranes (Amro et al., 2000; Sondi and Salopek-sondi, 2004). Similar pattern of results were also observed by Kim et al. (2011). There is no significant change in the amount of protein leaked from the cell membrane of all the untreated bacterial strains (Fig. 5). The protein leakage from the bacterial strains treated with chitosan nanoparticles was considerably increased after 6 h of treatment. The amount of protein leaked from the Gram negative bacteria was found to be higher than that of Gram positive bacteria. Kim et al. (2011) also observed similar results while testing the antibacterial activity of nanoparticles against E. coli and S. aureus. The results were in consistent with the results of Feng et al. (2000) and Jung et al. (2008). Next to protein leakage studies, the nucleic acid leakage pattern of control and the cells treated with chitosan nanoparticles was evaluated. There is no significant difference in the amount of nucleic acid leakage from nanoparticles treated cells and the control cells at the beginning. But after 6 h of incubation, we observed a significant difference between the cells treated with nanoparticles and the control group (Fig. 6). Ghosh and Ramamoorthy (2010) also evaluated the nucleic acid leakage pattern of E. coli after treating with silver nanoparticles and observed a similar pattern of results. The antibacterial properties of chitosan nanoparticles functionalized cotton fabrics were evaluated against the selected Gram positive and Gram negative bacteria. The nanoparticles treated fabrics displayed good antibacterial activity (Table 3) with a clear zone of inhibition around the cotton fabrics against all the tested pathogens; however, the untreated (control) fabrics did not show any inhibition zone. Similarly, Balakumaran et al. (2016b) have also shown antibacterial activity for silver coated cotton fabrics against pathogenic bacteria and fungi. Thus, it is apparent that the present result showed better antibacterial activity as the inhibition zone was found to be higher than previous reports (Sathianarayanan et al., 2010). The possible mechanism of antibacterial activity of chitosan nanoparticles treated cotton fabrics were probably due to the formation of chemical bonds between the functional groups of chitosan nanoparticles and the functional groups of the cotton fabrics and the physical adsorption of chitosan nanoparticles on the surface of the fabrics (Perelshtein et al., 2008). The worldwide cereal production is threatened by adverse environmental condition and pathogen attacks resulting in delayed seed germination, stunted seedling growth and decreased productivity. Chemical and biochemical modes of seed treatment were practiced for maintaining the seed quality (Boszoradova et al., 2011).

Fig. 5. Protein leakage studies on chitosan nanoparticles treated bacterial cells.

Fig. 6. Nucleic acid leakage studies on chitosan nanoparticles treated bacterial cells.

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Table 3. Antibacterial activity of chitosan nanoparticles embedded cotton fabrics.

Bacterial strains Zone of inhibition (mm) Staphylococcus aureus 0.9 Proteus mirabilis 1.9 Escherichia coli 2.4 Pseudomonas fluorescens 2.1 Klebsiella pneumoniae 2.3 Enterobacter aerogenes 2.9



Due to the growing demand of organic food era, customers are demanding food/crop free from chemicals which lead to the exploration of alternative eco-friendly and effective strategies for maintaining seed quality (Chandra et al., 2015). The germination is the key step of agriculture as it reflects in the productivity of the food crop. Hence, the germination test in laboratory is essential to determine the germination potential or viability of the seeds. In the present study, the germination rate of the seeds treated with chitosan and chitosan nanoparticles were assessed by paper towel germination test. The results revealed that the germination rate of seeds treated with chitosan nanoparticles was found to be more efficient when compared to the control and the seeds treated with chitosan (Table 4). The application of chitosan nanoparticles also serves as an appropriate option for the control of pests which might eliminate the toxic pesticide use and also enhances the yield of soybean (Kendra et al., 1984). Conclusion The results of the present study revealed that chitosan nanoparticles synthesized by the ionic gelation method possess strong antibacterial activity against Gram negative bacteria when compared to Gram positive bacteria. The morphological changes on bacterial cells by chitosan nanoparticles were observed by SEM which indicates that chitosan nanoparticles can be used as effective antibacterial agents against the bacterial pathogens. The underlying mechanisms behind the antibacterial activity of the nanoparticles were addressed by membrane leakage studies. Chitosan nanoparticles functionalized cotton fabrics exhibit higher antibacterial activity. The unique properties of Chitosan nanoparticles encouraged its usage as antimicrobial agent and also as a carrier to deliver various therapeutic drugs. Acknowledgements Authors thank the Management, D.G. Vaishnav College, Arumbakkam, Chennai for their support. The Metallurgy Department, IIT Madras; National Centre for Nanoscience and Nanotechnology (NCNSNT), University of Madras and SAIF, IIT-Madras are gratefully acknowledged for TEM, SEM and FT-IR analyses respectively. References 1. Ahmad, M.B., Lim, J.J., Shameli, K., Ibrahim, N.A., Tay,

M.Y. and Chieng, B.W. 2012. Antibacterial activity of silver bionanocomposites synthesized by chemical reduction route. Chem. Cent. J. 6(1): 101-109.

2. Aliasghari, A., Rabbani Khorasgani, M., Vaezifar, S.,

Rahimi, F., Younesi, H. and Khoroushi, M. 2016. Evaluation of antibacterial efficiency of chitosan and chitosan nanoparticles on cariogenic streptococci: an in vitro study. Iran. J. Microbiol. 8(2): 93-100.

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Table 4. Evaluation of seed germination using wet paper towel method.

Seeds tested Seed germination before and after treatment (mm) Untreated Chitosan nanoparticles treated Chitosan treated

Cow pea (Vigna unguiculata L.) 7 mm 9 mm 10 mm Black gram (Phaseolus mungo L.) 6 mm 20 mm 10 mm Fenugreek (Trigonella foenum-graecum L.) 8 mm 3 mm 5 mm



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