Designing chitosan–silver nanoparticles–graphene oxide nanohybrids with enhanced antibacterial activity against Staphylococcus aureus

Accepted Manuscript Title: Designing chitosan–silver nanoparticles-graphene oxide nanohybrids with enhanced antibacterial activity against Staphylococcus aureus Author: Bogdan Marta Monica Potara Maria Iliut Endre Jakab Teodora Radu Florica Imre Lucaci Gabriel Katona Octavian Popescu Simion Astilean PII: DOI: Reference:

S0927-7757(15)30234-X http://dx.doi.org/doi:10.1016/j.colsurfa.2015.09.046 COLSUA 20182

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

17-6-2015 9-9-2015 16-9-2015

Please cite this article as: Bogdan Marta, Monica Potara, Maria Iliut, Endre Jakab, Teodora Radu, Florica Imre Lucaci, Gabriel Katona, Octavian Popescu, Simion Astilean, Designing chitosanndashsilver nanoparticles-graphene oxide nanohybrids with enhanced antibacterial activity against Staphylococcus aureus, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2015.09.046 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.

Designing chitosan-silver nanoparticles-graphene oxide nanohybrids with enhanced antibacterial activity against Staphylococcus aureus

Bogdan Martaa*, Monica Potaraa*, Maria Iliuta,b, Endre Jakabc,d, Teodora Radue,f, Florica ImreLucacig, Gabriel Katonah, Octavian Popescuc,i, Simion Astileana#

a

Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute in BioNano-Sciences and Faculty of Physics, Babes-Bolyai University, M. Kogalniceanu Str 1, 400084,ClujNapoca, Romania b School of Materials and National Graphene Institute, The University of Manchester, Oxford Road, Manchester, M13 9PL, UK c Molecular Biology Center, Interdisciplinary Research Institute in Bio-Nano-Sciences and Faculty of Biology, Babes-Bolyai University, M. Kogalniceanu Str 1, 400084,Cluj-Napoca, Romania d Hungarian Department of Biology and Ecology, Faculty of Biology and Geology, Babes-Bolyai University, Clinicilor 5-7, 400006 Cluj-Napoca, Romania e Nanostructured Materials and Bio-Nano-Interfaces Center, Interdisciplinary Research Institute in BioNano-Sciences and Faculty of Physics, Babes-Bolyai University, M. Kogalniceanu Str 1, 400084,ClujNapoca, Romania f National Institute for Research and Development of Isotopic and Molecular Technologies, 67-103 Donat Street, 400293 Cluj-Napoca, Romania g Physico-Chemical Analysis Center, Interdisciplinary Research Institute in Bio-Nano-Sciences, BabesBolyai University, M. Kogalniceanu Str 1, 400084,Cluj-Napoca, Romania h Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University,11 Arany Janos, RO-400027 Cluj-Napoca, Romania i Institute of Biology, Romanian Academy, Spl. Independentei 296, 060031 Bucharest, Romania

# Corresponding author. Tel.: +40 264 454554x115; fax: +40 264 591906. E-mail address: [email protected] (Prof. Dr. S. Astilean). * These authors contributed equally to the work

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Graphical abstract

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Highlights  Fabrication of silver nanoparticles-graphene oxide nanohybrids  Fabrication of chitosan-silver nanoparticles-graphene oxide nanohybrids  Testing of antibacterial effects of nanohybrids against Staphylococcus aureus  Optimizing the composition of nanohybrids to enhance the antibacterial effect Abstract Designing hybrid nanomaterials that exhibit multiple mechanisms of antibacterial action provides a new paradigm in the fight against resistant bacteria. Herein, we present such a new hybrid nanomaterial which integrates the antibacterial and physico-chemical properties of silver nanoparticles, graphene oxide and chitosan biopolymer. The formation, stability and structure of the integrated three-component chitosan-silver nanoparticles-graphene oxide (chitAgNPs-GO) nanomaterial is

analyzed by UV-Vis extinction

spectroscopy,

transmission

electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and zeta potential measurements. The antibacterial activity is evaluated against two representative methicillinresistant Staphylococcus aureus (MRSA) strains (UCLA 8076 and 1190R) and relative to individual components (GO, chit-GO, AgNPs-GO) by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The three-component nanocomposites (chit-AgNPs-GO) exhibit higher antibacterial activity than most of the antibacterial agents based on AgNPs or AgNPs-GO reported so far and, more interestingly, their activity can be controlled by the ratio between the amount of chitosan and AgNPs-GO. The results presented in this study demonstrate the promising potential of the chit-AgNps-GO hybrids as effective nanomaterial to fight against the bacterial infections. Keywords: Graphene oxide; Silver nanoparticles; Chitosan; Nanohybrids; Antibacterial activity

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Introduction The emergence of drug-resistant bacterial strains has put medicine in great difficulty as some of them are harmful with potential life-threatening effects. This is the case of Staphylococcus aureus (S. aureus), a bacterium usually found on skin. Many people are long term carriers of S. aureus without any side effects. The skin acts as a barrier preventing the bacterium to enter the body, but in some cases the Staphylococcus manages to enter the body, causing medical issues ranging from light infections such as pimples, abscesses or furuncles, to serious conditions like sepsis, meningitis or bacteremia, with life-threatening potential [1]. Due to the interaction with many antibiotics, over time, the bacterium has developed new strains which show increased resistance to antibiotics and usual treatment methods. Such strains are the methicillin resistant S. aureus (MRSA), which usually develop in hospitals and show a broad resistance against many antibiotics from the penicillin family. There are still antibiotics that work against these particular bacterial strains such as tetracyclines or Vancomycin, but there is an acute need to develop new drugs or treatment methods which can attack bacteria through different mechanisms or render it unable to resist the treatment. Much effort and investment has been made to develop new drugs and treatment methods against these kinds of bacteria. The antibacterial effect of noble metals such as silver is well known and documented throughout the history [2]. Silver nanoparticles (AgNPs) exhibit a great promise for biomedical applications due to their unique potential to combine the intrinsic antibacterial effects with multiple other functionalities [2-4]. As for instance AgNPs were explored as drug carriers, probes, or even investigated in photothermal and photodynamical therapies [5-8]. Moreover, when combined with other antibacterial agents, they show a good synergistic antibacterial effect and enhanced properties [9,10]. As many as the advantages might be seen, there are still some problems and side effects that need to be addressed when taking AgNPs into consideration for implementing treatment methods. As AgNPs are usually prepared as colloidal solutions stabilized through electrostatic repulsion, when transferred into biological media, they could become unstable, leading to aggregation and decreased antibacterial efficiency. In this regard, a practical approach is to anchor them on films or support matrices which not only can increase the stability but enhance their antibacterial properties [11, 12]. Such an appropriate material is graphene oxide (GO) produced in solution by chemical exfoliation of graphite. Upon exfoliation, hydrophilic groups such as hydroxyl, carbonyl, and epoxy groups are 4

introduced onto the surface to make GO sheets soluble in water. This represents the most suitable method for easy preparation and at low cost of graphene in the form of reduced graphene oxide (r-GO). Since its discovery, graphene, a nanomaterial consisting from 1 to a few layers of carbon atoms, densely packed in a regular honeycomb structure with sp2 bonds between carbon atoms, has attracted the attention in many fields of expertise due to its extraordinary properties. Such properties are high electron mobility, good heat transfer, transparency, high shear strength, which make graphene viable for various applications in the fields of optics, electronics, material reinforcement, etc. [13,14]. In particular, GO has found its way into various medical applications including drug delivery or photo-thermal therapy [15,16]. Recent studies have also proven the antibacterial effects of GO, making it a somehow unusual antibacterial agent [17]. Furthermore, a number of reports have demonstrated lately that graphene–AgNPs hybrid structures can act synergistically to offer a number of unique physicochemical properties leading to stable nanocomposites with new and enhanced antibacterial properties [18-20]. To further increase the stability of AgNPs, they can be coated with a shell of biocompatible polymers. Chitosan is a good example of biocompatible polymer which favors coating and stabilization of AgNPs, and more interestingly, exhibits a strong bactericidal and homeostatic effect by itself [21,22]. Chitosan is obtained through the deacetylation of chitin, a natural occurring polymer, usually found in the exoskeleton of arthropods or in the cell walls of yeast and some fungi. Its main function is to reinforce and strengthen these structures. Chitosan has a lot of applications in the field of medicine including drug and gene delivery, tissue engineering, wound healing etc. [23,24]. An enhanced antibacterial effect and stability has been previously reported by our group for AgNPs-chitosan nanocomposites against two strains of MRSA [10]. Taking into account the properties of the three fore discussed materials, the present study is focused on investigating the antibacterial effect of chitosan-silver nanoparticles- graphene oxide nanohybrids (chit-AgNPs-GO). Our findings demonstrate a clear synergy between the three components in chit-AgNPs-GO nanocomposite, expressed by better bacteriostatic and bactericidal effect against two MRSA strains than any component alone or any two of them together.

2. Experimental 2.1. Materials 5

Chitosan flakes (310 to 375 kDa molecular weight, > 75% deacetylated), silver nitrate (AgNO3), potassium permanganate (KMnO4) 99% and sodium nitrate (NaNO3) 99% were purchased from Sigma-Aldrich. Graphite powder, 325 mesh, 99.9995% was supplied by Alfa Aesar. Hydrochloric acid (HCl) 35% was purchased from Lach-Ner. Sodium borohydride (NaBH4) was supplied by Merck. Sulfuric acid (H2SO4) 96%, hydrogen peroxide (H2O2) 30% and glacial acetic acid (CH3COOH) 99,8% were obtained from local manufacturers. Glacial acetic acid was diluted to a 1% aqueous solution before use. Chitosan was dissolved in 1% acetic acid solution. All chemicals were used without further purification. All reagents employed were of analytical grade and the solutions were prepared using ultrapure water with a resistivity of at least 18 MΩ·cm. All glassware used was cleaned with aqua regia solution (HCl:HNO3 3:1) followed by rinsing with ultrapure water.

2.2. Preparation of GO sheets Graphene oxide (GO) sheets were prepared through oxidation/exfoliation of graphite according to Hummers and Offeman’s method [25]. Briefly, graphite powder (1 g) was mixed with concentrated H2SO4 (25 ml) and vigorously stirred at room temperature for 30 min. Subsequently the mixture was cooled in an ice bath and NaNO3 (0.5 g) was added, followed by the slow addition of KMnO4 (3g) under continuous stirring. The ice bath was then removed, and the mixture was warmed at 35o C and stirred continuously for another 2 hours. The mixture gradually thickened becoming pasty and brownish grey in color. To this paste, bi-distilled water (46 ml) was added slowly under continuous stirring. The diluted solution was then stirred for another hour, keeping a constant temperature of 35o C. The suspension was further diluted by adding 140 ml of warm, distilled water and treated with 10 ml of H2O2 to reduce residual permanganate and manganese dioxide to manganese sulfate. After another 20 minutes of stirring, the resulted bright yellow mixture was filtered, and the filter cake was washed with a solution of 5% HCl (300 ml) and warm water 6-8 times. The obtained graphite oxide was further exfoliated by sonication in distilled water for 2 hours followed by centrifugation at 4000 rpm for 30 min. to remove unexfoliated graphite oxide. The colloid was then kept in an ultrasonic bath for 4 hours in order to exfoliate stacking graphene oxide sheets. The obtained GO sample was centrifuged 3 times at 18000 rpm for 30 min, each time removing the sediment and re-centrifuging the supernatant, in order to eliminate un-exfoliated graphite layers and large graphene flakes. The 6

supernatant obtained after the third centrifugation step (denoted GO) was kept and used for the preparation of the AgNPs-GO nanohybrids.

2.3. Preparation of AgNPs-GO and chit-AgNPs-GO nanohybrids In a typical procedure for the preparation of the AgNPs-GO nanohybrids, 5 ml GO solution at a concentration of 1mg/ml was mixed with 0.0034 g AgNO3 for 30 minutes, followed by the addition of 15 ml bi-distilled water. To this mixture, an aqueous solution of NaBH4 (5 ml, 3.35 x 10-3 M) was added dropwise at a speed of 1 ml/minute and stirred continuously for 4 hours at room temperature. The color of the solution slowly turned black-dark yellow indicating the reduction of silver ions and the formation of the AgNPs. The synthesized AgNPs-GO composites were purified with bi-distilled water by centrifugation at 18000 rpm for 30 minutes. The chitosan biopolymer was grafted onto the AgNPs-GO nanohybrids sheets through a self-assembly method. In brief, 2 ml of AgNPs-GO colloidal solution was stirred at room temperature. The desired amount of chitosan solution (0.25, 0.5 or 1 ml) was then quickly added to the AgNPs-GO solution under vigorous magnetic stirring, thus avoiding the aggregation of chit-AgNPs-GO nanohybrids. The final solution was stirred for 4 hours at room temperature to ensure that the system had reached the equilibrium. The obtained chit-AgNPs-GO nanohybrids were centrifuged at 18000 rpm for 30 minutes and re-suspended in bi-distilled water to remove any unused reactants. For determination of the antibacterial activity the samples were purified by centrifugation and re-suspended in bi-distilled water to remove any unused reactants.

2.4. Characterization UV-vis-NIR extinction spectra were recorded with a Jasco V-670 spectrophotometer, over a range between 190 and 1000 nm. The concentration of GO was determined based on BeerLambert law using their absorbance at 230 nm and an extinction coefficient of 65 µg/ml·cm [26]. The morphology of the AgNPs-GO nanohybrids was examined using a using a HITACHI, H7650 transmission electron microscope (TEM). TEM images of chit-AgNPs-GO nanohybrids were obtained with JOEL- JEM 1100 microscope. The samples used for TEM measurements were prepared by placing a drop of colloidal dispersion onto carbon-coated copper grids. Samples were left to dry at room temperature. The zeta potential of the prepared colloidal 7

samples was measured at a temperature of 25 ºC by laser Doppler micro-electrophoresis technique using a Malvern Zetasizer Nano ZS-90. The Nano ZS contains a He-Ne laser operating at a wavelength of 633 nm and an avalanche photodiode detector.The concentration of the silver was determined by atomic absorption spectroscopy (Avanta PM, GBC- Australia). The analysis was performed using Ag atomic spectroscopy standard 1000 mg/L acidic solution (AgNO3 in 0.5 M HNO3) from LGC Standards GmbH (Wesel, Germany). Each sample was measured in triplicate. XPS measurements were performed with a SPECS PHOIBOS 150 MCD instrument, equipped with monochromatized Al Kα radiation (1486.69 eV) at 14 kV and 20 mA, and a pressure lower than 10-9 mbar. A low energy electron flood gun was used for all measurements to minimize sample charging. High-resolution spectra of the elements of interest were recorded in steps of 0.05 eV using analyser pass energy of 30 eV. The spectra deconvolution was accomplished with Casa XPS (Casa Software Ltd., UK). XPS spectra are, for the most part, quantified in terms of peak intensities and peak positions. The peak intensities measure how much of a material is at the surface, while the peak positions indicate the elemental and chemical composition. Other values, such as the full width at half maximum (FWHM) are useful indicators of chemical state changes and physical influences. Broadening of a peak may indicate a change in the number of chemical bonds contributing to a peak shape. The spectra deconvolution was accomplished with Casa XPS (Casa Software Ltd., UK).

2.5. Bacterial strains and culture conditions Two methicillin-resistant Staphylococcus aureus (MRSA) strains were used in this study, UCLA 8076, originating from the University of California, Los Angeles, and 1190 R, obtained from the Semmelweis University, Budapest. Cultures were maintained on Mueller Hinton agar (Fluka, Buchs, Switzerland). The bacteria were cultured overnight in 5 ml Mueller Hinton broth (Fluka, Buchs, Swizerland) in a Certomat BS-T incubation shaker (Sartorius Stedim Biotech, Aubagne, France) at 37 °C, 150 rpm until the culture reached an OD600 of 1.0 (Spekol UV VIS 3.02, Analytic Jena, Jena, Germany), corresponding to 109 CFU ml-1. Prior to incubation with colloidal suspensions the cultures were diluted to 107 CFU ml-1 with sterile broth. 2.6. Antibacterial activity tests The plates used in the microdilution experiments were prepared by mixing different quantities of cation-adjusted Mueller Hinton broth supplemented with 2% NaCl (from 45 to 90 μl, in ten steps 8

of 5 μl) with different quantities of the antibacterial agent (from 50 to 5 μl, in ten steps of 5 μl) in order to achieve a final volume of 95 μl in all samples. The wells were inoculated with 5µl bacterial suspension of 107 CFU ml-1. The final bacteria concentration was 105 CFU ml-1. We tested six antibacterial agents (GO, chit-GO, AgNPs-GO and chit-AgNPs-GO with different ratio of chitosan to AgNPs-GO) for their bacteriostatic and bactericidal effects. Two control samples were also prepared, the negative control, containing sterile growth medium only, and the positive control containing 5µl bacteria suspension and 95 µl sterile broth. The inoculated microdilution plates were covered with a plastic lid and incubated at 37 °C for 24 h. Additionally, to exclude the role of free chitosan and silver ions fraction of AgNPs suspensions on the measured antibacterial effect, samples containing aqueous solutions of supernatant collected from purified chit-AgNPs-GO hybrids (1:4) instead of colloidal solutions were prepared in similar way. Bacterial growth was evaluated by measuring optical density (OD) at 600 nm and the minimum inhibitory concentration (MIC) was recorded as the lowest concentration that completely inhibits bacterial growth. For evaluation of the minimum bactericidal concentration (MBC), 100 µl aliquots were taken from wells without any detectable bacterial growth and plated onto Mueller Hinton agar plates. After incubation, colony forming units (CFUs), corresponding to the number of surviving cells, were counted and digital images of the plates were captured. The MBC was determined as the lowest concentration of the active agent that inhibits colony formation.

3. Results and discussion 3.1. Characterization of chit-AgNPs-GO nanohybrids The formation of colloidal chit-AgNPs-GO nanohybrids was monitored by combining the UVVis extinction spectroscopy, TEM images, zeta potential and XPS measurements. Figure 1 illustrates the UV-Vis extinction spectra of GO, AgNPs-GO and chit-AgNPs-GO colloidal suspensions. As shown in Figure 1a (green curve), the optical extinction spectrum of GO solution exhibits two characteristic peaks which are similar to those already reported in literature for GO [27]. The band located at 230 nm is assigned to the π-π* transitions of C=C bonds, and the shoulder band situated at about 300 nm is ascribed to the n–π* transitions of C=O bonds [27]. The extinction spectrum depicted in Figure 1b (black curve) features the typical plasmon resonance at 430 nm which indicates the presence of spherical silver nanoparticles in the 9

nanocompsite AgNPs-GO [28]. Figure 1c (red curve) illustrates the extinction spectrum of chitAgNPs-GO nanocomposites recorded after 4 hour of incubation at room temperature. We noticed that the initial extinction spectrum of AgNPs-GO solution was fully recovered which rule out any possible aggregation of the AgNPs-GO nanocomposites during the formation of chit-AgNPs-GO nanohybrids. However, a 6 nm blue shift accompanied with a narrowing of the plasmon resonance of AgNPs was observed after the addition of chitosan, which is most probably due to an etching process which leads to a small decrease of the size. The morphology of the AgNPs-GO and chit-AgNPs-GO nanohybrids was investigated by TEM measurements. As can be seen in Figure 2A spherical Ag nanoparticles with a diameter ranging from 10 to 30 nm are anchored onto the GO surface and no particles are observed outside the GO sheets. As evidenced from Figure 2B after biopolymer grafting AgNPs remain segregated onto the GO surface which is consistent with UV-Vis measurements. Adsorption of chitosan onto the surface of the AgNPs-GO nanohybrids was further demonstrated by zeta potential measurements. While AgNPs-GO have a negative surface charge of – 39.7 mV, it switches to positive (+ 39.8 mV) after the addition of biopolymer (the ratio of chitosan to AgNPs-rGO = 1:8) due to the protonated amino groups (NH3)+ of chitosan polymeric coating. Increasing the amount of chitosan (the ratio of chitosan to AgNPs-GO = 1:4 and 1:2) increases also the positive surface charge to + 44.1 and + 63.6 mV, respectively. The above result indicate that the chitosan biopolymer was attached onto the AgNPs-GO hybrid nanosheets through electrostatic attraction between positively charged chain of biopolymer and negatively charged AgNPs-GO sheets with some contribution from hydrogen bonding. Actually –NH2 groups in each molecular unit of polymer can be protonated which makes chitosan a polycationic material in acidic media. On the other side, AgNPs-GO nanohybrids have a strong negative surface charge of – 39,7 mV due to the ionization of carboxylic acid and phenolic hydroxyl groups onto the GO sheets [29]. The obtained chit-AgNP-GO nanohybrids exhibit high stability, confirmed by zeta potential measurements (> + 30 mV), due to the strong electrostatic repulsion between the hybrid GO flakes [30]. The high stability of our chit-AgNPs-GO nanohybrids is in good agreement with other observations which reported that chitosan biopolymer enhances the strength and dispersibility of GO sheets [29, 31]. Furthermore, X-ray photoelectron spectroscopy (XPS) was also performed to further analysis the elemental composition as well as the chemical bonds in the sample. The C 1s 10

spectrum reveals a component at around 284.7 eV in all three samples attributed to the sp2 bound carbon in graphene (C=C). The peak occurring at 285.8 eV in the GO, 285.4 eV in the AgNPsGO, and 285.5 eV in chit-AgNPs-GO sample can be attributed to C-O bonds. There is another peak that appears at 288.1, 288 and 288.5 eV in GO, AgNPs-GO and chit-AgNPs-GO respectively, which represents the C=O component. The core-level deconvolution for C1s reveals a significant decrease in the percentage of C-O and C=O bonds in the chit-AgNPs-GO sample in report to GO and AgNPs-GO, accompanied by an increase in percentage of C=C bonds in report with the fore mentioned samples. The XPS spectra also reveal the presence of chitosan on the surface of the chit-Ag-GO nanohybrids. Indeed, the deconvolution of the carbon core-level envelope for carbon reveals a new component attributed to N-C=O at 286.3 eV with a percentage of 8.6% (Table 1) which is not present in the case of GO and AgNPs-GO samples and supports the conclusion that chitosan in the chit-Ag-GO nanohybrids binds on the graphene nanosheets.

3.2. The antibacterial activity of the prepared chit-AgNPs-GO nanohybrids against MRSA Two representative strains of MRSA (UCLA8076 and 1190R) were selected for antibacterial tests because they usually lead to misidentification and cause therapeutic problems [32].The differences between these two strains reside in their cells composition. For example, 1190R strain is composed by a single population of cells which expresses a highly uniform resistance, and therefore, can grow in high concentrations of drug. In contrast, UCLA 8076 strain is composed by several subpopulations with a high proportion of cells (typically 99.9 % or more) that is susceptible to low concentration of drug, and a small proportion of cells (e.g. 1 in 106) that exhibits high resistance to methicillin. MRSA cells (107 CFU ml-1) were incubated at 37 ºC for 24 h with the same concentrations of GO, chit-GO, AgNPs-GO and chit-AgNPs-GO. The loss of viability of MRSA cells was evaluated using the minimal inhibitory (MIC) and minimum bactericidal concentrations (MBC) which are the standard microbiological measures to evaluate the bacteriostatic and bactericidal properties of antimicrobial agents. To ensure the reproducibility of the results, each experiment was conducted in duplicate and was repeated after several days. Table 2 presents the mean MIC values obtained for each antibacterial agent tested and the MBC values of chit-AgNPs-GO nanohybrids prepared with the optimal chitosan concentration. As shown in Table 2 significant differences were found in their bacteriostatic 11

activities among the materials tested. For example, in the case of GO dispersion there is no obvious inhibition of the bacterial growth even at the highest concentration tested (7.5 µg/ml). This result is in good agreement with other reported observations which point out the weak and controversial antimicrobial effect of GO and the fact that the degree of oxidation and the size of the GO flakes play a decisive role onto their antibacterial activities [17,18]. On the contrary, AgNPs-GO nanohybrids exhibit a significant antibacterial activity and inhibit the bacterial growth in a concentration dependent manner after 24 h of incubation. The MIC value obtained for AgNPs-GO nanohybrids (1.90 µg/ml Ag and 1.5 µg/ml GO) is lower than those observed previously [18]. However, we have previously demonstrated a smaller MIC value in the case of chitosan-coated AgNPs as result of synergistic activity of chitosan and AgNPs [10]. Based on this result, here we explore the potential of increasing the antibacterial effect of AgNPs-GO nanohybrids by coating them with chitosan. Therefore, the antibacterial activity of new hybrid nanocomposites called chitosan-coated AgNPs-GO (chit-AgNPs-GO) with different ratios between the amount of chitosan and AgNPs-GO were systematically investigated. As shown in Table 2, chit-AgNPs-GO nanohybrids (the ratio of chitosan to AgNPs-GO = 1:8) exhibit an increased bacteriostatic effect (MIC 1.19 µg/ml Ag and 1.41 µg/ml GO) as compared with AgNPs-GO nanohybrids. This enhanced effect can be partially attributed to the surface charge modification of the AgNPs-GO nanocomposites by chitosan as confirmed by their positive zeta potential (+ 39.8 mV relative to – 39.7 mV for AgNPs-GO), which makes the nanohybrids more likely to bind to the negatively charged membrane of the bacterium cells. In addition, chitosan itself possesses antibacterial activity against Gram-negative and Gram-positive bacteria [21]. It was found that chitosan as a polycation could either interact with anionic groups on the cell surface leading to increased permeability [33] or chelate trace elements or nutrients essential for enzyme activity [21]. Moreover, it was previously reported that the interaction between chitosan and AgNPs facilitates the release of silver ions from AgNPs which contribute to the antimicrobial effect of AgNPs via silver ion activity [34]. Increasing the amount of chitosan (the ratio of chitosan to AgNPs-GO = 1:4) increases also the antibacterial activity of chit-AgNPs-GO nanohybrids (MIC 1.09 µg/ml Ag and 1.35 µg/ml GO) relative to AgNPs-GO nanohybrids. However, the effect is not linear and higher amount of chitosan decreases considerably the bacteriostatic effect of the nanohybrids. Notably, chit-AgNPs-GO nanohybrids prepared with the ratio of 1:2 (chitosan to AgNPs-GO) exhibit a reduced antibacterial activity (MIC 3.81 µg/ml Ag 12

and 3 µg/ml GO) which is lower not only than the nanohybrids prepared with lower chitosan concentrations but also than the AgNPs-GO nanohybrids prepared without chitosan. To evaluate the possible antibacterial effect of free chitosan and silver ions fraction of AgNPs suspensions the supernatant collected from purified chit-AgNPs-GO hybrids (1:4) was also tested for antimicrobial activity. We found that the obtained MIC value of the prepared chit-AgNPs-GO rule out any contribution of supernatant on the antibacterial effect of chit-AgNPs-GO hybrids (1:4). The above results suggest that the ratio of chitosan to AgNPs-GO plays a decisive role onto the antibacterial activities of chit- AgNPs-GO nanohybrids. Preliminary evidence from the literature suggested that anchoring of chitosan onto GO sheets significantly improved their antibacterial properties [31]. We wonder whether this remarkable enhancement of antibacterial activity is a simple addition of individual contributions from GO, chitosan and AgNPs or it is the synergistic expression of antibacterial activity of new material formed by including an optimal concentration of biopolymer into the composition of nanohybrids. To address this question, MRSA cells were treated for 24 h with chit-GO dispersion at the same concentrations of GO as in the working concentrations of chit- AgNPs-GO nanohybrids. As shown in Table 2, chit-GO dispersion exhibits a weaker bacteriostatic activity (MIC 2.25 µg/ml GO) compared with chitAgNPs-GO nanohybrids. Therefore, the significant antibacterial activity of chit-AgNPs-GO nanohybrids is not due to the enhanced effect of GO after the modification with chitosan, but rather due to the unique physical and chemical characteristics of these nanocomposites which contribute all together in a synergistic manner to provide a unique nanointerface for interacting with MRSA cells through a capturing-killing process. This might explain why the better effect is expressed for a well defined chitosan concentration in the composition to chit-AgNPs-GO nanohibrids. In fact it is rationally to consider that insufficient chitosan onto the surface of AgNPs-GO nanohybrids lowers the affinity on nanohybrids toward cell membrane, whereas high amount of chitosan leads to a thick layer which in turn reduces the activity of AgNPs by altering the diffusion rate of metallic ions through the nanointerface. Next, we evaluated the bactericidal effect of chit-AgNPs-GO nanohybrids prepared with the optimal chitosan concentration. Fig. 4 shows representative images of MRSA cultures in the presence of different concentrations of chit-AgNPs-GO nanohybrids (1:4). The MBC value (3.28 µg/ml Ag and 4.05 µg/ml GO) is much smaller than those reported previously for AgNPs, GO, and chitosan, or any combination between the components [10,18, 31]. In addition, as prepared chit- AgNPs-GO nanohybrids 13

show the same strong bacterostatic and bactericidal effect on both the homogeneous and heterogeneous MRSA strains. The enhanced antimicrobial activity of the nanohybrids might result from a combination of different mechanisms of toxicity exhibited by the individual components. For example, while the chitosan facilitates the adhesion of the nanohybrids onto the lipid, other factors such as liposolubility and cell permeability mechanisms are influenced by the presence of graphene oxides (GO) sheets. Among the three components of the fabricated nanointerfce, AgNPs were proven to exhibit the strongest antibacterial activity [17-20, 31]. The chemical origin of the antibacterial effect of AgNPs is still under debate in literature and it has not been concluded whether the nanoscale or the ionic form of silver dictates on the cytotoxicity. It was reported that the antimicrobial activity of AgNPs results from a combination of their specific properties such as nanoparticle size, stability and surface chemistry [35,36]. For example, smaller particles were found to have a strong antibacterial effect due to their large surface area and high reactivity [35,36]. On the other side, it has been claimed that the toxicity of silver nanoparticles is brought by the chemical release of elemental silver Ag0 atoms from nanoparticle surface followed by successive transformation in Ag++ ions and then in Ag-S- or Ag-O- species which induce the oxidative stress [37,38]. Some other researchers have proposed as mechanism of action the cell membrane disruption or inactivation of osmosis by the presence of AgNP on the surface of bacteria cells [39]. Studies have shown that AgNPs present a more pronounced effect on Gram-negative bacteria than on Gram-positive ones, owing to the thick peptidoglycan layer in the latter [38,40]. Recently, it was found that the contact killing is the predominant antibacterial effect and surface immobilized nanoparticles exhibit increased antimicrobial activity [12,19]. As regards to above considerations, our chit-AgNPs-GO nanocomposites combine several beneficial characteristics such as small size, high density, monodispersity and multiple mechanisms of toxicity as well increased contact killing surface to make them interesting antibacterial materials.

4. Conclusions In conclusion, we have been successfully demonstrated the applicability of a new composite nanomaterial based on chitosan-AgNPs-GO as highly effective antibacterial agent against two strains of MRSA. The antibacterial properties of newly synthesized agents have been carefully assessed and compared with that of the individual components. The effects of different ratios of 14

chitosan to AgNPs-GO on the antibacterial activity of chit- AgNPs-GO nanohybrids have been systematically investigated. Distinct advantages of using chitosan-AgNPs-GO have been found and explained. The well know, strong antibacterial effect of AgNPs was here combined with GO sheets’ abilities to provide a scaffold structure for AgNPs and chitosan adsorption properties to maximize the interaction with MRSA cells through a capturing-killing process. When the ratio of chitosan to AgNPs-GO nanocopmposites is 1:4 the as prepared nanohybrids exhibit the best antibacterial performance, which is superior to that of the antibacterial agents based on AgNPs, GO, and chitosan, or any combination between the components reported so far. The unique physical and chemical characteristics of these nanocomposites contribute all together in a synergistic manner to reveal a novel, highly efficient antibacterial nanointerface. Acknowledgements This work was supported by CNCS – UEFISCDI, project number PN-II-PT-PCCA-2013-4-1232. Monica Potara gratefully acknowledges the financial support of the Sectoral Operational Programme for Human Resources Development 2007-2013, co-financed by the European Social Fund, under the project POSDRU/159/1.5/S/137750 -“Doctoral and postoctoral programs support for increasing research competitiveness in the field of exact Sciences”. The authors are grateful to Dr Adriana Vulpoi for TEM measurements of chit-AgNPs-GO samples.

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19

Figure captions Fig. 1. UV-Vis extinction spectra of: (a) GO, (b) AgNPs-GO and (c) chit-AgNPs-GO nanohybrids (chit:AgNPs-GO = 1:2).

20

Fig. 2. Representative TEM images of: (A) AgNPs-GO and (B) chit-AgNPs-GO nanohybrids

21

Fig. 3. Deconvolution of XPS high resolution C1s spectra for GO, AgNPs-GO and chitAgNPs-GO nanohybrids.

22

Fig.4. (A)-(C) Agar plates inoculated with MRSA (UCLA 8076 strain) and different concentrations of chit-AgNPs-GO nanohybrids (1:4) after 24 h of incubation. (A) 1.64 µg/mL Ag, (B) 2.73 µg/mL Ag and (C) 3.28 µg/mL Ag. The white dots represent the colonies of bacteria formed (CFUs). (D) Negative control (without bacteria and without colloidal solutions)

23

Table 1 Concentrations (at %) and binding energies (eV) of chemical bonds present in the GO, AgNPs-GO and chit-AgNPs-GO nanohybrids determined from deconvolution of the corresponding high resolution XPS spectra. 1s

C=C

C-O

C=O

GO

284.8 eV 46.3%

285.8 eV 38.6%

288.1eV 15.1%

O-C-O and N-C=O -------

AgNPsGO

284.7 eV 39.2%

285.4 eV 41%

288 eV 19.8%

-------

chitAgNPsGO

284.9 eV 50.4%

285.5 eV 25.8%

288.5 eV 9.6%

286.3 eV 8.6%

Table 2. MIC values (μg/mL) of GO, chit-GO, AgNPs-GO, and chit-AgNPs-GO and MBC value (μg/mL) of chit-AgNPs-GO (1:4) against two strains of MRSA. Strain

GO

chit-GO AgNPs-GO chit-AgNPs-GO (1:2)

chit-AgNPs-GO (1:4)

chit-AgNPs-GO (1:8)

MIC

MIC

MIC

MIC

MIC

MBC

MIC

UCLA 8076

> 7.5

2.25

1190

> 7.5

2.25

1.90 Ag + 1.5 GO 1.90 Ag + 1.5 GO

3.81 Ag + 3 GO 3.81 Ag + 3 GO

1.09 Ag + 1.35 GO 1.09 Ag + 1.35 GO

3.28 Ag + 4.05 GO 3.28 Ag + 4.05 GO

1.19 Ag + 1.41 GO 1.19 Ag + 1.41 GO

24