Inhibition of Herpes Simplex Virus Type 1 Infection by Silver

Bioconjugate Chem. 2009, 20, 1497–1502

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Inhibition of Herpes Simplex Virus Type 1 Infection by Silver Nanoparticles Capped with Mercaptoethane Sulfonate Dana Baram-Pinto,†,‡ Sourabh Shukla,† Nina Perkas,† Aharon Gedanken,† and Ronit Sarid*,‡ Department of Chemistry and Kanbar Laboratory for Nanomaterials at Bar-Ilan University Center for Advanced Materials and Nanotechnology, Bar-Ilan University, 52900 Ramat-Gan, Israel, and The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 52900 Ramat-Gan, Israel. Received January 22, 2009; Revised Manuscript Received June 6, 2009

Interactions between biomolecules and nanoparticles suggest the use of nanoparticles for various medical interventions. The attachment and entry of herpes simplex virus type 1 (HSV-1) into cells involve interaction between viral envelope glycoproteins and cell surface heparan sulfate (HS). Based on this mechanism, we designed silver nanoparticles that are capped with mercaptoethane sulfonate (Ag-MES). These nanoparticles are predicted to target the virus and to compete for its binding to cellular HS through their sulfonate end groups, leading to the blockage of viral entry into the cell and to the prevention of subsequent infection. Structurally defined Ag-MES nanoparticles that are readily redispersible in water were sonochemically synthesized. No toxic effects of these nanoparticles on host cells were observed. Effective inhibition of HSV-1 infection in cell culture by the capped nanoparticles was demonstrated. However, application of the soluble surfactant MES failed to inhibit viral infection, implying that the antiviral effect of Ag-MES nanoparticles is imparted by their multivalent nature and spatially directed MES on the surface. Our results suggest that capped nanoparticles may serve as useful topical agents for the prevention of infections with pathogens dependent on HS for entry.

INTRODUCTION The application of nanotechnology in therapeutics, biological imaging, drug delivery, biosensors, and cell labeling is being intensively explored (1-6). Biologically important entities, such as proteins, antibodies, antigens, and DNA, which possess nanometer size dimensions, may serve as good candidate targets for interactions with such nanoparticles (1, 7, 8). Viruses are common nanoelement pathogens that can infect and cause diseases in plants, animals, and humans, while viral diseases present challenging problems with worldwide social and economic implications. Development of antiviral drugs able to target the virus while maintaining host cell viability is challenging (9, 10). Herpes simplex virus type 1 (HSV-1) is a common infectious agent that occurs worldwide and infects humans of all ages (11). The outcome of HSV-1 infection includes a wide variety of clinical manifestations, ranging from asymptomatic infection to oral cold sores and severe encephalitis. The HSV-1 virion consists of a 152-kbp double-stranded DNA genome enclosed by an icosahedral capsid, which is surrounded by a lipid bilayer envelope that accommodates 11-12 virally encoded glycoproteins (11, 12). The envelope diameter ranges from 170 to 200 nm and contains an array of protruding glycoprotein spikes, making the full diameter of the virion about 225 nm on average. Each virion contains 600-750 spikes, with variable packing densities (12). The mechanism of HSV entry into the cell, involving its virus-receptor interaction, is one of the most comprehensively understood routes among members of the Herpesviridae family (13). HSV entry occurs when extracellular virions attach to the cell surface via glycoprotein C (gC) and gB, promoting the binding of gD to one of three alternative cellular receptors. In turn, membrane fusion machinery com* Corresponding author. Tel: 972-3-5317853. Fax: 972-3-7384058. E-mail: [email protected]. † Department of Chemistry and Kanbar Laboratory for Nanomaterials at Bar-Ilan University Center for Advanced Materials and Nanotechnology. ‡ The Mina and Everard Goodman Faculty of Life Sciences.

prised of gB, gH, and gL is activated to mediate fusion with plasma or endocytotic membranes (11, 14). During the attachment phase, gC and gB interact independently with cellular heparan sulfate (HS). This reversible interaction likely creates multiple points of adhesion and occurs in both wild-type and laboratory viral strains (14). The affinity of the binding of gC to HS is on the order of 10-8 M and is considered to be the major binding interaction during attachment (13). When gB and gC are absent, viral binding to the cell surface is severely reduced, signifying the important role of this step for viral entry (11, 15). Accordingly, cells that are defective in HS exhibit a dramatic reduction in susceptibility to infection (13). It is worth noting that the interaction with cell surface HS has been found to be a common pathway for attachment by several other human and animal viruses as well (15). HS is a ubiquitous constituent of cell plasma membranes and extracellular matrices, which mediates various physiological processes, such as development, cell adhesion, tumorigenesis, and viral and bacterial infections (16). It is a structurally diverse, highly sulfonated polysaccharide belonging to the family of glycosaminoglycans. HS is composed of alternating sequences of glucosamine and uronic acid. The amino sugars may be N-acetylated or N-sulfated (17-19). The glucosamine residues may, in some cases, carry O-sulfated groups at C6 or C3, and the uronic acid moieties may be O-sulfated at C2 (17). HS is characterized by great structural heterogeneity in terms of chain length and size and the extent of sulfation and epimerization within the modified segments (19). Topical, oral, or intravenous nucleoside derivatives (e.g., acyclovir) have been approved for the treatment of HSV infections and are widely used. However, the emergence of resistant viral strains, mainly after prolonged treatment in immunocompromised patients, is one of the main reasons for the continuous search for new antiviral drugs that can prevent or inhibit infection by both wild-type viruses and drug-resistant strains (13, 20). Compounds that mimic HS, such as sulfated polysaccharide (e.g., dextran sulfate, pentosan polysulfate, a

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Figure 1. Representative TEM images of Ag-MES nanoparticles. (A, B) with selected area electron diffraction pattern along with the schematic representation of MES binding on the surface of silver nanoparticles (A). The scale bars in TEM images correspond to 10 nm. The size distribution of the Ag-MES nanoparticles was obtained from the TEM analysis and determined by measuring the diameter of 103 Ag-MES nanoparticles (C).

mixture of highly sulfated mannose containing di- to hexasaccharides (PI-88)) and sulfated nonpolysaccharides (e.g., lignin sulfate, poly(sodium 4-styrene sulfonate) (T-PSS)), were shown to inhibit HSV attachment to cells, suggesting that these compounds act via competition with HS chains for the binding to viral post attachment proteins. Interference with post attachmentstepsbythesecompoundshasalsobeensuggested(14,21-24). Antiviral potencies of candidate compounds are usually enhanced with an increasing degree of sulfation and the size of the oligosaccharide chain. These allow an increased likelihood of successful recognition of the saccharide with the HS-binding domain on the HSV and a simultaneous interaction of the saccharide with numerous copies of viral attachment proteins. Yet, inhibition of HSV during its cell-to-cell spread requires penetration of these compounds into a narrow intercellular space and in this case the size of the inhibitor might be a limiting factor (14). Therefore, one should consider the characteristics of the different modes of virus infection and spread during the design of antiviral compounds. There are only a few reported nanoparticle-based antiviral therapies. Yacaman and co-workers have used silver nanoparticles for the inhibition of human immunodeficiency virus (HIV) infection (25), whereas other groups have used modified silver nanoparticles to inhibit monkeypox virus (26) and hepatitis B virus (27). More recently, Melander and co-workers have used functionalized multivalent gold nanoparticles for the prevention of HIV infection (28). Here, we describe the application of a structurally defined aqueous colloidal solution of silver nanoparticles that are capped with mercaptoethane sulfonate group (Ag-MES) as a novel inhibitor of HSV-1 infection. To the best of our knowledge, the antiviral properties of ω-sulfonated

nanoparticles remain an undeveloped area, which based on our findings, is of potential medical interest.

EXPERIMENTAL PROCEDURES Particle Synthesis. Mercaptoethane sulfonate-protected silver nanoparticles (Ag-MES) were synthesized by a modified procedure with a sonochemical reaction based on that described by Zou et al. (29). AgNO3 (0.102 g, Aldrich) in 50 mL of ethanol was mixed with 0.25 g of sodium 2-mercaptoethane sulfonate (MES, Fluka Analytical) dissolved in 25 mL of ethanol and 25 mL of a double-distilled water (DDW) solution. After purging the reaction mixture with argon gas for 30 min, sonochemical irradiation (Ti horn from Sonics and Materials VCX 600, 20 kHz, 600 W at 60% efficiency) was applied for 15 min in argon atmosphere. NH3 (0.1 mL, 25%) was injected into the flask 4 min after the start of the sonication. The reaction was held at a 10 °C, using a water-ice bath. Characterization of Ag-MES Nanoparticles. The morphology of the nanostructures was characterized with a Philips CM-120 transmission electron microscope (TEM), operating at 120 kV. Images were recorded by a Gatan Ultrascan 1000 2kx2k CCD camera. The nanoparticle size distribution was further characterized using Scion Image software (Alpha 4.0.3.2) based on the TEM images. X-ray photoelectron spectroscopy (XPS) was performed using an Axis HS with monochromatic Al KR source (Kratos Analytical). Thermogravimetric analysis (TGA) measurements were conducted using a TGA model Q500 (TA Instruments), equipped with a Pt crucible, for the estimation of the amount of the MES surfactant on the surface of the silver nanoparticles.

Inhibition of HSV-1 Infection by Ag-MES

Figure 2. Thermogravimetric analysis (TGA) of Ag-MES nanoparticles depicting percentage weight loss of the sample as a function of temperature.

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Figure 4. Cell toxicity assay. Cell viability was determined by the XTT assay. The viability of the cells was calculated as percentage relative to control cells, which were not treated with Ag-MES nanoparticles. Average results obtained for control batches were defined as 100% viability, and average values obtained for cells treated with Ag-MES nanoparticles were calculated accordingly. Measurements were taken after 24, 48, and 72 h at different Ag-MES nanoparticle concentrations (25-800 µg/mL). Similar results were obtained in two independent experiments.

plaque forming units (pfu)/well) in the presence of different concentrations of Ag-MES or MES at 37 °C. After 45 min incubation to allow for virus adsorption, the cells were overlaid with 500 µL of MEM-Eagle’s medium supplemented with 2% heat-inactivated FCS and 0.1% human γ-globulin (ZLB Behring GmbH, Marburg, Germany) with Ag-MES nanoparticles or MES for maintaining final concentration in each well. Controls included mock-infected cell monolayers with and without Ag-MES nanoparticles or soluble MES. After 2-3 days of incubation, the cell monolayers were stained with Giemsa stain, and the plaques were counted. All determinations were performed in four replicas. Figure 3. XPS spectra of the S 2p region of Ag-MES nanoparticles. Binding energy (BE) of 161.98 eV corresponds to the thiol group of MES, and BE of 167.77 eV corresponds to the sulfonate group of MES.

Cells and Virus. Vero African green monkey kidney epithelial cells were grown in minimum essential media (MEM)Eagle’s medium (Biological Industries, Israel) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Biological Industries), L-glutamine (Biological Industries), and penicillinstreptomycin-amphotericin (PSA) (Biological Industries). Cells were maintained at 37 °C under 5% CO2. The virus used was a wild-type HSV-1 McIntyre strain. Cell Toxicity Assay. The toxicity of the Ag-MES nanoparticles to Vero cells was examined using a cell proliferation kit with an XTT-based colorimetric assay that measures mitochondrial activity, according to the manufacturer’s instructions (Biological Industries). Optical density (OD) measurements for calculating cell viability percentage were taken using a TECAN Spectrafluor Plus (NEOTEC Scientific Instrumentation Ltd.) spectrophotometer at 405 nm wavelength. For the assay, Vero cell monolayers were grown in 96-well plates (2.5 × 104 cells/well) with 5% FCS in MEM-Eagle’s medium. Ag-MES nanoparticles were added into each well at different concentrations (25-800 µg/mL in a total volume of 150 µL/well). The control consisted of Vero cell monolayers with no Ag-MES nanoparticles. HSV-1 in vitro Assays. Viral infection was evaluated by plaque assay on Vero cells. Cells were plated in 24-well dishes (4.5 × 104 cells/well). Confluent monolayers of cells were inoculated with 250 µL of virus suspensions (25, 250, or 2500

RESULTS AND DISCUSSION Synthesis and Characterization of Ag-MES Nanoparticles. An aqueous dispersion of silver nanoparticles capped with sodium 2-mercaptoethane sulfonate was synthesized as described in the Experimental Procedures section. Transmission electron microscope (TEM) images of the Ag-MES nanoparticles revealed moderately polydispersed spherical nanoparticles with an average size of 4 ( 1 nm, while the selected area electron diffraction confirmed the presence of metallic silver (Figure 1). MES is expected to bind to the silver surface through the thiol (-SH) group, while the sulfonate end remains free and faces the solvent to target the virus (Figure 1). Thermogravimetric analysis (TGA) measurement on Ag-MES nanoparticles was conducted at a temperature range of 25-900 °C to estimate the amount of the MES on the surface of the silver. Weight loss (%) as a function of the temperature is shown in Figure 2. A total of 10.74% of the initial weight loss was observed over a range of 50-100 °C. We attribute this loss to access of MES molecules that were merely physically adsorbed to the surface of the nanoparticles and to solvent water molecules. A further weight reduction of 30.86% was detected at the temperature range of 320-550 °C. This loss occurred in two main steps: 22% of the weight was lost in the range of 320-370 °C and ∼10% between 320 and 550 °C. We assign this weight reduction, at such high temperatures, to the removal of MES molecules that were chemically bonded to the silver core. Based on the particles size obtained using TEM images and TGA analysis, we calculated that an average single Ag-MES nanoparticle covered with a full monolayer of MES contains 141 MES molecules. These calculations are based on previous

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Figure 5. Microscopic and quantitative analysis of effects of Ag-MES nanoparticles toward infection with HSV-1. (A) Optical microscopic image (×10 magnification) demonstrating the effect of Ag-MES nanoparticles on HSV-1 infectivity. Cells were infected with HSV-1 in the absence or the presence of 400 µg/mL Ag-MES nanoparticles. Mock cells were similarly treated, but no virus was added. Pictures were taken 48 h following infection. (B) Quantitative analysis of the Ag-MES inhibition efficiency; a, b, c, and d represent the number of plaques in cell cultures not treated with Ag-MES nanoparticles (mock, 25, 250, and 2500 pfu, respectively); a*, b*, c*, and d* refer to the number of plaques in cell cultures infected with similar viral loads in the presence of 400 µg/mL Ag-MES nanoparticles (mock, 0, 0, and 67 ( 23, respectively).

Figure 6. Effect of soluble MES on HSV-1 infection. Cells were infected with HSV-1 in the absence (a) or the presence (b) of 96 µg/mL MES. Pictures were taken 48 h following infection. Results represent four repeats for each condition. A t test for the two independent conditions (a and b) showed no significant difference in the diameter of the plaques; p ) 0.372, n ) 112.

Figure 7. Schematic representation of a proposed mode of HSV-1 inhibition by Ag-MES nanoparticles.

surface coverage measurements carried out with alkanethiolates on metallic nanoclusters (30-32). The calculated mass percentage of a full MES monolayer on a silver core nanoparticle is therefore 24.1%. TGA measurements showed a 30.86% weight reduction, suggesting that synthesized Ag-MES nanoparticles were fully covered with a MES monolayer. Figure 3 illustrates the XPS spectrum of S 2p region of Ag-MES nanoparticles. Sulfur appears in two main oxidation states: thiol group binding energy appears as a doublet (2p3/2 and 2p1/2) at 161.98 eV and sulfonate group binding energy appears as a doublet (2p3/2 and 2p1/2) at 167.77 eV. A third, relatively negligible (5.81%), oxidation state appears as well. Assuming that MES molecules were not altered during Ag-MES

nanoparticle synthesis, each molecule carries one thiol group and one sulfonate group. Therefore, a 1:1 stoichiometric relation between the two sulfur groups would be expected. However, the XPS spectrum indicates a different ratio, with a larger sulfonate peak area than that of the thiol. This result indicates that most of the MES molecules are bonded to the silver core through thiol groups. Cytotoxocity Assay. The toxicity of the Ag-MES nanoparticles to mammalian cells was of high concern. We tested the toxicity of the particles on kidney epithelial Vero cells at a concentration range of 25-800 µg/mL using the colorimetric XTT assay. Results were compared with cells that were not treated with Ag-MES nanoparticles (control). Figure 4 presents the results calculated as the percentage of viable cells relative to control cultures for cells incubated with Ag-MES nanoparticles for 24-72 h. As indicated by near 100% cell viability, Ag-MES nanoparticles did not affect the mitochondrial activity of the cells at any time point or concentration. Inhibition of HSV-1 Infectivity by Ag-MES Nanoparticles. Vero cells show a typical cytopathic effect when infected with HSV-1. Therefore, to evaluate the antiviral activity of the nanoparticles, we used a plaque reduction assay in these cells. Cells were infected with virus suspensions (25-2500 pfu/well) in the absence or presence of different Ag-MES concentrations. Partial inhibition of plaque formation was observed when 200 µg/mL Ag-MES nanoparticles was used. Increased inhibition

Inhibition of HSV-1 Infection by Ag-MES

was observed in the presence of 400 µg/mL Ag-MES nanoparticles, and no further inhibition was obtained when 800 µg/ mL Ag-MES nanoparticles was added. As shown in Figure 5A, infection with HSV-1 was almost completely blocked in the presence of 400 µg/mL nanoparticles. At low virus loads (25 and 250 pfu), infected cell cultures that were treated with Ag-MES nanoparticles demonstrated a similar morphology to control mock infected cells, with no evidence of any plaque formation. In contrast, infected cells that were not treated with Ag-MES nanoparticles demonstrated plaques, with increasing diameters during the experiment. At high virus loads (2500 pfu), very small plaques were observed in cultures treated with Ag-MES nanoparticles, while complete destruction of the culture was observed in the absence of Ag-MES nanoparticles, indicating that Ag-MES nanoparticles act as inhibitors of infection. Figure 5B presents quantitative analysis of the Ag-MES inhibition efficiency calculated according to three independent experiments. The number of plaques was counted in infected cell cultures that were treated with Ag-MES nanoparticles and compared with plaque numbers extrapolated from virus titrations in cell cultures not treated with Ag-MES nanoparticles. It is evident that at low virus loads (25 and 250 pfu), viral infection was completely blocked by the Ag-MES nanoparticles. At higher virus loads (2500 pfu), an average of 67 ( 23 plaques/well was observed, which is nearly a 97% decrease in the number of plaques relative to cell cultures that were not treated with Ag-MES nanoparticles. Furthermore, the observed plaques were tiny compared with the plaques obtained in the absence of Ag-MES nanoparticles. This suggests the efficiency of the Ag-MES particles in blocking HSV-1 infection. To compare the effect of the soluble MES with that of the nanoparticles, infection was carried out in the presence of 96 µg/mL MES, which is equivalent to the amount of MES incorporated in 400 µg/mL Ag-MES nanoparticles (Figure 6). However, soluble MES was found to be completely ineffective as an HSV-1 inhibitor after 48 or 72 h as indicated by the similarities in the number of plaques and their diameter in comparison to infected cells that were not treated with MES (t test for the two independent conditions showed no significant difference in the diameter of the plaques, p ) 0.372, n ) 112). Taken together, using viral plaque reduction assay in Vero cells, we show that infection with HSV-1 was almost completely blocked in the presence of the silver nanoparticles capped with MES. In contrast, equivalent concentration of soluble MES molecules, a clinically approved detoxifying agent for inhibition of hemorrhagic cystitis (33), which constitute our nanoparticle capping molecule, was found ineffective in preventing HSV-1 infection. We believe that the nanoparticles decorated with MES serve as multivalent inhibitors that mimic HS on the host cell membrane and, therefore, that the nanoparticles efficiently interact with HSV-1. This in turn prevents the infection of HSV-1 by blocking viral attachment to the cells as illustrated in Figure 7. Thus, one silver nanoparticle containing numerous functional groups can potentially target more than one virus. This spatial arrangement substantially enhances the effectiveness of the coated nanoparticles. Multivalent particles present the additional advantage of creating a high local concentration of binding molecules, thereby favoring the formation of more virus-particle pairs (28, 34, 35). In addition, we suggest that the free rotation of the soluble MES considerably reduces the interaction of the sulfonate groups with the target virus, whereas the sulfonate groups in the colloidal solution of the Ag-MES nanoparticles are directed toward the viral glycoproteins. Furthermore, steric effects involving shielding of other viral

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components that may assist in the attachment process could contribute to the inhibition observed. The exact inhibitory mechanism will be determined in future studies. Since several pathogens have been shown to exploit cell-surface HS as primary attachment sites during host invasion, Ag-MES nanoparticles may potentially be applied for the topical prevention of other infectious agents, such as genital HSV-2 and HIV. Moreover, alternative nanoparticles constructs, comprised of different core materials and sulfonated surfactants, may be designed based on our prototype and assayed for their antiviral activity.

ACKNOWLEDGMENT This work will be submitted to Bar Ilan University, Ramat Gan, Israel, by D. Baram-Pinto in partial fulfillment of the degree of Doctor of Philosophy.

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