Synthesis, characterization and biocompatibility of “green” synthesized silver nanoparticles using tea polyphenols

Abstract

Since ancient times, people have taken advantage of the antimicrobial effects of colloidal silver particles. Aside from the medical prospects, silver nanoparticles are found in a wide range of commercially available consumer products ranging from cosmetics to household cleansers. Current synthetic methods for creating silver nanoparticles typically call for potentially hazardous chemicals, extreme heat, and produce environmentally dangerous byproducts. Therefore, it is essential that novel “green” synthesis of nanoparticles becomes a reality, and it is imperative to fully analyze the potential toxic effects of these nanoparticles. In this study, we have shown that by reducing silver nitrate in solutions of tea extract or epicatechin of varying concentrations, spherical silver nanoparticles were formed that had controllable size distributions depending on the concentration of tea extract or epicatechin in the samples. Our ultra-resolution microscopy demonstrated that the nanoparticles were in fact interacting with the keratinocytes. Furthermore, evaluation of mitochondrial function (MTS) to assess cell viability and membrane integrity (LDH) in human keratinocytes showed that the silver nanoparticles were nontoxic. These results demonstrated that these nanoparicles are potentially biocompatible and warrant further evaluation in other biological systems.

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Introduction

Nanostructured noble metals have found widespread use in several technological applications,^1–5 and various synthetic methods have been exploited to meet these demands.^6–14 Synthesizing metal and semiconductor nanoparticles is gaining interest due to their extraordinary properties, which differ from when their bulk counterparts. Recently, there has been renewed interest in applying green chemistry principles to producing noble metal nanoparticles.^8,15–24 For example, silver and gold nanoparticles produced from vegetable oil can be used as antibacterial paints.²⁴ Green chemistry is the design, development, and implementation of chemical products and processes to reduce or eliminate the use and generation of substances hazardous to human health and the environment.²⁵ Strategies to address mounting environmental concerns with current synthetic approaches include: the use of environmentally benign solvents, biodegradable polymers, and nontoxic chemicals. In the synthesis of metal nanoparticles by reduction of the corresponding metal ion salt solutions, there are three areas of opportunity to engage in green chemistry: (i) choice of solvent, (ii) the reducing agent employed, and (iii) the capping agent (or dispersing agent) used. In this context, there has been increasing interest in identifying environmentally friendly materials that are multifunctional. For example, the tea/catechin used in this study functions as both a reducing and capping agent for Ag nanospheres. In addition to its high water solubility, low toxicity, and biodegradability, tea is the most widely used behaviorally active drug in the world. Green- tea catechins (GTCs) are groups of polyphenol compounds belonging to the flavonoid family. (GTCs) include (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC) and (−)-epigallocatechin gallate (EGCG), and possess various biological activities. However, until recently²⁶ there were no reports on the preparation of noble metals using tea extract, both of which play a crucial role in many medical applications.

With growing concerns on the biological and environmental impact of nanomaterials, the focus of creating nontoxic, “green” nanoparticles has increased significantly.^25,27–30 It is well-known that the ancient Greeks and Romans took advantage of silver's antimicrobial effects and used silver particles to fight infections. What is slightly less well-known is there are around 1000 commercially available products which contain some form of silver nanotechnology, ranging from topological creams and cosmetics, to antimicrobial socks and household cleansers. In addition, silver ions are the most used antimicrobial treatment for critical burns.⁶ However, previous studies have indicated that silver nanoparticles have a size-dependent cytotoxicity, smaller particles being the most toxic. Additionally, the mechanism of this cytotoxicity was shown to be reactive oxygen species (ROSs).³¹ Furthermore, it has been shown that silver nanoparticles exhibit increased cytotoxic effects with increased concentration.³² It has also been shown that surface modifications of silver nanoparticles can dramatically alter the toxicity.³³ In light of these studies and the ever-increasing probability of exposure to silver nanoparticles, it is imperative to develop nanoparticles that pose minimal risks to workers and consumers and that can be synthesized in an environmentally friendly and sustainable manner.

The goal of this study was to synthesize biocompatible, environmentally friendly silver nanoparticles. Silver nanoparticles were synthesized using a wet chemistry technique in which epicatechin and tea extract were used as reducing and capping agents. After evaluating the consumer products currently in the marketplace, we determined the most probable route for exposure was skin contact from various household cleaners and cosmetics. Therefore, we chose to use human keratinocytes (HaCaTs) as an in vitro model of exposure. In this study we have determined that synthesis using the “green” reducing agents epicatechin or tea extract is a viable way to create silver nanoparticles by reducing the silver nitrate salt. It was also found that the silver nanoparticles synthesized using epicatechin have a prolific response in mitochondrial function while minimally damaging the membrane integrity in the HaCaT exposure model. The silver nanoparticles synthesized using tea extract produced prolific responses in the mitochondrial function of the HaCaT cell models but caused damage to the cellular membrane.

Materials and methods

“Green” synthesis of silver nanoparticles

For the tea extract preparation, 1 g of tea powder (Red label from Tata (India) Ltd. 99%) was boiled in 50 ml of water and filtered through a 25 μl Teflon filter. Then 2 ml of 0.1 N AgNO₃ (AgNO₃, Aldrich, 99%) was mixed with 10 ml of tea extract and 10 ml of water. The solution was then shaken to ensure thorough mixing. The reaction mixture was allowed to settle at room temperature. The color of the mixture changed from light brown to green, indicating the formation of Ag nanoparticles, which were stable. The procedure was repeated with 5, 3, 1 and 0.5 ml of tea extract. We extended this strategy using a pure tea component, 0.01 N (−)-epicatechin (Aldrich, 99%), and the procedure was repeated exactly as described earlier for tea extract with 0.1 N AgNO₃. Throughout this paper we will refer to the particles based on the ratio of water content to reducing agent content (i.e. the 1 : 1 sample is that in which 10 ml water was combined with 10 ml tea extract, and so on).

Transmission electron microscopy (TEM) of Ag nanoparticles

The Hitachi H-7600 Transmission Electron Microscope (TEM) was used to measure the primary particle size (diameter) of all nanoparticles in this study. 10 μl of each sample was spotted onto a carbon-coated copper grid, which was purchased from Electron Microscopy Supply (product number 080612), and then viewed at 100 kV. The diameters of 100 particles were measured using the AMT Imaging software's point-to-point measurement function, which calculates the average and standard deviation in the measurements.

X-ray diffraction studies

XRD was used to identify crystalline phases of silver solids. A PANalytical Xpert Pro θ–2θ diffractometer using Cu Kα radiation at 45 kV and 40 mA was used. Scans were typically in the range 5–70° 2θ, with 0.02° step sizes that were held for 2 s each. Pattern analysis was performed using the Jade+ software v.7 or later (MDI, Inc., Livermore, CA), which generally followed the ASTM D934-80 procedure. Reference patterns were from 2002 PDF-2 release from the ICDD (International Center for Diffraction Data, Newtown Square, PA).

UV spectrometer measurements

Samples for UV spectroscopy measurements were reaction mixtures dispersed in distilled water. UV spectra were recorded using Varian UV-visible spectrometer (Model Cary 50 Conc).

Dynamic light scattering (DLS) of Ag nanoparticles

The agglomerate diameter measurements were performed using the Malvern Instruments Zetasizer Nano-ZS instrument, using the method previously described.³⁴ Samples were dispersed in water and HaCaT Exposure Media (HEM) at a concentration of 50 μg/ml.

Keratinocyte (HaCaT) cell culture

The human keratinocyte cell line, HaCaT, was received from Dr James Duiman at USAURICD.³⁵ The HaCaT cells were cultured with RPMI-1640 media with 10% fetal bovine serum and 1% penicillin/streptomycin. The HaCaT line was incubated at 37 °C, 100% humidity, and 5% CO₂. During exposure the HaCaT line was cultured with RPMI-1640 media with 1% penicillin/streptomycin and no fetal bovine serum (HaCaT exposure media (HEM)).

Treatment protocol

Cells were seeded to provide 60–80% confluence, in 96-well plates or chamber slides, within a growth period of 48–72 h. On reaching 80% confluence, typically 48 h, cells were treated with either 50 μg/ml or 100 μg/ml of nanoparticles suspended in exposure media. After 24 h exposure the nanoparticle biocompatibility was assessed using cytotoxicity assays and cellular interaction evaluation.

Cytotoxicity assays

The HaCaT cells were exposed to the Ag nanoparticles at 50 and 100 μg/ml in exposure media. After 24 h incubation with the nanoparticles, cell proliferation was measured using the CellTiter 96^® Aqueous One Solution Cell Proliferation Assay (Promega), and membrane leakage was evaluated using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay (Promega). The CellTiter 96^® Aqueous One Solution Cell Proliferation Assay contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS(a)] and an electron coupling reagent (phenazine ethosulfate; PES). The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture. The CytoTox 96^® Non-Radioactive Cytotoxicity Assay is a colorimetric assay that. quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. Released LDH in culture supernatants is measured with a 30 min coupled enzymatic assay that results in the conversion of a tetrazolium salt (INT) into a red formazan product. The amount of colored formazan product produced is proportional to the number of lysed cells. The amount of mitochondrial function and membrane leakage was then assessed spectrophotometrically with a SpectraMAX Plus 190 microplate reader. The data are represented as an average of three independent trials ± the standard deviation. The data is normalized as a percent of control.

Cellular interaction of Ag NP

The HaCaT cells were exposed for 24 h to a 15 μg/ml concentration of silver nanoparticles. This low concentration was used to increase imaging quality by decreasing the number of Ag nanoparticles refracting light and allowing a clear image of the cell for determining cellular interactions. After exposure, cells were washed with PBS to remove any excess Ag nanoparticles that were not interacting with the cells. Slides were observed with the CytoViva™ 150 Ultra Resolution Imaging (URI) Systems using the method previously described.³⁶

Statistical analysis

The data are represented as an average of three independent trials ± the standard deviation. The cytotoxicity assays were run and any group with a p value less than 0.05 are considered significant. Statistic analysis was performed using Microsoft Excel's t-test functions.

Results

Characterization

The formation of silver nanoparticles was confirmed using UV-vis spectroscopy and X-ray diffraction. The broad plasma resonance peak around 450 nm can be assigned to silver nanoparticles (see Fig. 1). In the XRD patterns, prominent Bragg reflections at 2θ values of 38.3 and 42.6 were observed (see Fig. 2), which correspond to the (111) and (200) Bragg reflections of face-centered cubic (fcc) Ag nanoparticles.

Fig. 1 UV spectra of silver nanoparticles synthesized using tea polyphenols.

Fig. 2 XRD pattern of silver nanoparticles synthesized using tea polyphenols: (a) control tea; (b) immediately after mixing with AgNO₃ (0.1 N); and (c) overnight reacted mixture.

Epicatechin-synthesized particles

TEM analysis revealed that using epicatechin as a capping and reducing agent for metal salts is a viable procedure to produce mono-dispersed spherical nanoparticles (Fig. 3A–E). The primary particle diameter of the Ag nanoparticles produced by reducing AgNO₃ with epicatechin of varying concentrations ranged from 11 nm to 30 nm and depended on the ratio of water and epicatechin in the solution. However, similar sizes of the particles were observed for the 1 : 1 (parts water to parts epicatechin) and the 10 : 3 ratios, which both produced particles of approximately 12 nm, as well as the 2 : 1 and the 20 : 1 ratios, which produced particles of approximately 25 nm.

Fig. 3 Morphology of the nanoparticles. A) 1 : 1 ratio of water to epicatechin (scale bar: 100nm), B) 2 : 1 ratio of water to epicatechin (scale bar: 100nm), C) 10 : 3 ratio of water to epicatechin (scale bar: 100nm), D) 10 : 1 ratio of water to epicatechin (scale bar: 100nm), E) 20 : 1 ratio of water to epicatechin (scale bar: 100nm), F) 1 : 1 ratio of water to tea extract (scale bar: 20nm), G) 2 : 1 ratio of water to tea extract (scale bar: 100nm), H) 10 : 3 ratio of water to tea extract (scale bar: 20nm), I) 10 : 1 ratio of water to tea extract (scale bar: 100nm), J) 20 : 1 ratio of water to tea extract (scale bar: 20nm).

DLS analysis of the epicatechin-synthesized nanoparticles was performed to examine the agglomerate size of the nanoparticles in HaCaT exposure media, and Millipore water. For the epicatechin silver nanoparticles dispersed in HEM the agglomerate size varied greatly. For the 1 : 1, 2 : 1, and 10 : 3 ratios the extent of agglomeration was measured at 65, 53, to 33 μm respectively and for the ratios of 10 : 1 and 20 : 1 the agglomerated sizes were measured at 1270 nm and 1320 nm. While dispersed in HEM the epicatechin silver nanoparticle agglomerate size generally decreased with decreasing concentration of epicatechin. In general, for the silver nanoparticles dispersed in Millipore water, the agglomerate size is fairly constant ranging from 33 μm to 48 μm, except for the 10 : 1 ratio which had an agglomerate size of 4640 nm. An interesting note is the ratio of 10 parts water to 1 part epicatechin produced the third largest primary particle size, but consistently produced the smallest agglomerate size in both dispersants examined (summarized in Table 1).

Table 1 Summary of the characterization of epicatechin and tea extract synthesized Ag nanoparticles^a

	Sample	TEM particle size distribution (nm)	DLS agglomerate size (nm)
			Exposure media	Water
a Primary particle size evaluated with transmission electron microscopy. Agglomerate size evaluated in HaCaT exposure media (HEM), MAC exposure media (MEM) and Millipore water, using dynamic light scattering (DLS). Estimated number of particles calculated using the TEM primary particle size distribution.
Epicatechin	9% AgNO3 in 1 : 1	11.5 ± 4.7	6.51 × 10^4	3.05 × 10^4
	12% AgNO3 in 2 : 1	25.8 ± 15.8	5.30 × 10^4	4.78 × 10^4
	13% AgNO3 in 10 : 3	11.9 ± 3.9	3.29 × 10^4	4.48 × 10^4
	15% AgNO3 in 10 : 1	17.3 ± 7.0	1270	4640
	16% AgNO3 in 20 : 1	24.2 ± 6.5	1320	3.03 × 10^4
Tea extract	9% AgNO3 in 1 : 1	A. 91.3 ± 20.9	805	510
		B. 6.7 ± 2.9
	12% AgNO3 in 2 : 1	A. 59.0 ± 19.7	1370	611
		B. 9.2 ± 1.9
	13% AgNO3 in 10 : 3	A. 71.1 ± 22.3	1340	661
		B. 6.1 ± 2.4
	15% AgNO3 in 10 : 1	A. 49.8 ± 14.7	1730	487
	16% AgNO3 in 20 : 1	A. 25.9 ± 6.8	1810	220
		B. 3.8 ± 0.88

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Particles synthesized using tea extract

TEM analysis of the tea extract synthesized silver nanoparticles revealed primarily spherical nanoparticles that were poly-dispersed, with most samples having two primary particle sizes. For the 1 : 1 (parts water to parts tea extract) ratio, the larger primary particle size was measured to be 91.3 ± 20.9 nm with a smaller primary particle size of 6.7 ± 2.9 nm (Fig. 3F). For the 2 : 1 sample a large primary particle size of 59.0 ± 19.7 nm was measured with a smaller primary particle size of 9.2 ± 1.9 nm present (Fig. 3G). For the 10 : 3 sample, a larger size was measured at 77.1 ± 22.3 nm, with a smaller size at 6.1 ± 2.4 nm (Fig. 3H). The ratio of 10 : 1 was the only sample synthesized using tea extract that was found to be mono-dispersed, with a primary particle size of 49.8 ± 14.7 nm (Fig. 3I). Lastly, the 20 : 1 sample was measured to have a larger primary particle size of 25.9 ± 6.8 nm, with a smaller primary particle size of 3.8 ± 0.88 nm (Fig. 3J). In general, the primary particle size decreased when the concentration of tea extract in the solution was decreased.

DLS analysis of the tea extract synthesized silver nanoparticles was performed in HaCaT exposure media and Millipore water (Table 1). While dispersed in HEM the agglomerate sizes ranged from 805 nm (1 : 1 sample) to 1.81 μm (20 : 1 sample). Unlike the epicatechin-synthesized particles, generally the agglomerate size increased with decreasing concentration of tea extract when dispersed in HEM. When dispersed in Millipore water the agglomerate size ranged from 220 nm (20 : 1 sample) to 661 nm (10 : 3 sample). The agglomerate size increased with decreasing concentration of tea extract until the 10 : 3 ratio, and then decreased with decreasing tea extract concentration. In general the particles synthesized using tea extract had the least aggregation when dispersed in Millipore water.

Cellular uptake evaluation with CytoViva URI

Cellular interactions with epicatechin-synthesized nanoparticles. The HaCaT control (Fig. 4A) revealed healthy HaCaT cells that consisted of a confluent monolayer. When exposed to the 1 : 1 (water:epicatechin) sample, the HaCaT cells showed minimal interactions with the silver nanoparticles (Fig. 4B). The particles that were observed interacting with the cells appeared as agglomerates on the cellular membrane and had no significant effect on the HaCaT cellular morphology. When the HaCaTs were exposed to the 2 : 1 sample, more silver nanoparticles were seen interacting with the cells, but again they appeared on the membrane boundary, and caused no significant change in the morphology of the cells (Fig. 4C). Exposure to the 10 : 3 sample exhibit the most interaction between the HaCaT cells and the silver epicatechin nanoparticles. Numerous particles were observed interaction with the cell but most still remain on the cell membrane causing little to no change in cell morphology (Fig. 4D). When exposed to the 10 : 1 sample a minimal number of nanoparticles are seen, and most are on the cell membrane. However, there is a slight change in the cellular morphology; cells appear smaller and slightly elongated (Fig. 4E). When the HaCaT cells were exposed to the 20 : 1 sample the cells appear identical with the control cells in morphology and integrity. The only noticeable difference is the occasional nanoparticle seen on the cellular membrane (Fig. 4F). With all samples the silver epicatechin nanoparticles were observed interacting with the cell; it is believed that this interaction is limited to the cellular membrane and that these particles are not taken into the cell, something that we intend to confirm by future studies using confocal microscopy and TEM imaging.

Fig. 4 Morphological evaluation of HaCaT cells after treatment with epicatechin-synthesized Ag nanoparticles. A) Control cells, B) 1 : 1 ratio of water to epicatechin, C) 2 : 1 ratio of water to epicatechin, D) 10 : 3 ratio of water to epicatechin, E) 10 : 1 ratio of water to epicatechin, F) 20 : 1 ratio of water to epicatechin. Arrows indicate locations of nanoparticles.

Cellular interactions with nanoparticles synthesized using tea extract. The HaCaT control (Fig. 5A) shows cells that form a confluent monolayer. When the HaCaT cells were exposed to the 1 : 1 sample (Fig. 5B), extensive interaction was observed between the HaCaT cells and the silver tea nanoparticles, where many particles are believed to have crossed the membrane barrier but have induced no significant change in cellular morphology. Observations of the HaCaT exposure to the 2 : 1 sample revealed drastic cellular interactions with the silver nanoparticles (Fig. 5C). As in the 1 : 1 sample, the particles appear to have crossed the membrane barrier but appear to have no adverse effects on the cellular integrity. When exposed to the 10 : 3 sample the HaCaT cells showed extensive interaction with the silver nanoparticles; most particles again being internalized (Fig. 5D). When exposed to the 10 : 1 sample there was a notable decrease in the number of nanoparticles interacting with the cells (Fig. 5E). However, these particles still appear to be internalized into the cell membrane. When the HaCaT cells were exposed to the 20 : 1 sample, the cells showed a dramatic decrease in interaction with the silver nanoparticles (Fig. 5F). The few particles that are seen are still believed to be internalized by the cell with a few interacting with the membrane barrier; however, the cells appear to have a loss of connectivity with the surrounding cells. In general, interaction with silver nanoparticles decreased with decreasing concentrations of tea extract.

Fig. 5 Morphological evaluation of HaCaT cells after treatment with Ag nanoparticles synthesised using tea extract. A) Control cells, B) 1 : 1 ratio of water to tea extract, C) 2 : 1 ratio of water to tea extract, D) 10 : 3 ratio of water to tea extract, E) 10 : 1 ratio of water to tea extract, F) 20 : 1 ratio of water to tea extract. Arrows indicate locations of nanoparticles.

In vitro biocompatibility

Epicatechin-synthesized biocompatibility cytotoxicity assays. The cellular viability assay showed that in most cases the nanoparticles synthesized using epicatechin induced an increase in the mitochondrial function in the HaCaT cell line at both 50 μg/ml and 100 μg/ml concentrations (Fig. 4 A). The highest increase in mitochondrial function was measured in the 100 μg/ml concentrations, with increases between 20–30% above the control. For most of the 50 μg/ml concentration the increase in HaCaT mitochondrial function was found to be 10–20% above the control. However, the HaCaT mitochondrial function was reduced for the 1 : 1 sample at 50 μg/ml in the HaCaT cell culture, but this reduction was not seen when the concentration was increased to 100 μg/ml.

Along with cell viability, membrane integrity was examined by measuring the amount of LDH leaked by the cell. The HaCaT cells displayed minimal membrane leakage in all samples at both the 50 μg/ml and 100 μg/ml concentrations. When exposed to the 50 μg/ml of the 1 : 1, 2 : 1 and 10 : 3 samples, a decrease of 1–10% leakage below the control was measured. No significant difference was measured between the control cells membrane leakage and the 10 : 1 and 20 : 1 samples at 50 μg/ml. At a 100 μg/ml concentration of the 1 : 1 and 10 : 3 samples, the HaCaT cells exhibited an increase in membrane leakage of 10–20%. For the ratios of 2 : 1, 10 : 1, and 20 : 1 there was no significant membrane leakage at the 100 μg/ml concentration when compared to the control (Fig. 6B).

Fig. 6 A. Mitochondrial function of the HaCaT cell line exposed to epicatechin-synthesized Ag nanoparticle; B. Membrane leakage of the HaCaT cell line exposed to epicatechin-synthesized Ag nanoparticle; C. Mitochondrial function of the HaCaT cell line exposed to Ag nanoparticles synthesized using tea extract; D. Membrane leakage of the HaCaT cell line exposed to Ag nanoparticles synthesized using tea extract.

Biocompatibility cytotoxicity assays for Ag nanoparticles synthesized using tea extract. The cellular viability assay demonstrated that the nanoparticles synthesized using tea extract induced a prolific response in the mitochondrial function in the HaCaT cell line at concentrations of 50 μg/ml and 100 μg/ml (Fig. 4C). In general, the sample’s ability to stimulate mitochondrial function increased with decreased concentration of tea extract at both 50 μg/ml and 100 μg/ml concentrations. In the HaCaT cell line the highest increase in mitochondrial function was measured in the 20 : 1 ratio at 100 μg/ml, with increases of approximately 55% above the control. For the higher concentrations of tea extract the increase in mitochondrial function was between 4–20% for the 50 μg/ml concentration and 25–40% for 100 μg/ml concentration. Each sample showed a higher increase in the HaCaT mitochondrial function when exposed to 100 μg/ml than the 50 μg/ml concentration.

Again, membrane integrity was examined by measuring the amount of LDH leaked by the cell. The highest increase in membrane leakage for the HaCaT cell line was measured 20% above the control, when exposed to 50 μg/ml concentration of the 1 : 1 sample. When exposed to 50 μg/ml and 100 μg/ml the 10 : 3 and 10 : 1 samples, the HaCaT cells showed no significant increase in membrane leakage. At 100 μg/ml concentration, the other samples induced a 5–20% increase in membrane leakage (Fig. 4D).

Discussion

For nearly 5000 years tea has been championed for its antioxidant and health benefits. Many studies have suggested that the consumption of tea offers protection from developing many different types of cancer. It has been shown that, in cell-free environments, tea preparations effectively trap reactive oxygen species, reducing damage to lipid membranes, proteins, and nucleic acids .^13,37 Epicatechin is a flavonoid (Flavan) that is widely distributed in nature and is present in tea as gallocatechin, and is thought to aid against the risk of stroke, heart failure, cancer, and diabetes.³⁸ Furthermore, it is an antioxidant that protects the skin from UV damage and tumor formation.³⁹ Therefore, since nanoparticles have been shown to induce the formation of ROSs,^36,40 the antioxidant capabilities of epicatechin and tea could be used to counteract this adverse reaction.

CytoViva observations revealed few interactions between the HaCaT cells and the epicatechin-synthesized silver nanoparticles with no nanoparticles internalized by the cells. In the HaCaT cell model, no evidence of cytotoxicity was observed for the epicatechin-synthesized silver nanoparticles. Instead, there was a general increase in the levels of mitochondrial function which was inversely related to the concentration of the epicatechin. The protective and prolific reactions to these nanoparticles is likely due to the “green” synthesis method. The trends from the MTS and LDH data illustrated that cell viability was positively affected, potentially through the antioxidants protecting the cells.

In contrast to the epicatechin-synthesized nanoparticles, CytoViva observations revealed that the nanoparticles synthesized using tea extract were internalized by the cells, and that in general, the higher the concentration of tea extract the greater the cellular interaction and internalization. Despite the cells being internalized, there were no changes in cellular morphology, and there was not an apparent cytotoxic effect from the presence of the nanoparticles. When exposed to 50 μg/ml of nanoparticles synthesized using tea extract, the HaCaT cell line generally showed an increase in mitochondrial function of 0–40%, with greater increases seen in samples with lower concentrations of tea extract. At 100 μg/ml the HaCaT cell line exhibited an increase of mitochondrial function of 25–50%, with no trend based on the concentration of tea extract. The amount of LDH leakage showed an increase of less than 10% for the 10 : 3, 10 : 1, and 20 : 1 samples at both 50 μg/ml and 100 μg/ml. Exposure to the 1 : 1 and 2 : 1 samples produced an increase in LDH leakage of approximately 20% at 50 μg/ml and 15% at 100 μg/ml. Overall, the nanoparticles synthesized using tea extract appear less toxic than previously studied Ag nanoparticles.³⁶

We hypothesize that the protective and prolific reactions of these nanoparticles are a direct result of the synthetic method, and that as an artifact of the synthesis process, a coating of the epicatechin or tea extract remains on the surface of these nanoparticles. When the epicatechin is used during synthesis, residual amounts remain on the surface of these nanoparticles and can in turn upregulate superoxide dismutase, which will protect the cells from ROS damage. Similarly, the nanoparticles synthesized using tea extract can protect against ROSs, since tea has been shown to trap reactive oxygen species, which reduces their ability to damage proteins. The formation of reactive oxygen species is a normal by-product of cellular reactions, and in addition, it has been demonstrated to be a mechanism of cell death by nanoparticles. In fact, a previous study with titanium dioxide nanoparticles demonstrated that the disruption in cell viability could be prevented by pre-treatment or co-treatment with an antioxidant.⁴⁰ Therefore, it is believed that the epicatechin or tea extract present on the surface of these nanoparticles effectively inhibits the ROSs inside the cell, which has been shown to be the mechanism of cellular death, when exposed to silver nanoparticles of a similar size and shape.³⁶ Furthermore, since these cells demonstrated greater viability than the control cells, this suggests that the presence of the antioxidants also protected them from the ROSs produced normally by the cell. We have observed similar relative non-toxicity and biocompatibility profiles for the iron particles synthesized using tea polyphenols.⁴¹

Conclusions

In this study we have shown that the green synthesis of silver nanoparticles using epicatechin or tea extract as a reducing and capping agent not only produced nanoparticles in an environmentally benign process, but that these particles were non-toxic even at high concentrations (100 μg/ml). In fact, in most cases these nanoparticles created a prolific response, which is most likely a result of antioxidants being present on the surface of the nanopaticle. These preliminary in vitro studies will need to be followed up by future in vivo studies before we can accurately say they are more biocompatible, but this method of synthesis appears to be promising based on the initial in vitro studies.

Acknowledgements

We thank Col. Riddle and Col. Reilly for their strong support and encouragement for this research. Electron microscopy work was performed at the Nanoscale Engineering Science and Technology (NEST) facility at the University of Dayton. Research was funded by the Bioscience and Protection Division, Air Force Research Laboratory.

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Footnote

† Present address: Pegasus Technical Services, 46 E Hollister Street, Cincinnati, 45219, Ohio , USA.

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