In Vitro Evaluation of Colloidal Silver on Immune Function : Antilymphoproliferative Activity

Colloidal silver (AgC) is currently used by humans and it can be internalized through inhalation, injection, ingestion, and dermal contact. However, there is limited information about immunological activity; more investigations using colloidal silver are needed. In the present study, the effects of AgC (17.5 ng/mL) on immunological parameters (proliferation and immunophenotyping) using human peripheral bloodmononuclear cells (PBMC) andmacrophages (phagocytosis) and cytotoxicity on leukemia and lymphoma cancer cell lines (1.75 to 17.5 ng/mL) were investigated. AgC was observed to significantly (p < 0.05) decrease interleukin-2 (IL-2) production and proliferation induced by phytohemagglutinin or concanavalinA in PBMCwithout affecting its cell viability butwith cytotoxic effect on cancer cells. IL-2, IL-4, IL-6, IL-10, INF-γ, and IL-17A cytokines production andCD3, CD3CD19, CD3CD4, CD3CD8, and CD16CD56 PBMC phenotypes were not affected by AgC. The present study demonstrates that colloidal silver is harmless and nontoxic to the immune system cells and its ability to interfere with the immune response by decreasing cell proliferation when stimulated with mitogens demonstrated the antilymphoproliferative potential of AgC.


Introduction
Bioactivity of substances has been screened to identify properties related to the antitumoral or antiproliferative action [1].Products with this activity have been used in clinical practice for the treatment of cancer or diseases related to inflammation such as autoimmunity.Silver products have been used for thousands of years for hygiene and as antimicrobial agents.Since 1964, colloidal silver (AgC) has been registered as a biocidal material in the United States [2]; in Mexico, AgC is commonly used as disinfectant of water and food for human consumption [3].Generally, these products are a mix of metallic nanoparticles with oxidation state of zero (Ag +0 ) and silver salts with oxidation states of Ag +1 , +2, or +3 [4].There are studies indicating that the antibacterial [5] and antitumor properties depend on the oxidation states of silver [3].Silver nanoparticles (AgNPs) and silver ions (Ag + ) have different levels of toxicity due to their superficial charge interactions.Silver ions are more toxic because they can interact with negative groups of proteins, producing structural changes on the cellular membrane and cytoplasmic proteins, while AgNPs can interact with DNA, causing damage and structural blocking; some studies have proposed that these nanostructures can produce increased toxicity at long exposition times due to the fact that AgNPs in aqueous solutions can oxidize and release silver ions; the successful use of colloidal silver solutions can be explained by this action mechanism [6,7].The anticancer activity of AgC on MCF-7 cancer cells is probably due to induced apoptosis by decreasing lactate dehydrogenase and increasing superoxide dismutase activities; in contrast, in PBMC, lactate dehydrogenase activity decreased with AgC, but it did not correlate with cell death [3].There is scarce scientific information about immunological and cancer parameters in humans regarding the effects of colloidal silver, as a mix of nanoparticles and ions, compared to silver nanoparticles

Colloidal Silver (AgC).
Grenetine-stabilized colloidal silver was purchased from MICRODYN (México) as a 0.35% stock solution.It was sterilized by filtration (0.2 m filter, Millipore, USA) and diluted to a concentration of 10.5 g/mL with RPMI-1640, supplemented with 10% FBS for in vitro assays.

Reagents.
Phytohemagglutinin (used at doses of 5 g/mL) and concanavalin A (used at doses of 5 g/mL) were purchased from Sigma-Aldrich (St. Louis, MO, USA).Doxorubicin (LEMERY S.A. de C.V., México) was stored as 3.4 mM stock solution in RPMI 1640 at room temperature and LPS (E. coli 0111:B4, Sigma-Aldrich) was prepared at 10 ng/mL to treat human peripheral blood mononuclear cells.

AgC Characterization.
The morphology of colloidal silver was investigated with field emission scanning electron microscope (SEM) (Nova NanoSEM200 FEI).Colloidal solution (50 L) was placed on a carbon tape and dried at room temperature.Nanoparticle size and distribution measurements were determined using a dynamic light scattering (DLS) performed by a nanosizer NS 90 Malvern Instruments (Malvern Instruments, UK); size distributions given by DLS were reported as percentage of intensity, and results were presented as the mean value of at least three measurements.Resonance plasmon measured by UV-vis was observed in a range between 300 and 600 nm using NanoDrop Spectrophotometer 2000 (Thermo Fisher Scientific).

Isolation of Peripheral Blood Mononuclear Cells (PBMC).
Blood from healthy human volunteers was obtained with heparinized syringes and placed into sterile polypropylene tubes.PBMC were further isolated with Histopaque 1.077 density gradient centrifugation at 400 g for 30 min at 25 ∘ C (Sigma-Aldrich).PBMC were washed twice with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (referred as complete RPMI medium) at 250 g for 10 min at 25 ∘ C. 2.5.Cell Viability.PBMC were adjusted to 1 × 10 6 cells/mL in complete RPMI medium, and 17.5 ng/mL of colloidal silver was added and incubated for 72 h at 37 ∘ C and 5% CO 2 atmosphere.Thereafter, the supernatant was removed by centrifugation at 250 g.Cells were then washed twice with complete RPMI medium.Cell viability was determined by the colorimetric MTT reduction assay, and optical densities resulting from formazan production were read at 570 nm.Cell viability was expressed as percentage viability compared with the untreated control.Results were given as the mean ± SD of three independent experiments.K562 (chronic myelogenous leukemia), MOLT-4 (acute lymphoblastic leukemia), Ramos (Burkitt's lymphoma), and L5178Y (lymphoma) cancer cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in complete RPMI medium.Cells were grown at 37 ∘ C and 5% CO 2 atmosphere.Cancer cell lines (5 × 10 3 cells/well) were plated on 96-well plates and incubated for 24 h at 37 ∘ C in 5% CO 2 atmosphere.
After incubation, the culture medium was removed, and colloidal silver was added at concentrations ranging from 1.75 to 17.5 ng/mL.The plates were then incubated for 5 h at 37 ∘ C and 5% CO 2 atmosphere.Thereafter, the supernatant was removed and cells were washed twice with RPMI 1640 medium.Cell viability was determined by the trypan blue exclusion method, and cytotoxicity was expressed as 50% (LD 50 ) and 100% (LD 100 ) cell growth inhibition, compared with untreated control.Results were given as the mean ± SD of three independent experiments.2.6.Cytokines Assay.Supernatants from AgC-treated PBMC were analyzed for cytokines levels.IL-2, IL-4, IL-6, IL-10, IFN-, TNF-, and IL-17A human cytokines were determined by flow cytometry (Accuri C6, BD Bioscience, CA, USA), using CBA Th1/Th2/Th17 Cytokine Kit BD6 (BD Bioscience, Bedford, MA, USA), following manufacturer's instructions.CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA).Protein values were converted to NIBSC/WHO protein standards for further comparisons.

Proliferation and IL-2 Assay. PBMC were isolated by
Histopaque density gradient centrifugation, washed three times with PBS, and resuspended in Diluent C at 10 6 cells/mL.The cell suspension was then diluted with the same volume of 2.5 mM PKH26 dye stock (Sigma, St. Louis, MO) prepared in Diluent C, incubated for 3 min at room temperature, then added to an equal volume of FBS, and incubated at room temperature for a further 2 min to stop the labeling, according to " Rimaniol et al., 2003 [8]."Thereafter, PBMC were adjusted at concentrations of 1 × 10 6 cells/mL, treated with colloidal silver at a concentration of 17.5 ng/mL, PHA or Con A, and incubated for 72 h at 37 ∘ C and 5% CO 2 atmosphere; the cellular proliferation was determined by flow cytometry.The amounts of IL-2 content in supernatants of PBMC were measured by Human IL-2 High Sensitivity ELISA kit (limit of detection 0.4 pg/mL) and used according instructions of manufacturer (eBiosciences, Vienna, Austria).

Determination of Nitrite/Nitrate
Production.Nitrites and nitrates levels were measured by the colorimetric Griess reaction using nitrate reductase (Nitrate/Nitrite Colorimetric Assay Kit Cayman, USA) according to the manufacturer's instructions.Serum nitrite/nitrate levels were expressed as nM/200 L.

Flow Cytometry Analysis of Cell Surface Antigens.
PBMC were adjusted at concentrations of 1 × 10 6 cells/mL and treated with colloidal silver at the concentration of 17.5 ng/mL, and the plates were incubated for 72 h at 37 ∘ C and 5% CO 2 atmosphere.Thereafter, cells were collected by centrifugation at 600 g for 10 minutes and one set of antibodies CD3 + FITC/CD8 + , PE/CD45 + , and PerCP/CD4 APC or another set of antibodies CD3 + FITC/CD16 + , CD56 PE/CD45, and PerCP/CD19 APC (BD Multitest IMK Kit) were added and incubated 15 minutes at room temperature in the dark.The erythrocytes were then lysed by adding 450 L of 1x BD FACS6 lysing solution and incubating 15 minutes at room temperature in the dark.Samples were then ready to be analyzed on the cytometer.The acquisition was performed on Accuri C6 BD instrument using BD Multiset software with BD Worklist Manager software.Automated gating was used for easy analysis of each subset population.
2.10.FITC-Dextran Uptake.Dendritic cells (DCs) were generated from PBMC and allowed to adhere to 0.22 m filter-capped culture flasks (TPP, Germany).After 2 h at 37 ∘ C nonadherent cells were removed, and adherent monocytes were subsequently cultured for 5 days in RPMI-1640 supplemented with 10% FBS and 800 U/mL rhuGM-CSF (Peprotech, México) and 50 ng/mL IL-4 (Peprotech, México).Thereafter, cells were treated with colloidal silver at concentrations of 17.5 ng/mL and incubated for 72 h at 37 ∘ C and 5% CO 2 atmosphere.DCs endocytosis was evaluated by incubating 1 × 10 6 cells with 1 mg/mL FITC-Dextran (BD) for 30 min at 37 ∘ C.After washing, cells were analyzed by flow cytometry.Controls included tubes incubated with FITC-Dextran at 4 ∘ C to inhibit the endocytic process and a basal uptake performed at the 0-time point.Uptake was quantified by FACS analysis (10,000 cells per point) [5].Viability was determined by the trypan blue exclusion technique.

Statistical Analysis.
The results were evaluated by ANOVA and median cytokines levels between treatments were compared using the Mann-Whitney  test.

Colloidal Silver Characterization.
The characterization of colloidal silver dissolved in water and cell culture medium was analyzed by dynamic light scattering (DLS); colloidal silver dissolved in water showed an average size of 100 nm with a polydispersity index of 0.2; colloidal silver dissolved in cell culture medium showed an average size of 155 nm and polydispersity index of 0.23 (Figures 1(a) and 1(b)).SEM image showed particles population dissolved in water with particle size between 50 and 190 nm (Figures 1(c) and 1(d)); colloidal silver dissolved in cell culture medium showed a combination of nanoparticles and some silver salts (Figures 1(e) and 1(f)); however, the average size obtained by DLS correlated with histogram of particles and characteristic plasmon resonance (Figures 1(g), 1(h), and 1(i)).The analysis of colloidal silver suggests that this solution has semispherical shape and heterogeneous population, formed by nanoparticles and cluster formed by silver salts.Once the AgC was characterized, we proceeded to evaluate the different biological effects.

Determination of Cellular Proliferation and IL-2 Production.
First, we determined whether the cytotoxic dose previously used in breast cancer cells [3] affects lymphocytes or macrophages functions.The results showed that colloidal silver treatment did not affect significantly ( = 0.231) PBMC cell proliferation and cell count, and the treatments with mitogens Con A or PHA significantly ( = 0.05) increased the cell proliferation and cell count, compared with the untreated control; interestingly, the combined treatments with AgC/Con A or AgC/PHA significantly ( = 0.05) decreased the cell proliferation and cell count (Figures 2  and 3) of PBMC.Similar results were observed by flow cytometry and fluorescence microscopy using the fluorescent dye PKH26 (control (94%), Con A (165%), PHA (156%), AgC (91%), AgC + Con A (80%), and AgC + PHA (77%)) (Figures 3, 4, and 5).In addition, AgC treatment did not affect the IL-2 production when compared with the control (15 pg/mL) ( = 0.85), but an increase in IL-2 production was found when PBMC were stimulated with PHA (176 pg/mL) or Con A (150 pg/mL), and treatments with AgC + Con A (15 pg/mL) or AgC + PHA (13 pg/mL) decreased IL-2 production ( = 0.05) (Figure 4).

Cytotoxic Effect of AgC on Lymphoid and Leukemia Cancer
Cell Lines.Our results confirmed the antiproliferative and cytotoxic properties of AgC on leukemic (Molt-4 and K562) and lymphoma cell lines (Ramos and L5178Y) in a dosedependent manner (1.75 to 17.5 ng/mL) ( = 0.03) (Figure 6).

Peroxynitrites and Uptake of FITC-Dextran Determinations.
The colloidal silver treatment (99%) did not affect the viability of dendritic cells (Figure 7(a)) or the levels of peroxynitrites evaluated (Figure 7(b)) but increased the phagocytosis percentage (Figure 7(c)), compared with the control ( < 0.001).

Discussion
It is known that several chemotherapeutic agents used in cancer treatment (cyclophosphamide, vinblastine, vincristine, bleomycin, and cisplatinum) have antiproliferative and cytotoxic effects; but at low doses they can stimulate the immune response [9].The effect on the immune system of any substance should be tested because any damage   Figure 3: Cellular count of PBMC treated with colloidal silver and mitogens.PBMC (1 × 10 6 cells/mL) were treated with Con A (5 g/mL), PHA (5 g/mL), AgC (17.5 ng/mL), AgC (17.5 ng/mL) + Con A (5 g/mL), or AgC (17.5 ng/mL) + PHA (5 g/mL) and incubated for 72 h at 37 ∘ C and 5% CO 2 atmosphere.Cell count was analyzed by flow cytometry.The data represent the mean ± standard deviation of three independent experiments.*  < 0.05.AgC, colloidal silver; PHA, phytohemagglutinin; and Con A, concanavalin A. Notes.Representative flow cytometric analysis of lymphoid subsets from PBMC.The data represent the mean ± standard deviation of three independent experiments.AgC, colloidal silver.
in this system could affect the body homeostasis.In the present study the analysis of colloidal silver suggests the presence of a heterogeneous population of nanoparticles and clusters formed by silver salts, probably originated from interactions with proteins contained in complete culture medium.The aggregation effect of the silver particles in biological fluids due to the presence of proteins and lipids has been reported [7].In addition our results showed that AgC (17.5 ng/mL) can inhibit the production of IL-2 induced by mitogens.Hence, the immunosuppression induced in PBMC could be mediated by the inhibition of IL-2 release.The immunosuppressant activity of some drugs such as cyclosporine and azathioprine has been corroborated by the inhibition of proliferation of lymphocytes and production of cytokines (INF-, IL-2, RANTES, TGF-, and TNF-), when lymphocytes are stimulated by reagents such as PHA, Con A, or pokeweed mitogen [10].IL-2 is an autocrine growth factor for T cells [11] and its production has been selectively inhibited by the treatment with the immunosuppressants tacrolimus (FK506) or mycophenolic acid [10].It is known that immunosuppressant agents (corticosteroids) are used for the treatment of lymphoid malignancies because they induce apoptosis of malignant lymphoid cells [12].We demonstrated the antiproliferative and cytotoxic properties of AgC (155 nm) (doses ranging from 1.75 to 17.5 ng/mL) on leukemic and lymphomatous cell lines despite previous reports which mentioned that silver particles in the range of 10-100 nm of diameters are more cytotoxic than bigger size particles [13].It is necessary to know the mechanism of selectivity between PBMC and myeloid and lymphoid origin cancer cells and future studies should be developed in the area of receptor-mediated apoptosis such as discussed by "Vega and De Maio, 2005 [14]."Because of glucocorticoid resistance in lymphoma treatment, the tumor cells continue their expansions in the presence of glucocorticoids [12].For this reason AgC could offer a new clinical option to be considered but more studies related are needed.On the other hand, our results indicate that cytokines production and the percentage of surface markers expressed by T, B, and NK cells are not affected in response to AgC (17.5 ng/mL), opposite to other immunosuppressors such as rapamycin or soraphen, for which the immunosuppressor effect is attributed to the interference of a signaling pathway and the metabolism of fatty acids [15].Furthermore, there is evidence that immune system cells can be activated in response to biomaterials although MHC/peptide/TCR induced signaling pathways are not expected.However, alternative noncognate pathways can lead to activation by cross-linking glycoproteins on the plasma membrane surface such as mitogen induced lymphocyte proliferation [16].With regard to peroxynitrites, colloidal silver (17.5 ng/mL) did not induce their production, but phagocytosis activity increased probably due to the uptake of colloidal silver in an unspecific manner.Other authors have described that silver nanoparticles in a range of 5, 10, 15, and 100 nm increase the reactive oxygen species affecting the mitochondrial function [17] and that phagocytosis of silver particles blocks cell cycle in the S-phase and stimulates inflammatory signaling through generation of reactive oxygen species, followed by secretion of TNF-, which decreased the viability of rat liver cells [18].
In conclusion, the present study demonstrates the nontoxicity of colloidal silver over immune system cells and its ability to interfere with the immune response by decreasing cell proliferation when stimulated with mitogens, suggesting that colloidal silver can be considered as an immunosuppressive agent, but more studies must be performed to establish its effectiveness and mechanism of action.

Figure 1 :
Figure 1: Size distribution of colloidal silver.(a) DLS of colloidal silver dissolved in water showed an average size of 100 nm with a polydispersity index of 0.2.(b) DLS measurements of colloidal silver dissolved in cell culture medium with an average size of 155 nm and polydispersity index of 0.23.(c, d) SEM image of particles populations dissolved in water.(e, f) SEM image of colloidal silver dissolved in cell culture medium.(g, h) Histogram of particles dissolved in water and culture medium, respectively.(i) UV-vis spectra of colloidal silver.(j) Silver salts formed in samples of colloidal silver dissolved in culture medium.DLS, dynamic light scattering; SEM, scanning electron microscopy; and a.u., arbitrary units.

Figure 6 :Figure 7 :
Figure 6: Cell viability of cancer cell lines treated with colloidal silver and mitogens.K562, Molt-4, Ramos, and L5178Y cancer cell lines (5 × 10 3 cells/well) were treated with several doses of AgC and incubated for 5 h at 37 ∘ C and 5% CO 2 atmosphere.Cell viability was analyzed by MTT assay.The data represent the mean ± standard deviation of three independent experiments.*  < 0.05.AgC, colloidal silver.

Table 2 :
Phenotypic characterization of lymphoid human PBMC subsets.