Physicochemical investigations of biogenic chitosan-silver nanocomposite as antimicrobial and anticancer agent

Accepted Manuscript Title: Physicochemical investigations of biogenic chitosan-silver nanocomposite as antimicrobial and anticancer agent Author: Arjunan Nithya Henry Linda Jeeva Kumari Chandra Mohan Singaravelu Ruckmani Kandasamy Jothivenkatachalam Kandasamy PII: DOI: Reference:

S0141-8130(16)30704-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.07.003 BIOMAC 6277

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

27-4-2016 16-6-2016 1-7-2016

Please cite this article as: Arjunan Nithya, Henry Linda Jeeva Kumari, Chandra Mohan Singaravelu, Ruckmani Kandasamy, Jothivenkatachalam Kandasamy, Physicochemical investigations of biogenic chitosan-silver nanocomposite as antimicrobial and anticancer agent, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.07.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Physicochemical

investigations

of

biogenic

chitosan-silver

nanocomposite as antimicrobial and anticancer agent Arjunan Nithyaa, Henry Linda Jeeva Kumarib,c, Singaravelu Chandra Mohana, Kandasamy Ruckmanib,c,*, Kandasamy Jothivenkatachalama,* a

Department of Chemistry, Bharathidasan Institute of Technology, Anna University,

Tiruchirappalli 620024, Tamil Nadu, India b

National Facility for Drug Development for Academia (NFDD), Pharmaceutical and Allied

Industries, Bharathidasan Institute of Technology, Anna University, Tiruchirappalli 620024, Tamil Nadu, India c

Department of Pharmaceutical Technology, Centre for Excellence in Nanobio Translational

REsearch (CENTRE), Bharathidasan Institute of Technology, Anna University, Tiruchirappalli 620024, Tamil Nadu, India ∗Corresponding authors. Tel.: +91 431 2407961/ +91 431 2407978; fax: +91 431 2407333/ +91 431 2407910.

E-mail address: [email protected], [email protected] (K. Jothivenkatachalam), [email protected] (K. Ruckmani).

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HIGHLIGHTS 

Biocompatible chitosan was used for the synthesis of CS-Ag nanocomposites.



The physicochemical characteristics of the nanocomposites were elucidated.



The highest antimicrobial activity was shown against Salmonella sp.



The anticancer effect of composite exhibited significant IC50 value 29.35 µg mL-1.

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ABSTRACT Chitosan (CS), a seaweed polysaccharide is a natural macromolecule which is widely being used in medical applications because of its distinctive antimicrobial and anticancer properties. Silver, a noble metal, is also receiving wide attention for its potential usage in antimicrobial and anticancer therapeutics. In this study, an effective way of reduction of silver using chitosan at varying reaction temperatures and an optimized concentration of silver were performed. The optical, structural, spectral, morphological and elemental studies of the biosynthesized chitosan-silver (CS-Ag) nanocomposites were characterized by several techniques. The synthesized CS-Ag nanocomposites exhibit particle size around 20 nm and were further exploited for potent biological applications in nanomedicine due to their nanometric sizes and biocompatibility of chitosan. The antimicrobial activity of the biosynthesized CS-Ag nanocomposites exhibits zone of inhibition ranged between 09.666 ± 0.577 and 19.000 ± 1.000 (mm). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were from 8 to 128 µg mL-1 and 16 to 256 µg mL-1 respectively, with the highest antimicrobial activity shown against Gram-negative Salmonella sp. The synergistic effect of chitosan and silver as a composite in nanometric size revealed significant IC50 value of 29.35 µg mL-1 and a maximum of 95.56 % inhibition at 100 µg mL-1 against A549 lung cancer cell line, resulting in potent anticancer effect. Keywords: Chitosan Nanocomposite Antibacterial Anticancer

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1. Introduction Debilitating illnesses occur due to various disease-causing pathogens. Crude estimates suggest that more than 10 million people die every year with diverse infections, and the statistics is not substantially declining [1]. Rapid evolution of drug resistance in the microorganisms due to uncontrolled usage of antibiotics possesses serious impediment to disease management with a need for alternative safe and effective antimicrobial agents [2]. Cancer is the second largest non-communicable disease which causes death worldwide. According to the World Health Organization, the number of cancer cases is envisaged to increase from 8.2 million to 13 million annually, and is expected to increase to 22 million by 2030 [3]. Lung cancer is the most predominant type of cancer with a morbidity of 1.6 million which is 19.4 % of the total deaths occurred due to cancer [4]. In the recent days, a promising strategy for cancer treatment is chemotherapy. Incorporation of natural products in the treatment of diseases has gained considerable attention owing to their potential biological activities when compared to synthetic molecules that impose serious adverse reactions. Therefore, intensive research has been focused on the discovery of effective anticancer agents from natural sources that could exert minimal toxicity on normal cells [5]. Marine seaweeds are natural and cheap sources of potential drug candidates. Over 15,000 novel compounds have been isolated from marine seaweeds so far, of which, interesting functional properties of polysaccharides have been discovered [6]. Among these polysaccharides, chitosan, an interesting biopolymer offers a unique set of characteristics which include hydrophilicity, biocompatibility, renewability, film forming ability and biodegradability [7-9]. Chitosan is an N-deacetylated product of chitin, the second most abundant polysaccharides found in nature. Chitosan has been considered an important biomaterial due to its chemical reactivity since it contains many –OH and –NH2 groups [10]. Chitosan also exhibits a number of notable biological properties such as antimicrobial, biosensing and anticancer activities with its major applications in medicine, pharmaceuticals, biochemistry, food science and agriculture [11,12]. Technologies are being largely used in the development of drug resistant antimicrobials and anticancer agents, and one being nanotechnology. An important aspect of nanotechnology is to accomplish a controllable synthesis of nanoparticles in different compositions to achieve different sizes and shapes. Nanotechnology is a growing field that incorporates the usage of biomaterials in nanoscale for wide medical and pharmaceutical applications. Nanomedicine deals with the incorporation of nanomaterials for medical applications. One of the facets of nanomedicine is the development of nanoscale materials for antimicrobial drug delivery [13] and anticancer therapy [14]. Nanomaterials have potential

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applications in cancer therapeutics, biosensing, diagnostic imaging and targeted drug delivery [15]. They are advantageous due to their surface plasmon resonance (SPR), surface enhanced Raman scattering (SERS) and enhanced Rayleigh scattering in metal nanoparticles. Metal nanoparticles have characteristic physical, chemical, electronic, electrical, magnetic, mechanical, dielectric, thermal, optical and biological properties [16], owing to their high surface-to-volume ratio, surface energy, spatial confinement and reduced imperfections, as opposed to bulk materials [17,18]. Studies on silver nanoparticles have confirmed their potential application in major fields of science and technology. Among other noble metals, silver is widely used due to their biological properties such as antibacterial, antifungal, larvicidal, antiparasitic and anticancer properties [19,20]. Silver nanoparticles (AgNPs) have gained attention to combat a broad spectrum of microorganisms like multidrug resistant bacteria and fungi [21]. Silver nanoparticles synthesis methods are an important perspective for environmental protection. There are extensive conventional methods have been reported in the synthesis of silver nanoparticles by using sodium borohydrate, citrate, hydrazine, ascorbate and starch as reducing agents [22,23]. Greener methods and eco-friendly means are rapidly being developed in the synthesis of nanoparticles due to their cost-effectiveness and safe production [24-27]. The selection of capping agent for synthesis of silver NPs is to increase the stability, also cover less bioavailability and less toxic to environment and human. Chitosan has also been reported to be a mild reducing agent for the reduction of silver ions and as a capping agent to avoid aggregation by controlling the growth of nanoparticles [28]. Due to the incredible properties of chitosan, it is accepted as the most effective and promising support for nanobiocomposite synthesis [29]. The functional groups of chitosan may allow it to interact with other materials such as CS/TiO2, CS/Cu2O, CS/CdS, CS/zeolite, CS-ZnO, CS-Ag and CS-Au for the application in the removal of dyes, toxic organics, biosensors and antimicrobial agents [30-34]. Among other nanocomposites, silver has been much application because of its broad spectrum of toxicity against microbes as well as a limited toxicity to humans. Chitosan-silver (CS-Ag) composite is one of the composite materials widely used in the field of environmental remediation [35], biological application [36-40], wound dressing [41] and biosensing activity [42]. In this study, we report the synthesis of biogenic CS-Ag nanocomposite and its physico-chemical characterizations. Furthermore, its exploitation as potent antimicrobial and anticancer agents in the field of nanomedicine has been explored. 2. Materials and methods

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2.1. Materials Silver nitrate, chitosan and glacial acetic acid were used as of analytical grade. All the solutions were prepared with deionized water. Nutrient broth (NB), nutrient agar (NA) and Mueller Hinton agar (MHA) media used for the cultivation of bacteria were obtained from Hi media, Mumbai, India. Standard microbial strains Staphylococcus aureus MTCC 3160 and Pseudomonas aeruginosa MTCC 1688 (equivalent ATCC 9027) were procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. Clinical isolates Staphylococcus sp., Streptococcus sp., Enterococcus sp., Salmonella sp., and Shigella sp., were obtained from local hospitals. Human adenocarcinoma cell line A549 was obtained from National Centre for Cell Science (NCCS), Pune, India. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were procured from Invitrogen, USA. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Hi media, Mumbai, India. 2.2 Synthesis of CS-Ag nanocomposites

The typical synthesis of CS-Ag nanocomposites at varying concentrations of silver nitrate were carried out by stirring 10 mL of 0.7 % (w/v) chitosan in 0.1 M glacial acetic acid. The viscous solution was stirred continuously overnight to dissolve chitosan. Different concentrations of silver nitrate such as 52 mM, 104 mM and 156 mM were used for the synthesis. A volume of 4 mL of each concentration was added to the viscous solution of chitosan and stirred. At a low concentration of 52 mM, fast reduction of AgNps takes place using chitosan as a reducing agent. An optimised concentration of silver nitrate was then used for the reduction at different temperatures like room temperature, 90 ⁰C and 120 ⁰C which were labelled as CS-AgA, CS-AgB and CS-AgC respectively. 2.3. Characterization of CS-Ag nanocomposites UV-Visible spectra were recorded on an integrated sphere assembly Shimadzu UV2450 Spectrophotometer using barium sulphate as a reference sample. X-ray diffraction (XRD) is a versatile, non-destructive analytical method for the detection and quantitative determination of various crystalline phases. Powder XRD data were collected via Philips PW 1710 diffractometer with Cu Kα radiation (λ = 1.5406 A⁰) and graphite monochromator, operated at 45 kV; 30mA and 25 ⁰C. Fourier transform infrared spectroscopy (FTIR) spectra were obtained with JASCO 460 plus spectrometer using potassium bromide pellets within the range of 400 - 4000 cm−1. BET analysis provides specific surface area of materials by nitrogen

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multilayer adsorption was measured at 77K on a Belsorp mini, BEL Japan system. The samples were pre-treated at 473 K for 2 h under N2 atmosphere. The surface charge of the nanocomposite was measured by zeta potential analyser using Nano ZS series, Malvern, UK instrument. Field emission scanning electron microscopy (FE-SEM) with energy dispersive xray spectroscopy (EDX) was taken using Gemini Zeiss Supra 55 to determine the morphology of CS-Ag nanocomposite. Surface topography of the nanocomposite was characterized by atomic force microscopy (AFM) using Park system AFM XE 100. The size and shape of the nanocomposite were analyzed using TECNAI T20 High-resolution transmission electron microscopy (HRTEM) operating at 200 keV and the phase analysis was done by observing the selected area electron diffraction (SAED) pattern. X-ray photoelectron spectroscopy (XPS) data were collected with Alpha 110 instrument from Thermo Fischer Scientific (East Grinstead, UK) with monochromatic Al-kα (hv=1486.7 eV) radiation and a pass energy of 20 eV. The calibration of binding energy of the spectra was performed with C 1s peak of carbon due to atmospheric contamination at 284.8 eV. 2.4. Evaluation of antimicrobial activity The pathogens tested were standard strains of clinical origin procured from MTCC and the clinical strains were isolated from cutaneous skin infections obtained from local hospitals. CS-Ag nanocomposites were completely dispersed in sterile deionised water using an ultrasonicator (Digital ultrasonic cleaner, Equitron) at a stock concentration of 1000 µg mL-1. Antibiotic streptomycin (100 µg mL-1) and sterile deionised water were used as positive and negative controls respectively. Well diffusion technique was followed to perform antimicrobial susceptibility test as described in our previous study [35]. MHA was used for the cultivation of bacteria. Briefly, freshly grown overnight bacterial cultures were used and the turbidity of suspension was adjusted to 1×108 CFU mL−1. 100 µL of them were spread plated uniformly on 25 mL of solidified and dried agar plates. Wells were punctured at equidistance using sterile pipette tips. 100 µL each of streptomycin and CS-Ag nanocomposites were dispensed into the wells to obtain 10 µg per well. The plates were incubated at 37 ⁰C for 24 h and the zone of inhibition was observed and measured in mm. The assays were performed in triplicates. 2.5. Statistical analysis and pictorial bar representation The data of antibacterial activities were analyzed for statistical significance using student’s t-test, and the correlation coefficient (R) was used to detect the significance level of two variables. The analysis was carried out using statistical package of social sciences (SPSS

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package software, Version 14.0, Chicago, ILA, USA). The percentages of antibacterial efficacy of the biosynthesized CS-Ag nanocomposite on both Gram-positive and Gramnegative standard and clinical bacterial strains were pictorially described by a bar representation to show the highly susceptible organisms. 2.6. Determination of MIC and MBC The minimum inhibitory concentration (MIC) of CS-Ag nanocomposite was further determined by broth macro-dilution method based on Clinical and Laboratory Standards Institute (CLSI) guidelines. CS-Ag nanocomposite was completely dispersed in sterile deionized water to obtain a stock concentration of 512 µg mL-1. 500 µL of the stock solution was incorporated with 500 µL of sterile NB medium to obtain a concentration of 256 µg mL-1. Two-fold serial dilutions were performed to obtain the concentrations 128, 64, 32, 16 and 8 µg mL-1. 50 µL of 1 x 106 CFU mL-1 bacterial suspensions were transferred to each tube of varying concentrations of CS-Ag nanocomposite. Inoculums and NB medium were used to compare the turbidity visually. All the tubes were incubated at 37 ⁰C, 250 rpm shaking for 24 h. The lowest concentration that inhibited the growth of bacteria determines the MIC. A loopful of culture from each tube was inoculated onto sterile MHA plates and incubated at 37 ⁰

C for 24 h. The lowest concentration at which 99.9 % bacteria is killed determines minimum

bactericidal concentration (MBC). All the assays were done in triplicate. 2.7. Cell culture A549 lung cancer cells were maintained at 1 x 106 cells mL-1 in DMEM, supplemented with 10% heat-inactivated FBS and incubated at 37 °C in a humidified atmosphere with 5% CO₂. Cells were seeded in 96 well plates at a density of 1 x 10⁴ cells/well and allowed to attach overnight. The medium was discarded and the cells were incubated with different concentrations (10-100 μg mL-1) of the synthesized CS-Ag nanocomposite for 24 h. Untreated cells were used as control wells and epirubicin (10 μg mL-1), a standard anticancer drug was used as a positive control. All the assays were performed in triplicate. The morphology of the cells was observed under an inverted microscope (TiE, Nikon, Japan) and the phase-contrast images at 10 X magnification were captured. 2.8. Cytotoxicity assay MTT dye reduction assay was performed to determine the percentage of cell viability and anticancer effect of CS-Ag nanocomposite at various concentrations [43]. The assay

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depends on the reduction of MTT by mitochondrial dehydrogenase, an enzyme present in the mitochondria of viable cells to a purple colored formazan product. After 24 h incubation at 37 ⁰

C in 5% CO2 humidified atmosphere, 10 μL of MTT (5 mg mL-1) was added and incubated

for 4 h. The medium was discarded and 100 μL of DMSO was added to dissolve formazan crystals. The absorbance was read at 570 nm in a multimode microplate reader (EnSpire, PerkinElmer, Singapore). The assays were performed in triplicate. The percentage of cell viability and the percentage of inhibition at various concentrations were calculated using the following formulae: % viability = (At / Ac) x100 % inhibition = (Ac – At)/Ac x100 where, At is the absorbance of the test sample and Ac is the absorbance of the control A graph was plotted between the concentration of the sample and percentage of inhibition, thereby determining the IC50 value of CS-Ag nanocomposite which is the maximal concentration of the drug to cause 50 % inhibition of biological activity of cancer cells. 3. Results and discussion 3.1. Optical analysis The UV-Visible absorption spectra of the biosynthesized CS-Ag nanocomposites exhibited a surface plasmon resonance (SPR) of silver at 420 nm suggesting that the reduction of Ag is by chitosan. The color of the solution progressively changes from colorless to light yellow and then dark brown indicating the change in the structure of Ag when combined with chitosan. Various concentrations of silver nitrate such as 52 mM, 104 mM and 156 mM were used for the reduction of AgNPs. As the concentration of silver increases, absorption peak decreases, which might probably be due to the formation of clusters, thereby, decreasing the formation of AgNps [44]. Thus, the optimised concentration of 52 mM silver nitrate was used throughout the study. Reduction of silver was carried out at three different temperatures such as room temperature, 90 ⁰C and 120 ⁰C as represented in Fig. 1(a-c). As the reaction temperature increases, the SPR absorption peak of silver also increases, which might occur due to the rapid reduction of silver at higher temperatures. 3.2. Structural analysis The structural properties of CS-Ag nanocomposites (CS-AgA, CS-AgB and CS-AgC) were analyzed using XRD technique as shown in Fig. 2(a). The peaks were exhibited at 38.2⁰ and

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44.2⁰ with the corresponding planes (111) and (200) of face centered cubic geometry, and the lattice parameters were determined to be a = 4.0580 which matches with the JCPDS card no. 87-0720. The formation of broad peak around 20⁰ was due to the presence of CS in the nanocomposite. Intense peaks were observed for the nanocomposites with an increase in temperature. The crystallite size was calculated by using Debye-Scherrer’s formula and it was around 5 nm for all the samples.

3.3 Spectral analysis The FTIR spectral analysis of pure chitosan shows common bands at 3436 cm-1, 2912 cm1

, 1648 cm-1, 1377 cm-1 and 1076 cm-1 that belong to the stretching vibration of –OH and –NH

groups, –CH group, amide group, COO- group of carboxylic acid salt and stretching vibration of C-O-C in glucose unit [45]. The nanocomposites show all the corresponding bands of chitosan and also a band around 590 cm-1. This confirms the presence of metallic silver in the nanocomposites as depicted in Fig. 2(b). As compare to the spectrum of pure chitosan with CS-Ag nanocomposite, observed peaks are shifted towards the lower wave number with high intense confirms the strong interaction between Ag and CS. CS-AgA shows strong peak when compared with other samples. Even though fast reduction of Ag takes place at a higher temperature, CS-AgA composite was stable and reduced slowly at room temperature and hence, CS-AgA was taken for further analyses. 3.4. Surface area and charge analysis The N2 adsorption-desorption isotherms were measured using N2 adsorption-desorption analyzer. CS-AgA nanocomposite shows a type III isotherm indicating the formation of micropores and the hysteresis loops were observed in Fig. 3(a). Surface area was calculated by multiple BET analysis and is found to be 128 m2 g-1. Total pore volume obtained at P/Po is 0.33 cc g-1 and the pore size is 1.9 nm as calculated by BJH method. CS-Ag nanocomposite exhibits higher surface area when compared with AgNPs [46]. Surface charge of CS-Ag nanocomposite was analyzed for the stability of nanocomposite. The zeta potential analysis is shown in Fig. 3(b) and is found to be +12.5 mV. Higher the surface area and positive charge on CS-Ag nanocomposite are requisites for higher antimicrobial activity which is further discussed in section 3.7. 3.5. Morphological study

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The surface morphology of the optimized CS-AgA composite was analyzed using FESEM with EDX technique as represented in Fig. 4 (a) and (b). The metal nanoparticles are dotted over the surface of chitosan matrix. The existence of metal and chitosan in the composite was also confirmed by EDX analysis. The surface morphology and particle size were further characterized by AFM technique as shown in Fig. 4 (c), and the particle size distribution graph was plotted for AFM image and resulted with the size of around 20 nm as given in Fig. 4 (d). HRTEM results (Fig. 4e) imply that spherical silver nanoparticles are present in the chitosan suspension in concordance with the result of FE-SEM image. The average particle size of around 10 nm was found from the particle size distribution graph of HR-TEM image in Fig. 4 (f). The three diffraction patterns are clearly seen in Fig. 4 (g) and it can be indexed as a face centred cubic lattice. The first strongest rings are the combination of both (111) and (200) planes, the second ring corresponds to (220) crystallographic plane, and the third ring belongs to (311) plane of Ag in accordance with JCPDS card no. 87-0720. 3.6. Elemental analysis The wide XPS spectrum contains the main elements of CS-AgA composite such as carbon, oxygen, nitrogen and silver, whose peaks are fitted by multiple Gaussians as shown in Fig. 5 (a-d). The C 1s can be deconvoluted into C–C/C=C (284.4 eV), C–OH/C–N (285.9 eV) and the binding energy (BE) of 282.9 eV can be assigned to the bond between C and Ag during the formation of CS-Ag nanocomposite [47-50]. The O 1s peak appeared in the range of 530–533 eV, which can be deconvoluted into the major peak appear at C=O (530.8 eV) and the fewer peak at C-OH (533 eV) [47]. The peak of N 1s reveals the presence of –NH2 (397.8 eV) in chitosan, and the protonated –NH3+ group (399.9 eV) which related to the stronger interaction of Ag NPs with –NH3+ of chitosan [51]. The less intense peak at 405 eV may be due to the existence of nitrate ions from the precursor. The characteristic peak of Ag 3d was observed that corresponds to Ag 3d3/2 and Ag 3d5/2 levels. The peaks at the binding energy (BE) of 368.2 eV and 374.2 eV denote the presence of metallic silver, whereas, in the case of CS-Ag composite, two lines appeared with lower BE as 366.6 eV and 372.2 eV might be due to the presence of Ag ions [51]. The binding energies such as Ag-C (282.9), Ag-O (533 eV) and the protonated amine group (399.9 eV) also an evident for the interaction of Ag with chitosan. Thus, the XPS result reveals that the CS-Ag composite contain both Ag 3d and N 1s, confirming the existence of metal and chitosan in the composite in agreement with XRD and EDX results.

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3.7. Antimicrobial activity and mechanism of action The synthesized CS-Ag nanocomposites show effective antimicrobial activity against both standard and clinical pathogens (Figs. 6-8). The zones of inhibition (mm) are recorded in Table 1 as mean and standard deviation (SD). The positive control streptomycin showed a maximum zone of inhibition 18.000 ± 2.000 (mm), whereas, there was no activity for the negative control. The zone of inhibition for CS-Ag nanocomposites ranged from 09.666 ± 0.577 to 19.000 ± 1.000 (mm) against various pathogens. The antibiotic streptomycin was resistant against the clinical strain Streptococcus sp. whereas, the synthesized CS-Ag nanocomposite proves effective against a multi-drug resistant bacterium. The plausible mechanism of antibacterial activity is deciphered as: chitosan and silver are reported to have antibacterial activity [35]. Therefore, the enhanced antibacterial effect of CS-Ag nanocomposites is due to the synergistic effect of chitosan and silver. Adherence of bacteria to the surface of CS-Ag nanocomposites is rapid within 30 min, attributing to their antibacterial property. Moreover, the nanometric size and larger surface area of CS-Ag nanocomposites owe to greater adsorption onto the surface of bacterial cells and can easily penetrate the bacterial cell wall causing cell death. Bacterial interactions will be more with larger surface area nanocomposites. Higher surface area leads to an increase in the contact surface resulting in greater antimicrobial activity when compared with bulk metals [46]. The bactericidal effect is also due to the surface charges on the composites and bacteria. The presence of negatively charged teichoic acids and lipopolysaccharides of the Gram-positive and Gram-negative bacteria respectively results in a negatively charged bacterial cell wall. As the zeta potential analysis of the synthesized CS-Ag nanocomposite gives a positive charge, this strongly interacts with the negatively charged cell membrane of the bacteria, causing intracellular leakage and disruption of nuclear functions [52]. Therefore, the synthesized CS-Ag nanocomposite exhibiting a higher surface area of 128 m2 g-1 and surface charge of +12.5 mV result in excellent antibacterial effect with the combined action of chitosan and AgNPs. 3.8. Statistical analysis and pictorial bar representation One sample t-test analysis was also carried out to evaluate the significance of CS-Ag nanocomposites and is found to be highly significant (p < 0.001) with a true effect (Table 1). The highest zone of inhibition of CS-Ag nanocomposite is recorded against a Gram-negative Salmonella sp. as 19.26 mm, followed by Gram-positive S. aureus MTCC 3160 and Gramnegative P. aeruginosa MTCC 1688 as 17.91 mm and 16.55 mm respectively. The

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percentages of efficacy against Staphylococcus sp., Shigella sp., Enterococcus sp., and Streptococcus sp., are found to be 13.18 mm, 12.84 mm, 10.47 mm and 9.8 mm respectively (Fig. 9). Thus, the antibacterial activity of the synthesized nanocomposite proves effective against both standard and clinical Gram-positive and Gram-negative bacteria. 3.9. Determination of MIC and MBC The MIC and MBC values of CS-Ag nanocomposite against various bacterial pathogens ranges from 8 to 128 µg mL-1 and 16 to 256 µg mL-1 respectively (Table 2). CS-Ag nanocomposite shows the highest mean zone of inhibition 19.000 ± 1.000 (mm) and lowest MIC (8 µg mL-1) and MBC (16 µg mL-1) against Salmonella sp., followed by S. aureus with a zone of inhibition 17.667 ± 0.577 (mm), MIC (16 µg mL-1) and MBC (32 µg mL-1). The results interpret that CS-Ag nanocomposite possesses remarkable antibacterial activity against both standard and clinical Gram-positive and Gram-negative bacteria, proving its broad spectrum of antimicrobial activity. 3.10. Cytotoxicity assay The morphological assessment of cells (Fig.10) reveals that the treatment of CS-Ag nanocomposite on A549 cells reduced the cell viability by altering cellular morphology similar to that of epirubicin treated cells. The morphological characteristics include shrinkage and blebbing of the cells, reduced cell density, reduction in cell to cell contact when compared to that of untreated cells (control). This positively indicates a pronounced cytotoxic effect of CS-Ag nanocomposite on A549 cells, showing a great selectivity to cancer cells with potential application in cancer chemoprevention and chemotherapy. The percentage of cell viability was determined and a graph was plotted with concentration vs. percentage cell viability (Fig. 11a). The cytotoxic effect of the nanocomposite is found to increase with increase in concentration, resulting in a direct dose-response relationship when plotted with concentration vs. percentage inhibition (Fig. 11b). A maximum of 95.56 % inhibition is observed at 100 µg mL-1 and the IC50 value is found to be 29.35 µg mL-1. This pronounced anticancer effect with minimal IC50 value is due to the synergistic effect of nano-silver and chitosan. Studies show that the anticancer effect of silver nanoparticles against A549 cells results with an IC50 value of 100 µg mL-1 and inhibition of about 85% at 500 µg mL-1 [53]. Chitosan also has significant anticancer effects against A549 cells with 20.28 % cell viability exerted at 500 µg mL -1 [54]. Therefore, the excellent anticancer effect of CS-Ag nanocomposite is due to the synergistic action of chitosan and silver in the synthesized nanocomposite with comparatively lesser IC 50 value and maximal inhibition than the individual counterparts.

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The plausible mechanism for the anticancer effect of synthesized nanocomposite is mainly due to the formation of reactive oxygen species or increase in intracellular oxidative stress, triggering apoptosis and necrosis. Chitosan and silver nanoparticles have antiangiogenic properties [55,56], which is known for their potential ability in blocking the activity of abnormally expressed signaling proteins. Oxidative stress, a phenomenon that is often considered as an epicentre for the induction of cytotoxicity, is induced by cytotoxic agents and this toxicity is exerted by transferring electron from molecular oxygen or by blockade of electron transport chain via an unknown mechanism [57]. This interprets that the synthesized nanocomposite exhibits excellent anticancer property which can possibly be used as a chemotherapeutic agent in nanomedicine. 4. Conclusion Chitosan, a natural polysaccharide has been widely used for various biological applications due to their prospective benefits. Incorporation of nanomaterials for medical applications is currently an emerging field to overcome the drawbacks of the existing therapies. The present study describes the facile synthesis and characterization of CS-Ag nanocomposites. The absorption spectra resulted with an SPR peak at 420 nm, which confirmed the reduction of Ag using chitosan. FTIR analysis confirmed the presence of chitosan and Ag in the nanocomposite. The XRD pattern of nanocomposites affirmed the face centred cubic structure, and the same diffraction pattern was observed in SAED analysis. The particle size of 20 nm was obtained by AFM technique and the spherical AgNps presented over the chitosan matrix was confirmed by HR-TEM techniques. The elemental composition of CS-Ag nanocomposite confirms the major elements carbon, oxygen, nitrogen and silver by XPS and EDX results. Surface analysis leads to higher surface area and positive charge of CSAg nanocomposite. The biosynthesized CS-Ag nanocomposites possess remarkable antibacterial activities against standard and clinical pathogens, amongst which Salmonella sp. shows higher antimicrobial activity when compared with other pathogens. The MIC (8 µg mL1

) and MBC (16 µg mL-1) of the nanocomposite were also comparatively lower for Salmonella

sp., a Gram-negative organism, although the antibacterial effect seems to be fairly balanced for both Gram-positive and Gram-negative organisms. CS-Ag nanocomposites proved effective than the standard antibiotic in the case of Gram-positive Streptococcus sp. The antibacterial property of the synthesized nanocomposites can further be exploited in the synergistic action of antibiotics targeting various bacterial infections, paving way for future therapeutics in nanomedicine. The anticancer activity of the synthesized CS-Ag nanocomposite was also studied with A549 lung cancer cell line. A significant IC50 value of

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29.35 µg mL-1 was obtained. The synergistic effect of chitosan and silver as a composite in nanometric size exhibited a maximum of 95.56 % inhibition at 100 µg mL-1, resulting in potent anticancer activity. Note This paper is dedicated to the memory of our mentor Prof. P. Natarajan, INSA senior scientist, NCUFP, University of Madras, Chennai. Acknowledgement The author K.J. acknowledges the Department of Science and Technology, Government of India for the financial support (Sanction No. SR/FT/CS-042/2008). The author K.R. gratefully acknowledges the Department of Science and Technology, Government of India sponsored National Facility for Drug Development (NFDD) for Academia, Pharmaceutical and Allied industries

(VI-D&P/349/10-11/TDT/1,

Dt.21.10.2010),

Bharathidasan

Institute

of

Technology, Anna University, Tiruchirappalli for its valuable and sophisticated facilities to carry out the present study. References [1] A.D. Lopez, C.C.J.L. Murray, The Global Burden Of Disease, 1990–2020, Nat. Med. 4 (1998) 1241-1243. [2] A.P. Magiorakos, A. Srinivasan, R.B. Carey, Y. Carmeli, M.E. Falagas, C.G. Giske, S. Harbarth, J. F. Hindler, G. Kahlmeter, B. Olsson-Liljequist, D.L. Paterson, L.B. Rice, J. Stelling, M.J. Struelens, A. Vatopoulos, J.T. Weber, D.L. Monnet, Multidrug-Resistant, Extensively Drug-Resistant And Pandrug-Resistant Bacteria: An International Expert Proposal For Interim Standard Definitions For Acquired Resistance, Clin Microbiol Infect. 18 (2012) 268–281. [3] T. Marudhupandi, T.T. Ajith Kumar, S. Lakshmanasenthil, G. Suja, T. Vinothkumar, In vitro anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines, Int. J. Biol. Mac. 72 (2015) 919–923. [4] S. Heinavaara, T. Hakulinen, Predicting the lung cancer burden: Accounting for selection of the patients with respect to general population mortality, Statist. Med. 25 (2006) 2967– 2980.

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Figure captions 1. Fig. 1. UV-Visible absorption spectra of CS-Ag nanocomposites synthesized at varying temperatures (a) room temperature, (b) 90 ⁰C and (c) 120 ⁰C. 2. Fig. 2. (a) XRD pattern and (b) FTIR spectra of biosynthesized CS-Ag nanocomposites at varied temperatures. 3. Fig. 3. (a) N2 adsorption-desorption isotherm and (b) Zeta potential analysis of CSAg nanocomposite. 4. Fig. 4. (a) SEM image, (b) EDX spectra, (c) AFM 2D image, (d) Particle size distribution by AFM image, (e) HR-TEM image, (f) Particle size distribution by HR-TEM image and (g) SAED pattern of the biosynthesized CS-Ag nanocomposite. 5. Fig. 5. XPS spectra of the biosynthesized CS-Ag nanocomposite, (a) C 1s spectra, (b) O 1s spectra, (c) N 1s spectra and (d) Ag 3d spectra,. 6. Fig. 6. Antimicrobial activity of CS-Ag nanocomposites against standard strains: (a) Gram-positive bacterium Staphylococcus aureus MTCC 3160 and (b) Gramnegative bacterium Pseudomonas aeruginosa MTCC 1688 – (A) Streptomycin 10 µg, (B) Deionized water 100 µL, CS-Ag nanocomposites 100 µg synthesized at (C) room temperature, (D) 90 ⁰C and (E) 120 ⁰C, (--) empty well. 7. Fig. 7. Antimicrobial activity of CS-Ag nanocomposites against Gram-positive clinical pathogens: (a) Staphylococcus sp. (b) Streptococcus sp. and (c) Enterococcus sp. – (A) Streptomycin 10 µg, (B) Deionized water 100 µL, CS-Ag nanocomposites 100 µg synthesized at (C) room temperature, (D) 90 ⁰C and (E) 120 ⁰C, (--) empty well. 8. Fig. 8. Antimicrobial activity of CS-Ag nanocomposites against Gram-negative clinical pathogens: (a) Salmonella sp. and (b) Shigella sp.– (A) Streptomycin 10 µg, (B) Deionized water 100 µL, CS-Ag nanocomposites 100 µg synthesized at (C) room temperature, (D) 90 ⁰C and (E) 120 ⁰C. 9. Fig. 9. Pictorial bar representation of the antibacterial efficacy of CS-Ag nanocomposite on various clinical pathogens. 10. Fig. 10. Morphological assessment of the anticancer effect of CS-Ag nanocomposite against A549 lung cancer cell line after 24 h incubation visualized under an inverted phase-contrast microscope at 40 X magnification (a) Control

22

(untreated) cells (b) Positive control epirubicin (10 µg mL-1) treated cells (c) CSAg nanocomposite (100 µg mL-1) treated cells. 11. Fig. 11. (a) In vitro cytotoxic effect of biosynthesized CS-Ag nanocomposite against A549 cells at various concentrations by cell viability (MTT) assay and (b) Dose-response effect of CS-Ag nanocomposite with concentration vs. inhibition (Mean ± SD., n=3).

Table captions 1.

Table 1. Measurement of zone of inhibition (mm) and t-test analysis of CS-Ag nanocomposite against bacterial pathogens.

2.

Table 2.

MIC and MBC of CS-Ag nanocomposite (µg mL-1) against various

microorganisms.

23

Fig. 1. UV-Visible absorption spectra of CS-Ag nanocomposites synthesized at varying temperatures (a) room temperature, (b) 90 ⁰C and (c) 120 ⁰C.

24

Fig. 2. (a) XRD pattern and (b) FTIR spectra of biosynthesized CS-Ag nanocomposites at varied temperatures.

25

Fig. 3. (a) N2 adsorption-desorption isotherm and (b) Zeta potential analysis of CS-Ag nanocomposite.

26

27

28

Fig. 4. (a) SEM image, (b) EDX spectra, (c) AFM 2D image, (d) Particle size distribution by AFM image, (e) HR-TEM image, (f) Particle size distribution by HR-TEM image and (g) SAED pattern of the biosynthesized CS-Ag nanocomposite.

29

30

Fig. 5. XPS spectra of the biosynthesized CS-Ag nanocomposite, (a) C 1s spectra, (b) O 1s spectra, (c) N 1s spectra and (d) Ag 3d spectra,.

31

Fig. 6. Antimicrobial activity of CS-Ag nanocomposites against standard strains: (a) Gram-positive bacterium Staphylococcus aureus MTCC 3160 and (b) Gram-negative bacterium Pseudomonas aeruginosa MTCC 1688 – (A) Streptomycin 10 µg, (B) Deionized water 100 µL, CS-Ag nanocomposites 100 µg synthesized at (C) room temperature, (D) 90 ⁰C and (E) 120 ⁰C, (--) empty well.

32

Fig. 7. Antimicrobial activity of CS-Ag nanocomposites against Gram-positive clinical pathogens: (a) Staphylococcus sp. (b) Streptococcus sp. and (c) Enterococcus sp. – (A) Streptomycin 10 µg, (B) Deionized water 100 µL, CS-Ag nanocomposites 100 µg synthesized at (C) room temperature, (D) 90 ⁰C and (E) 120 ⁰C, (--) empty well.

33

Fig. 8. Antimicrobial activity of CS-Ag nanocomposites against Gram-negative clinical pathogens: (a) Salmonella sp. and (b) Shigella sp.– (A) Streptomycin 10 µg, (B) Deionized water 100 µL, CS-Ag nanocomposites 100 µg synthesized at (C) room temperature, (D) 90 ⁰C and (E) 120 ⁰C.

34

18

Zone of Inhibition (mm)

16 14 12 10 8 6 4 2

St ap hy lo co Ps cc eu us do au m re on us as ae ru gi St no ap sa hy lo co cc us St sp re pt oc oc cu ss En p te ro co cc us sp Sa lm on el la sp Sh ig el la sp

0

Fig. 9. Pictorial bar representation of the antibacterial efficacy of CS-Ag nanocomposite on various clinical pathogens.

35

Fig. 10. Morphological assessment of the anticancer effect of CS-Ag nanocomposite against A549 lung cancer cell line after 24 h incubation visualized under an inverted phase-contrast microscope at 40 X magnification (a) Control (untreated) cells (b) Positive control epirubicin (10 µg mL -1) treated cells (c) CS-Ag nanocomposite (100 µg mL-1) treated cells.

36

Fig. 11. (a) In vitro cytotoxic effect of biosynthesized CS-Ag nanocomposite against A549 cells at various concentrations by cell viability (MTT) assay and (b) Dose-response effect of CS-Ag nanocomposite with concentration vs. inhibition (Mean ± SD., n=3).

37

Table 1 Measurement of zone of inhibition (mm) and t-test analysis of CS-Ag nanocomposite against bacterial pathogens. Zone of inhibition (mm) Streptomycin 10 µg

CS-Ag 100 µg

Microorganisms Std. t-test

Mean

Deviation

Std. t-test

Mean

Deviation

Staphylococcus aureus

49.000

16.333

0.577

53.000

17.667

0.577

Pseudomonas aeruginosa

15.588

18.000

2.000

18.520

16.333

1.528

Staphylococcus sp.

16.000

10.667

1.155

22.517

13.000

1.000

Streptococcus sp.

00.000

00.000

0.000

29.000

09.667

0.577

Enterococcus sp.

19.654

17.333

1.528

31.000

10.333

0.577

Salmonella sp.

23.000

15.333

1.155

32.909

19.000

1.000

Shigella sp.

37.000

12.333

0.577

38.000

12.667

0.577

38

Table 2 MIC and MBC of CS-Ag nanocomposite (µg mL-1) against various microorganisms CS-Ag nanocomposite (µg mL-1) Microorganisms

MIC

MBC

Staphylococcus aureus MTCC 3160

16

32

Pseudomonas aeruginosa MTCC 1688

32

64

Staphylococcus sp.

64

128

Streptococcus sp.

128

256

Enterococcus sp.

128

256

Salmonella sp.

8

16

Shigella sp.

64

128

39