Silver Nanoparticles Embedded in Natural Rubber Films: Synthesis, Characterization, and Evaluation of In Vitro Toxicity
Natural rubber (NR) films can reduce silver metal ions forming embedded metal nanoparticles, a process that could be described as green synthesis. The NR films acting as a reactor generate and incorporate silver nanoparticles (AgNPs). Organic acids and amino acids play a crucial role in the formation of AgNPs. The plasmon extinction obtained in the UV-visible spectrum shows the presence of nanoparticles in the film after dipping the NR film into a solution of silver nitrate at 80°C. Electron microscopic analysis confirms the presence of AgNPs in the NR film and characterization by atomic force microscopy shows a change in the roughness of the NR film with AgNPs. In addition, our preliminary results from in vitro toxicity studies (MTT and comet assays) of the NR films and NR films with silver nanoparticles (NR/Ag) show that they are not toxic to cell lineage CHO-K1 (cells from the ovary of a Chinese hamster), an important result for potential medical applications.
Plasmonic  films fabricated with metallic nanoparticles embedded in natural or synthetic polymeric materials are extensively studied, since they have potential for applications in new technologies [2, 3] and in the medical field [4, 5].
Natural rubber is a natural polymer used in the manufacture of various materials used in health care and thin film technologies [6–8]. It is a biocompatible polymer capable of inducing the angiogenesis process  and is being marketed in form of cream-gel REGEDERM® to aid in the healing process of cutaneous wounds. This natural polymer can also be used to induce the formation of metal nanoparticles [10, 11]. Metallic nanoparticles, in particular silver nanoparticles, are the subject of studies regarding the interactions of these particles in biological environments, such as interaction with bacteria and fungi [12–16], interaction with human cells [17, 18], and also the implications generated when AgNPs interact with DNA [19, 20]. Correspondingly, new material combining the properties of NR and silver nanoparticles opens possible avenues for new applications. A main concern is the biocompatibility of these new materials, since they could be toxic or produce genotoxicity depending on their concentrations and dimensions. The NR film provides an active substrate acting as reducing agent and stabilizer of the metallic nanoparticles, producing a material with encapsulated AgNPs and improved biocompatibility.
In this study we evaluate the in vitro biocompatibility of incorporated AgNP within a polymeric matrix. The work focuses on characterizing the NR film without and with silver nanoparticles. The appearance of plasmon absorption band in UV-visible attests to the presence of metal nanoparticles. Film characterization is carried out using FT-infrared spectra, electron microscopy images, atomic force microscopy (AFM), and elemental analysis. In addition, toxicity studies were performed through the techniques of MTT and comet assays.
2. Experimental Section
Natural Rubber Latex (NRL) was collected from different trees of Hevea brasiliensis tree RRIM 600 clone, at Indiana Farm (Experimental Farm), located in the Indiana city, near Presidente Prudente, São Paulo State, Brazil. The latex collected was stored in a dark vessel, with capacity of 1 liter and 20 mL of ammonia hydroxide, that is, 2% v/v, was added to prevent spontaneous coagulation. Silver nitrate salt (ACS reagent, ≥99.0%) was purchased from Sigma-Aldrich and used as received. Cell line used in this study was CHO-K1 (cells from the ovary of a Chinese hamster). Cells were cultured in 10 mL of DMEM/F10 supplemented by 10% fetal bovine serum in 25 cm2 flasks and kept in an incubator with CO2 at 37°C.
2.2. Natural Rubber Film (NR) Preparation
NR films were prepared by depositing 10 mL of NRL in a Petri dish measuring 95 mm in diameter and put in an oven for thermal treatment (annealing for 10 hours at 65°C), to complete the NRL polymerization. The films obtained have an average thickness of around 0.5 mm.
2.3. Synthesis of NR/Ag Film
The formation of silver nanoparticles was induced by direct reaction between NR film and silver nitrate solution. The natural rubber films were submerged into AgNO3 solution, with a concentration of M. Throughout the procedure, the silver nitrate solution containing the NR films was kept in a sand bath at a temperature of 80°C. The NR films were withdrawn from silver nitrate solution at different reduction times: 30 min (NR/Ag30), 60 min (NR/Ag60), 90 min (NR/Ag90), and 120 min (NR/Ag120). The results are presented in Figure 1.
2.4. Preparation of Liquid Extracts
To obtain the liquid extracts, equal surface areas of NR, NR/Ag30, NR/Ag60, NR/Ag90, and NR/Ag120 films were placed in glass Petri dishes with straight bottom 9.5 cm in diameter. To each plate 10 mL of distilled water was added. The plate was sealed with PVC film and carried to the oven heated at 37°C and maintained there for 24 hours. After this time the liquid was removed from the plate and transferred to a clean container and filtered and the pH was set to 7.4. The extracts were used to carry out the MTT and comet assays. A portion of this solution was set aside for quantification of silver content.
2.5. Exposure Protocol
For the MTT assay, the cells were seeded on a transparent 24-well plate at a density of 1.0 × 105 cells per well and taken to CO2 incubator at 37°C for stabilization during 48 h; then the cells were put in contact with the different liquid extracts (NR, NR/Ag30, NR/Ag60, NR/Ag90, and NR/Ag120) and with distilled water for negative control (CTR). The ratio is 1 : 1 (culture media : liquid extracts or distilled water) and the cells were incubated for 24 h. For the alkaline version of the comet assay, cells were seeded at a density of 5.0 × 105 cells in transparent 12-well plate. Cells were treated for 24 hours with distilled water (CTR) or with different liquid extracts in 1 : 1 ratio.
2.6. Cellular Toxicity Evaluation
2.6.1. MTT Assay
The cytotoxic potential of extracts was assessed using the MTT reduction method . Following a period of exposure (24 h), culture medium was removed, MTT solution of 0.3 mg/mL was added and cells were incubated for 4 hours. The culture medium was removed and added DMSO in order to precipitate formazan crystals. The corresponding absorbance reading was performed in a microplate reader at a wavelength of 492 nm to assess the number of viable cells in each well. The absorbance of the control was considered to represent 100% cell viability (CV). The CV of the rest of the samples was determined using the following formula: where CVE is cell viability of cells exposed to the extract; AE is absorbance of cells exposed to the extract; ACTR is absorbance of negative control cells; AB is absorbance of blank (well containing culture medium only).
2.6.2. Comet Assay
The levels of DNA damage in cells exposed to both extracts were evaluated by comet assay. Following exposure to the extracts, the adherent cells were trypsinized and were used for the preparation of the slides for the comet assay, according to the protocol described by Singh et al. , with a few modifications. The samples were mixed with low-melting-point agarose (0.5%), placed into glass slides, and covered with coverslips and the slides were placed in a lysis solution for a period of one hour. After the lysis step, all slides were transferred to an electrophoresis tank containing freshly prepared cold alkaline buffer. The electrophoresis was performed at 25 V and 300 mA during 20 minutes. After neutralization and fixation steps, the slides were then stained with diamidino-2-phenylindole (DAPI) (1 mg/mL DAPI H2O) solution and visualized by fluorescence microscopy with 400x of magnification. One hundred nucleoids were counted per slide, and the DNA damage was visually classified into four categories according to the migration of DNA fragments, in accordance with the method described by Kobayashi et al.[23, 24]. The results were compared by parametric analysis of variance (ANOVA) using the Student-Newman-Keuls method or the nonparametric Kruskal-Wallis test, in accordance with the distribution of the data (normality and homogeneity of variance). values < 0.05 were considered significant and the results were expressed as means ± SD (standard deviation).
2.7. Characterization Techniques
2.7.1. Characterization of NR/Ag Films
The extinction data were recorded using SHIMADZU UV-visible spectrophotometer, model UV-1800, with a scan range from 300 nm to 800 nm. The FT-IR/ATR spectral data were obtained using Bruker alpha-ATR FT-IR spectrometer, the scan range from 400 cm−1 to 4000 cm−1. The SEM images and the elemental analysis were recorded using the Scanning Electron Microscopy (SEM) model FEI Quanta 200 FEG high microscopy resolution with EDAX Energy Dispersive Spectroscopy (EDS) and X-Ray Detector, and all the samples were coated with a thin layer of conductive carbon to improve conductivity and these measurements were performed at the Great Lakes Institute Environmental Research (GLIER), University of Windsor. Atomic force microscopy (AFM) images were recorded using a Digital Instruments NanoScope IV, operating in tapping mode with Al-coated silicon tip (model TESPA, Bruker). Images were collected with high resolution (1024 lines per scan) at a scan rate of 0.5 Hz. Digital processing of the images used the free SPM data analysis software Gwyddion 2.30. Silver content of extracts was quantified via optical emission spectrometry using inductively coupled plasma (ICP-OES); the equipment detection limit (<LoD) is 0.029 μg/mL with an error per each measurement of 0.003 μg/mL. The samples were subject to acid digestion using concentrated nitric acid followed by ICP-OES quantification using an Optima 8000 ICP-OES spectrometer Perkin Elmer.
3. Results and Discussion
Figure 1 shows the UV-Vis spectra of NR and NR/Ag films obtained in different reductions times. The NR film does not have absorption peaks, facilitating the characterization of the films with silver nanoparticles. NR/Ag films synthesized at 30 min, 60 min, 90 min, and 120 min presented typical brood plasmon absorption peaks at ~430 nm. With increase in reduction time, the intensity of the peak is proportional to the AgNPs concentration  and also is likely to increase size of the silver nanoparticles in the polymer matrix [26, 27]. The symmetrical shape of the plasmon indicates low dispersion of nanoparticles size formed during the reduction process [22, 23].
The vibrational spectra of neat NR films and NR/Ag films can be seen in Figure 2; the spectra were recorded at different points of the samples (5 points), and Figure 2 shows the average spectra. The NR spectrum for neat NR film agreed with the characteristic FT-IR spectrum reported in the literature [10, 28], which corresponds to that of the cis-isoprene structure as the other components present in the latex, such as proteins and lipids [10, 28–30]. The vibrational bands of the NR film in the 3300 cm−1 to 3030 cm−1 region are associated with the stretching modes of C-H, N-H, and O-H bonds; vibrational bands at 2960 cm−1 to 2850 cm−1 are associated with C-H bonds in lipids and the isoprene chain; vibrational bands at 1660 cm−1 to 1650 cm−1 are related to C=O stretching associated with amide I and vibrational bands at 1545 cm−1 are associated with amide II (protein fraction from the film); vibrational bands at 1445 cm−1 to 1038 cm−1 are associated with amide III and symmetric and asymmetric bending of the isoprene; vibrational bands at 1040 cm−1 to 1038 cm−1 are associated with the C-N and C-O stretching from 890 cm−1 to 840 cm−1. A tentative assignment is presented in Table 1.
Notably, the intensity IR bands around 3290 cm−1, 1654 cm−1, and 1545 cm−1 decrease or disappear in NR/Ag films synthesized at 30 min, 60 min, 90 min, and 120 min. These vibrational bands are likely associated with the protein fraction of the film, playing a role in reducing the silver ions. The latter is supported by the appearance of the band at 1515 cm−1, and this band is observed, with low intensity, for the NR/Ag30 film; the intensity of it increases as the reduction time lengthens. Those changes are clearly noted in the spectra shown in Figure 2, where they are indicated by dotted line. This band, according to literature, is associated with amine group (R2-NH) [10, 28–30], and this could be a product of the synthesis, which could only come from protein denaturation or destruction of the amide bond; for this reason the bands associated with protein disappear after we put the film of latex in the solution of silver nitrate at 80°C, and the possible product of reaction between amide and Ag+ is silver nanoparticles and amines.
The SEM image of the surface of the NR/Ag120 film is shown in Figures 3(b) and 3(c); it is possible to observe the presence of structures that stand out of the matrix, which is the NR film; this image was chosen because we are able to see clearly the lighter dots representing the metal nanostructures in the sample (they are identified with a yellow arrow); those nanoparticles have a size between 20 nm to 60 nm. To confirm the nature of the observed particles an elemental analysis was performed, shown in Figure 3(a). The spectrum in Figure 3(a) shows the emission peak of the X-rays of silver (AgL) for the point indicated in Figure 3(b). The elemental composition values are shown in Table 2, and the percentage of silver is 3.24% mass, and this percentage refers to the point indicated by the yellow arrow in Figure 3(b). On the other hand, the size of particles observed in Figures 3(b) and 3(c) corroborates the results shown in Figure 1 (UV-Vis) where it is possible to observe plasmon band with maximum around 430 nm, which according to the literature  corresponds to silver nanoparticles that have this size.
AFM images are given in Figure 4 for neat NR film and films produced in different reduction times NR/Ag30, NR/Ag60, NR/Ag90, and NR/Ag120. Analyzing the AFM images is possible to observe film surface roughness changes. The values of root mean square average roughness (Rq) were 78.8 nm, 33.7 nm, 32.0 nm, 24.1 nm, and 20.1 nm for NR film, NR/Ag30, NR/Ag60, NR/Ag90, and NR/Ag120, respectively. These changes is directly associated with increasing the reduction time; in other words, the roughness of this surface decreases with increasing synthesis time. This fact could be associated with the growth of AgNPs at film structure; once the higher time of synthesis is related to the bigger amount of nanoparticles, then they occupy the spaces into the matrix, and the surface becomes smoother.
It is necessary to assess whether the NR/Ag films have biological characteristics compatible to be defined as a biomaterial. Two important characteristics of biomaterials are biocompatibility and biofunctionality . Among the various biocompatibility tests required, in vitro cytotoxicity and genotoxicity tests are the first two assays conducted to begin to assess the biocompatibility of a new material [32, 33]. Among the various existing in vitro cytotoxicity assays, the MTT test is the most commonly used; using this methodology the cell viability is quantified by conversion of tetrazolium into formazan by mitochondria viable cells . In this sense we test our films without and with silver nanoparticles with MTT assay. To support the essay, we analyzed whether extracts free any compound after 24 hours using inductively coupled plasma optical emission spectrometry (ICP-OES) (see Figure 5). The quantification results show that the total silver concentrations for NR/Ag30 and NR/Ag60 liquid extract are above the detection limit (0.029 μg/mL), and the amount is approximately 0.17 μg/mL and 0.07 μg/mL, respectively (see Figure 5). The latter means that the NR/Ag film releases silver in distilled water used to make the liquid extract. For the extracts NR, NR/Ag90, and NR/Ag120 the total silver released was not identified or was below the detection limit; that is, the films prepared with higher reduction times are not releasing silver when placed in solution. One explanation for the detection of silver in the extracts obtained from NR/Ag30 and NR/Ag60 may be related to the time of reduction of silver nanoparticles. In other words, the interaction between silver nanoparticles and natural rubber films is more effective when the high reduction times occurs. It should be emphasized that in the NR/Ag30 and NR/Ag60 extracts total silver has been identified, but the amount of silver found in the extract did not cause toxic effect for cell viability of CHO-K1 lineage.
The results of MTT assay are shown in Figure 6. The bar graph shows the results for all samples and control; in Figure 6(a) we show an image of the CHO-K1 cells exposed to liquid extract from NR film in time zero; in Figure 6(b) we show the same cell culture after 24 hours of exposition by liquid extract; it is possible to observe mild differences between the two images, because the liquid extract has low toxicity (<10%), and it can be seen that the extract made with NR film and the extracts of films with silver nanoparticles with different concentration were not cytotoxic to the cell lineage tested in comparative with negative control. When measuring the cytotoxicity of a material from its extract, it is expected to evaluate whether this material can release toxic compounds for extracting vehicle . It is reasonable to assume that the low toxicity associated with silver nanoparticles released to extractor vehicle for the studied cell line could be related to the synthesis route used to obtain the nanoparticles, since the use the NR film itself as a reducing agent and stabilizer for nanoparticles does not use organic chemicals commonly used in literature. The most satisfying explanation for the low toxicity is that our material contains encapsulated AgNPs and prevents the release of silver to the liquid extract. Most toxicity studies developed with nanoparticles in solution [18, 36–39] show that the silver concentration is directly related to cytotoxicity, and our results are in agreement with these findings, since the silver concentration released by NR/Ag films is very low.
In the cytotoxicity tests, genotoxicity tests are also important to evaluate a biomaterial. The comet assay is a widely used and very sensitive method for the evaluation of damage to DNA molecule . The comet assay results are shown in Figure 7. It is found that there is no DNA damage for exposed cells to extract made from NR and the extract made from NR/Ag films compared with the negative control. For NR/Ag90 and NR/Ag120 extracts no cytotoxicity was expected, because the silver was not detected in the extracts by ICP-OES. de Souza et al.  conducted a study in which two cell cultures (CHO-K1 and CHO RS5) were exposed to different concentrations of AgNPs (0.025 to 5.0 μg/mL) for 24 hours and concluded that the increase of genotoxic damage is dose-dependent and the genotoxic effects are related to concentrations equal to or bigger than 1.25 μg/mL, and these results can be references to confirm the results presented in this paper, because the silver concentration in liquid extract is below toxic concentration. We could say that the key of low toxicity for our material is the fact that nanoparticles are incorporated into the film; for this reason the silver release is restricted and presents a low toxicity. In the future we must consider a prolonged period of incubation (longer than 24 hours) for film immersed in water, to reinforce the idea that films with AgNPs are not degraded and do not release toxic compounds to the cells. We must also study other cell lines to improve our biological approach. The results discussed here open the possibility for different redirection to our material. The NR/Ag30 and NR/Ag60 films can be directed to applications in the biomedical area, once those films release silver, from the matrix, and this characteristic gives them a bactericidal capacity, since the use of materials releasing silver as an antimicrobial agent is already well described in the literature . Already NR/Ag90 and NR/Ag120 films would be directed to technological applications, where you have the need for flexible polymer electrooptical properties .
The synthesis of flexible NR/Ag films with silver nanoparticles is demonstrated. Natural rubber films are used as reducing agent to form incorporated silver nanoparticles. Electron microscopy results show that homogeneity of the nanoparticles distribution on surface of the films and the size of those nanoparticles vary around from 65 nm to 85 nm. Plasmon absorption and elemental analysis confirm the presence of silver in the NR/Ag films. Preliminary cytotoxicity studies demonstrated that this new film material does not cause damage to the metabolism, nor genetic damage to the studied cell line, and these results may open the door for direct bioapplications.
The authors declare that they have no competing interests.
The authors acknowledge Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Universidade do Oeste Paulista (UNOESTE), and Agência Paulista de Tecnologia dos Agronegócios (APTA).
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