Silver-Chitosan Nanocomposite Prepared with Aqueous Sodium-Hydroxide and Aqueous Acetic Acid Solutions: Characteristics and Their Cytotoxic Effects

INTRODUCTION
Chitosan, a deacetylated derivative of chitin, is considered a food preservative due to its antimicrobial and antioxidant activities [1]. Chitosan has been applied in nanotechnology for packaging and coating due to its ability to form films [2]. The use of nanotechnology in the food industry is dramatically increasing due to the new properties of materials at the nanometer scale [3]. Nanoparticles are made of natural or synthetic polymers in a size range below 100 nm [4]. Chitosan nanoparticles, produced from the natural chitosan polymer, have displayed higher antimicrobial activity than chitosan [5]. In addition, chitosan nanocomposites can be used in developing drug delivery vectors and nanocomposites-based biosensors [6, 7]. Metal nanoparticles are also widely used in modern food coatings. Silver nanoparticles have been shown to possess antimicrobial properties because they degrade sulfur and phosphorus compounds in proteins and the genetic material of bacteria. The chitosan can be used as a matrix to place silver nanoparticles in coatings named silver-chitosan nanocomposites (SCNC) with antimicrobial properties [8-10].  Different chemical, physical, and biological techniques have been developed to synthetize SCNC [10-13]. Some studies have chemically prepared SCNC in an aqueous solution of acetic acid [10, 14] or aqueous sodium hydroxide [11, 15, 16].

The structures and sizes of SCNC can be characterized by Fourier transform infrared (FTIR) spectrophotometer, electron microscopy, UV spectrophotometer, X-ray powder diffraction (XRD), and surface-enhanced Raman spectroscopy (SERS) [10, 16, 17].
The toxicity of these nanoparticles, which are used as antimicrobial agents in food coatings, should be investigated because of their possibility of migration to foods and consumption by people. Various sizes of the nanoparticles and different types of cell lines affect their cytotoxicity. Accordingly, some reports showed the cytotoxicity, and anti-cancer effects of silver-chitosan nanocomposite on human umbilical artery endothelial cells (HUAECs) and A549 cells (Lung cancer cell line), respectively [18, 19]. In contrast, some publications advocated the non-toxicity of the chitosan-coated silver nanoparticles on healthy dermis cells (ATCC CRL-2522TM) and macrophages [8, 20, 21]. Furthermore, limited information is reported about different kinds and concentrations of the nanoparticles. 
With respect to the various type of nanoparticles and the different types of cell lines, as well as limited information about the toxicity of the different types of nanoparticles and the nanoparticle concentrations, our study aimed to synthesize and investigate the toxicity of chitosan nanoparticles, and silver-chitosan nanocomposites, used in food coatings. Two synthesis methods of silver-chitosan nanocomposites (SCNC) were conducted, including aqueous acetic acid (SCNC-AAAS) and sodium hydroxide solutions (SCNC-ASHS), and the properties of the synthesized nanoparticles were investigated using various methods such as FTIR and SEM. The cytotoxic effects of these synthesized nanoparticles have not been assayed on Vero cells (the epithelial cell class of African green monkeys) as normal cells, and HT-29 cells (colon cancer cells), simultaneously that were studied in this research.

MATERIALS AND METHODS
Preparation of silver-chitosan nanocomposite (SCNC)
Preparation of SCNC with aqueous acetic acid solution (SCNC-AAAS)
SCNC-AAAS was synthesized according to the method of Honary et al. with the following modifications [14]. At first, 100 ml of chitosan solution (0.5 mg/ml) (Aldrich Chemical, Germany) was prepared in acetic acid solution (1-2%) (Merck, Germany). Due to the poor solubility, chitosan was kept at room temperature for 24 hours. Second, the prepared solution was added to 1 liter of 6 mM silver nitrate solution and was stirred for one hour on a stirrer (IKA, Germany). Third, the 58 mM sodium borohydride (NaBH4) solution (Merck, Germany) was added dropwise until the color shifted from colorless to brown. Finally, the solution was heated at 50 °C in an oven (model CE.FH.151.4, Germany) to evaporate large amounts of water. The remained water was then completely removed by a freeze dryer (CHRIST, Germany).

Preparation of SCNC with aqueous sodium hydroxide solution (SCNC-ASHS)
SCNC-ASHS solution was synthesized according to Akmaz et al., as follows [11]. 100 mg of chitosan (Aldrich Chemical Company, Germany) was added to 50 ml of 95 °C water. One ml of 0.02 M solution of silver nitrate (AgNO3) (Merck, Germany) was added to the suspension during the sonication (Iranian Knowledge-Based Company of Nasir Research, Iran). Then, 100 μl of 0.3 M Sodium hydroxide (NaOH) solution (Merck, Germany) was added dropwise. At this stage, the color changed from colorless to reddish-yellow. The solution was sonicated for 10 minutes at 95 °C. The nanoparticles were then washed with distilled water and dried in an oven (Memmert, Germany) at 50 °C.

Examination of nanoparticle characteristic
Fourier-Transform Infrared Spectroscopy (FTIR)
FTIR method was used to determine the molecules and biological functional groups responsible for nanoparticle synthesis (NIRS XDS Process Analyzer, Metrohm) [22]. 

Scanning Electron Microscope (SEM)
SEM was applied to take images from the synthesized nanoparticles. The image details such as magnification and voltage were recorded in the related image (TESCAN, Czech Republic, model-TESCAN-Vega 3)

X-Ray Diffraction (XRD)
In the X-ray diffraction method, the SCNC-AAAS powder was placed on a glass slide and analysed using X-Ray diffractometer (Bruker D8 ADVANCE, Japan) which was set at photon energy of 40 KeV and 40 mA with CuKα1 beam with wavelength λ=1.54 at 2θ angle and angular range of 20 to 80 [23].

Cell culture
Two cell lines HT-29 and Vero were considered for the experiment. The cells initially freezed in cryotubes were transferred into sterile falcon tubes containing 10 ml of RPMI-1640 (for HT-29) or DMEM (for Vero) complete culture medium containing GlutaMax (Shell Max, Iran) supplemented with 10% fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin (100 IU/ml and 100 µg/ml) (Bio-idea, Iran), and 0.05% amphotericin B (2.5 µg/ml) (Sigma, USA). The cells were then centrifuged (Biosan, Latvia) at 1000 rpm for 10 minutes, and the supernatants were discarded. The sediments containing 4×105 cells were then cultured in T-25 cell culture flasks containing the complete culture medium (6 ml) and incubated at 37 °C in 5% CO2 and 95% humidity. The cell culture media were changed every 48 hours to achieve about 90% confluency [24]. The cells were then detached with 300 μl of 0.05% trypsin-versene solution (Bio-Idea Company, Iran) and were collected after the centrifugation (1000 rpm, 10 minutes) for the further cell treatment.  

Cytotoxicity test
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test (Bio idea-Iran) was used to evaluate cell toxicity. Initially, 100 μl of culture medium containing 104 cells was added to each well of 96-well microplates to reach the appropriate cell confluency. The cells were then treated with SCNC-AAAS and SCNC-ASHS, and the cytotoxicity was fulfilled after 24h and 48h incubations. Both SCNCs were used at the concentrations of 400 μg/ml to 1.56 μg/ml with series of 2-fold dilutions. Afterwards, the wells were evacuated and washed with phosphate-buffered saline (PBS) (100 μl/well) three times. Three wells were considered for each concentration and the control group, receiving no treatment.  MTT test was performed according to the instructions given by the manufacturer. Accordingly, 100 μl of RPMI-1640 without phenol red and 10 μl of MTT (12 mM) were added into 96-well plates containing cells and placed in a 37 °C incubator for 4 h. The wells contents were discarded and 50 μl of DMSO was added to each well. The plates were then incubated for 10 minutes at 37 °C. Finally, the microplates were measured at optical density (OD) value of 570 nm using a plate reader (BioTek, USA). This test was repeated three times, and the cytotoxicity percentage was calculated using the following equation [25, 26]:

Acridine orange/Ethidium bromide (AO/EB) fluorescent staining
To evaluate cell apoptosis induced by SCNC-AAAS, AO/EB fluorescent staining was applied for both HT-29 and Vero cells. Firstly, 1×105 cells/well were seeded into 12-well tissue culture plates. After 72h incubation, the supernatants were discarded, and the monolayer cells were washed with PBS and supplemented with 6.25 µg/ml, 12.5 µg/ml, and 25 µg/ml SCNC-AAAS for HT-29 cell line, and 1.56 µg/ml, 3.12 µg/ml, and 6.25 µg/ml SCNC-AAAS for Vero cells. Negative controls, without any treatment, were considered for both cell lines. The cells were incubated for 24h (37 ºC, 5% CO2) and then harvested using 200 µl of EDTA (1 mM). After adding 2 ml RPMI-1640 or DMED medium supplemented with 10% FBS, the cells were centrifuged, and 200 µl medium was added to the remained cells. 20 µl of the cell suspension was mixed with an ethidium bromide/acridine orange dye. Then, 10 μl of the dyed cells was transferred to a hemocytometer slide and assessed using a fluorescence microscope (CETI, 040856, Belgium) at ×20 magnification [24, 27]. 

Preparation of cells for microscopy
Preparation of cells for the phase-contrast microscopy
About 1.5×105 cells (HT-29 or Vero cells), suspended in 2 ml of RPMI-1640 or DMEM medium, were seeded into six-well plates and incubated (37°C, 5% CO2). After the cell monolayer formation, 400 μg/ml of each SCNCs-AAAS and SCNCs-ASHS were added to both HT-29 and Vero cell lines, and the plates were incubated (37°C, 5% CO2) for 24h. The cells were examined by phase-contrast microscopy (Optika, Italy) at ×10 magnification morphologically [28]. 

Preparation of cells for the scanning electron microscopy (SEM)
About 1.5 × 105 cells (HT-29 or Vero cells) in 2 ml of the media were initially cultured in six-well plates and incubated. After the formation of the cell monolayers, the cells were evaluated by adding different concentrations of SCNC-AAAS or SCNC-ASHS into the wells and were incubated for 24 hours. SCNC-AAAS concentrations were considered to be 100 μg/ml in Vero cells and 12.5 μg/ml in HT-29 cells, while SCNC-ASHS was added at a concentration of 100 μg/ml and 6.25 μg/ml in HT-29 cells and Vero cells, respectively. The cells were then washed with PBS (2 ml/well) and fixed with 3% glutaraldehyde (2 ml/well) (Dae Jung, Korea) for 2 hours at 4° C. To dehydrate the cells, alcohol solutions (2 ml/well) (Merck, Germany) with concentrations of 50%, 60%, 70%, 80%, 90%, and 100% were exerted for 30 minutes for each concentration. The plates were finally kept in a laminar flow hood for 2h at room temperature. The cell images were then taken by scanning electron microscopy [29]. 
Statistical analysis 
All data were analyzed as mean ± standard deviation (SD) in triplicate experiment. Statistical significance for MTT test was determined by one-way analysis of variance and independent t-test using SPSS v.19 software. P <0.05 was considered statistically significant. IC50 was calculated using the non-linear regression analysis method in GraphPad Prism v.8.3.0 software. 

RESULTS AND DISCUSSION
SCNCs Microscopy
FTIR spectroscopy
Fourier transform infrared spectroscopy analysis was carried out to identify the chemical structures of SCNC-AAAS and SCNC-ASHS. Details are given in Fig. 1. The spectral shifts of the diagram in the 3350 cm-1 frequency show the presence of amine and hydroxyl groups and their overlap. Also, the shifts that occurred in the 2880 cm-1 wavenumber resulted from the presence of CH2 in chitosan and nanocomposite. The peak observed at 1513 cm-1 indicates the bending vibration of the NH band. It is noticeable that the interaction of NH2 or O-H groups of chitosan with silver ions tends their vibrations peak to lower wavenumbers. The sharp peak observed in the 1284 cm-1 wavenumber represents the CH2 wagging vibrations. The changes in the frequency range from 1060 to1020 cm-1 usually correspond to C-N and C-O stretching vibrations in which the C-O stretching vibrations are formed in the frequency of the composite combined. The relatively sharp peak detected in the frequency of 800 cm-1 indicates the absorption of NO3- ions from silver nitrate salt. The peak observed in 670 cm-1 can result from reducing the activity of chitosan and metallic silver precipitation. Seemingly, the saccharide structure changes after adsorption of silver ions detectable at 1050 cm-1 peak [23, 30]. 

Scanning Electron Microscopy
The size and morphology of the nanocomposites recorded by SEM are shown in Fig. 2. SCNC-AAAS are detectable with cubic shapes and with both sizes of smaller and larger than 100 nm; while SCNC-ASAH are spherical-shape with a size smaller than 100 nm. 

X-Ray Diffraction (XRD)
To detect nanoparticle composition we used other techniques including X-Ray powder Diffraction (XRD) according to previous studies. According to the higher cytotoxicity effect of SCNC-AAAS, XRD was only used for this nanocomposite chitosan. Fig. 3 shows the pattern of silver chitosan nanocomposites with aqueous acetic acid solution. The peaks at the angles of 38.23, 44.44, 64.54 and 77.62 degrees are assigned to silver at 111, 200, 220, and 311 diffusers, respectively. The broad peak at 22.5 degrees is related to the presence of chitosan.

Cytotoxicity 
The cytotoxicity effects of SCNC-ASAH (from 400 to 1.56 μg/ml) on HT-29 and Vero cells are shown in Fig. 4. The cytotoxicity of SCNC-ASAH on HT-29 and Vero cells was significantly increased  between the time 24h and 48h, and the nanoparticle concentrations (p<0.05)

IC50 value of SCNCs in HT-29 and Vero cell lines
The IC50 values of SCNC-AAAS in HT-29 and Vero cells were determined as 4.4 and 13.40 μg/ml, respectively. Since HT-29 cell line showed a lower IC50 value, its toxicity to SCNC-AAAS was higher than that of Vero cells. Furthermore, IC50 values of SCNC-ASHS in HT-29 and Vero cells were calculated at 11.54 and 19.36 μg/ml, respectively. The lower IC50 value of HT-29 cells implied higher cell toxicity to SCNC-ASHS than that of Vero cells. 

Acridine orange/Ethidium bromide (AO/EB) fluorescent staining
The cell viability, early apoptosis, late apoptosis, and necrosis were examined in the presence of SCNC-AAAS and SCNC-ASHS by AO/EB staining (Fig. 5). The results showed that when SCNCs concentrations increased, cell viability rates decreased, and early apoptosis, late apoptosis, and necrosis increased.

Cell microscopy treated with different concentrations of SCNCs
Phase-contrast microscopy
Changes in morphology of both HT-29 and Vero cells after 24 hours of treatment with SCNC-AAAS and SCNC-ASHS nanoparticles were recorded by contrast phase microscopy. Changes such as decreased cell adhesion and increased floating cells were observed. 

Scanning Electron Microscopy                                                                                                                                
The cell morphology changes after 24h of treatment are illustrated in Fig. 6. The control group, which did not receive any treatments, showed a normal shape and surface, while the treated cells were changed to a honeycomb structure, and holes appeared in the cell membrane. Cell death resulted from leakage of intracellular contents throughout the cell membrane. 
The antimicrobial properties of chitosan have been enhanced by loading chitosan with various metals. Among all antimicrobial metals, silver possesses great toxicity against a wide range of microorganisms. Nanocomposites based on silver nanoparticles (SNPs) have been used as antimicrobial films for food packaging [30]. However, the toxicology of SNPs has remained unknown. Additionally, SNPs can be absorbed into the bloodstream via different routes of administration, leading to the deposition of silver in many organs, including the liver and spleen, and potentially can damage the organ. Previous studies have shown that different surface stabilizers have distinct impacts on SNPs cytotoxicity. Because of its good biocompatibility and antibacterial properties, chitosan is often employed as the active ingredient of topical wound materials in combination with SNPs [31]. Chitosan is also used as a stabilizer instead of a chemical reducing agent for protecting SNPs from agglomeration [11]. 
In the present study, chitosan was employed for producing SCNC in which sodium borohydride and sodium hydroxide were used as reducing agents for silver ions to produce SCNC-AAAS and SCNC-ASHS, respectively. They revealed cytotoxic effects on both HT-29 colon cancer cells and normal Vero cells, depending to the dose and time. Palem et al. reported a 5-7% cytotoxicity on normal 3T3 fibroblasts and cancer Hela cells in the presence of SCNC [32]. Their results were in accordance with our findings. In the present study, toxicity was observed on both normal (Vero) and cancer (HT-29) cells in the presence of both SCNCs. However, SCNC-AAAS showed more toxicity towards HT-29 cells with the IC50 of 4.4 µg/ml, representing the possibility of its anti-cancer effect. Another study found that chitosan/silver nonocomposite had cytotoxicity toward breast cancer (MCF-7) cell, however chitosan/silver/multiwalled carbon nanotubes showed higher effect [14]. SCNC is reported to have an anti-cancer effect on A549 lung cancer cells, with IC50 of 29.35 μg/ml [18]. This study also showed that SCNC-AAAS and SCNC-ASHS with IC50 of 4.40 and 11.54 μg/ml possessed anti-cancer effects on HT-29 cells, respectively. It is indicated that Ag-doped chitosan-poly vinyl alcohol nanocomposites impact more on human liver cancer (HEPG2) cells with IC50 of 43.7 µg/ml than breast cancer (MCF7) cells with IC50 of 52.5 μg/ml [33]. This result is in accordance with our findings. 
Tyliszczak et al. stated that chitosan-based hydrogels modified with SNPs produced by sodium borohydride in concentrations of 25, 50, 75, and 100 (wt%) showed no toxic effect on dermis cells BJ (CRL-2522TM) [8]. Wang et al. reported that silver immobilized in the sliver nanoparticle-doped chitosan composite films shows a significant influence on the cell adhesion and subsequent proliferation of human umbilical vein endothelial cells [19]. Jena et al. reported that chitosan-coated silver nanoparticles, using chitosan as stabilizing and reducing agent, showed no significant cytotoxic or DNA damage on the macrophages at the bactericidal dose [21]. The less toxic effects of SCNCs in former studies were likely due to the type of cells. A reason for the non-toxicity of SCNC in the mentioned studies compared to the present study is the difference in the type of investigated cells so that normal dermis cells, umbilical vein endothelial cells, and macrophages exerted more resistance to SCNCs compared to normal kidney epithelial cells. SCNC size is another reason.  SCNC size is a considerable aspect of different results, which can be varied from less than 10 nm to more than 100 nm, in our study. It seems that larger size (100 nm) of SCNCs causes more cell biological disorders in comparison with smaller particles (10 nm) [8, 34]. Another influential factor is the methods SCNC synthesis. In the current study, chitosan was alternatively used as a silver ion reducing agent instead of sodium borohydride, used in previous studies. Jena et al. showed that the same-size particles above 100 nm were not toxic to the macrophage [21]. Because of the cytotoxicity of our nanoparticles on the normal cells, their application is not recommended in food coatings. Our synthetized nanoparticles were highly toxic on the cancerous cells, and more investigations are recommended to survey their appropriate effects on treating cancers. 

CONCLUSION
In the present study, SCNC-AAAS and SCNC-ASHS showed more toxic effects on the cancerous cells than the normal cells. However, SCNC-AAAS showed a higher toxic effect on both normal and cancer cell lines compared to SCNC-ASHS. The results implied that the synthesis procedure of SCNCs plays a notable role in the cytotoxicity of the nanoparticles. Because of their highly toxic effects on the normal cell line, both types of SCNCs, are not recommended for the food industry. Nevertheless, due to their probable anti-cancer effects, found in cell culture assay, they may be applicable in the treatment of colon cancer which requires more subsequent studies. Furthermore, according to the results, exposure time, the nanoparticles concentrations, the procedure of nanoparticle synthesis, and the cell line types considerably can affect SCNCs cytotoxicities.  

ACKNOWLEDGMENTS
The authors gratefully acknowledge the Research Affair of Shiraz University. 

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.