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


 In this study cytotoxic effects of silver-chitosan nanocomposites with aqueous sodium-hydroxide solution (SCNC-ASHS), and aqueous acetic acid solution (SCNC-AAAS) were evaluated, in vitro. The morphology of the synthesized nanoparticles were characterized by Fourier-Transform Infrared Spectroscopy (FTIR), and Scanning Electron Microscopy (SEM). Their cytotoxicity were then evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) in concentrations of 1.56 to 400 µg/ml, and acridine orange/ethidium bromide (AO/EB) staining after 24h and 48h. Results showed the cytotoxicity of 400 µg/ml of SCNC-ASHS on Vero and HT-29 cells of 80.57% and 84.37% after 24h, and 82.20% and 84.84% after 48h. While, the values for SCNC-AAAS on Vero and HT-29 cell-lines were respectively 80.63% and 87.64% after 24h, and 83.60% and 87.44% after 48h. The most toxicity on HT-29 cells was belonged to SCNC-AAAS with IC50 of 40.4 µg/ml. In the staining procedure, cell viability for 25 µg/ml concentration of SCNC-AAAS was 41.84% in HT-29 cell and, for 6.25 µg/ml of SCNC-AAAS was 37.51% in Vero cells. A considerable decrease in cell viability was observed. Types of nanoparticles, synthesis methods, and different cell lines play role in inducing cytotoxicity. Anti-cancer effect of the nanoparticles on the colon cancerous cells (HT-29), of that SCNC-AAAS displayed higher effect than SCNC-ASHS.


Introduction
Chitosan, a deacetylated derivative of chitin, is considered as a food preservative due to its antimicrobial and antioxidant activities [1]. Its antimicrobial effect is due to the reaction between the positive charge of chitosan amino groups and the negative charge of microbial cell membranes, leading to the release of proteinases and other intracellular components of the microorganisms. The chitosan has been applied in nanotechnology for packaging and coating due to its ability to form lms [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 the size range below 100 nm [4].
Chitosan nanoparticles, produced from the natural polymer of chitosan, has displayed higher antimicrobial activity than the chitosan [5,6]. In addition, chitosan nanocomposites can be used in developing drug delivery vectors and nanocamposite based biosensors. For instance, chitosan nanocarriers can be developed by grafting chitosan to some hydrophobic amino acids and then employed as an appropriate drug delivery vector [7,8]. Metal nanoparticles are also widely used in modern food coatings. Silver nanoparticles have shown to have 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, that is, chitosan-silver nanocomposites that have antimicrobial properties [9].
The toxicity of these nanoparticles, which used as antimicrobial agents in food coatings, should be investigated because of their migration to foods and consumption by people. Various size of the nanoparticles, different types of cell lines affect the 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 [10,11]. In contrast, some publications advocated the non-toxicity of the chitosan-coated silver nanoparticles on healthy dermis cells (ATCC CRL-2522TM), and macrophages [9,12,13]. 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 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. So far, the cytotoxic effect of these nanoparticles were not assayed on Vero cells (the epithelial cell class of African green monkeys) as normal cells, and HT-29 cells (colon cancer cells), simultaneously.  [14]. At rst, 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 (NaBH 4 ) solution (Merck, Germany) was then added dropwise until the color shift from colorless to brown. Finally, the solution was heated at 50°C by 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 the method of Akmaz et al., as follows [15].

Cell culture
Two cell lines HT-29 and Vero were considered for the experiment. In order to thaw the cells, they transferred into a falcon tube containing 10 ml of RPMI 1640 complete culture medium containing GlutaMax (Shell Max, Iran) supplemented with 10% fetal bovine serum (Gibco, USA), 1% penicillinstreptomycin (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 supernatant was discarded. The sediment, containing cells, was then cultured in a T25 cell culture ask containing the complete culture medium and incubated at 37°C in the presence of 5% CO 2, and 95% humidity. The cell culture medium was changed every 48 hours to achieve about 90% con uency [17]. 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 10 4 cells) was added to each well of 96-well microplate to reach the appropriate cell con uency. The cells were then treated with SCNC-AAAS, and SCNC-ASHS, and the cytotoxicity was ful lled 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 three times with phosphate-buffered saline (PBS). Three wells were considered for each concentration and for 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 the 96-well plate containing cells and placed in a 37 ° C incubator for 4 h. Then, the contents of the wells were removed and 50 µl of DMSO was added to the each well and incubated for 10 minutes at 37°C. Finally, the microplate was 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 [18,19]:

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

Cytotoxicity
The cytotoxicity effects of SCNC-ASAH (from 400 to 1.56 µg/ml) on HT-29 and Vero cells are shown in

Acridine orange/ethidium bromide (AO/EB) uorescent staining
The cell viability, early apoptosis, late apoptosis, and necrosis were examined in the presences of SCNC-AAAS and SCNC-ASHS by AO/EB staining (Fig. 4). The results showed that when SCNCs concentrations increased, cell viability rates decreased, and early apoptosis, late apoptosis, and necrosis increased.

Scanning Electron Microscopy
The cell morphology changes after 24h of treatment are illustrated in Figure 5. The control group, which did not receive any treatments, showed a normal shape and surface, while the treated cells were changed to the honeycomb structure, holes appeared in the cell membrane. Cell death resulted from leakage of intracellular contents throughout the cell membrane.

Discussion
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 lms for food packaging [26]. However, the toxicology of SNPs has still remained unknown. Additionally, SNPs can be absorbed into the bloodstream via different routes of administration, leading to deposition of silver in many organs, including the liver and spleen, and potentially can damage the organ. Previous researches have shown that different surface stabilizers have distinct impacts on SNPs cytotoxicity. Chitosan, because of its good biocompatibility and antibacterial properties, is often employed as the active ingredient of topical wound materials in combination with SNPs [28]. Chitosan is also used as a stabilizer instead of chemical reducing agents for protecting SNPs from agglomeration [15].
In the present study, chitosan was employed for producing SCNC in which sodium borohydride and sodium hydroxide were used as the reducing agent for silver ions to produce SCNC-AAAS and SCNC-ASHS, respectively. They revealed cytotoxic effects, dependent to the dose and time, on both HT-29 colon cancer cells and normal Vero cells.
Palem et al. reported a 5-7% cytotoxicity on normal 3T3 broblasts and cancer HeLa cells in the presence of SCNC [29]. Their results were in accordance with our ndings. In the present study, a toxicity in both normal and cancer cells was observed in the presence of SCNC.
SCNC is reported to have anti-cancer effect on A549 lung cancer cells, with IC 50 of 29.35 µg/ml [10]. This study also showed that SCNC-AAAS and SCNC-ASHS with IC 50 of 4.40 and 11.54 µg/ml possessed anticancer 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 IC 50 of 43.7µg/ml than breast cancer (MCF7) cells with IC 50 of 52.5 µg/ml [30]. This result is in accordance with our ndings.
Tyliszczak et al. stated that chitosan-based hydrogels modi ed 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) [9]. Wang et al. reported that silver immobilized in the sliver nanoparticle-doped chitosan composite lms, shows a signi cant in uence on the cell adhesion and subsequent proliferation of human umbilical vein endothelial cells [11]. Jena et al. reported that chitosan-coated silver nanoparticles, using chitosan as stabilizing and reducing agent, showed no signi cant cytotoxic or DNA damage on the macrophages at the bactericidal dose [13]. The less toxic effects of SCNC in former studies was likely due to the type of cells. It appears that the reason of non-toxicity of SCNC in the mentioned studies compared to 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 SCNC compared normal kidney epithelial cells and they did not undergo cytotoxicity.
SCNC size is a considerable aspect of different results, which can be varied from the less than 10 nm to more than 100 nm, in our study. It seems that larger size (100 nm) of SCNC causes more cell biological consequences in comparison with smaller particles (10 nm) [9,31] Another in uential factor is type of synthesis procedure of SCNC. 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 [13].
Because of the cytotoxicity of our nanoparticles on the normal cells, the application of them is not recommended in food coatings. Our synthetic nano-particles were highly toxic on the cancerous cells, thus they could be used in treating cancers.

Conclusion
Finally, 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 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 nanoparticles cytotoxicity. Because of high toxic effects on normal cell line, both types of SCNCs, are not recommended for the food industry. Nevertheless, due to their proper anti-cancer effects, found in cell culture assay, they may be applicable in the treatment of the colon cancer that requires more subsequent studies. Furthermore, according to the results, exposure time, the nanoparticles concentrations, procedure of nanoparticles synthesis, and the cell line types considerably affect the cytotoxicity.    The sign * represents a signi cant difference in 24 hours between HT-29 and Vero cells, the sign ** represents a signi cant difference in 48 hours between HT-29 and Vero cells, the sign *** represents a signi cant difference between the two times of 24 and 48 hours in HT-29 cells, and the sign **** represents a signi cant difference between the two times of 24 and 48 hours in the Vero cell (p <0.05).