Tannic acid modified silver nanoparticles show antiviral activity in herpes simplex virus type 2 infection

Abstract

The interaction between silver nanoparticles and herpesviruses is attracting great interest due to their antiviral activity and possibility to use as microbicides for oral and anogenital herpes. In this work, we demonstrate that tannic acid modified silver nanoparticles sized 13 nm, 33 nm and 46 nm are capable of reducing HSV-2 infectivity both in vitro and in vivo. The antiviral activity of tannic acid modified silver nanoparticles was size-related, required direct interaction and blocked virus attachment, penetration and further spread. All tested tannic acid modified silver nanoparticles reduced both infection and inflammatory reaction in the mouse model of HSV-2 infection when used at infection or for a post-infection treatment. Smaller-sized nanoparticles induced production of cytokines and chemokines important for anti-viral response. The corresponding control buffers with tannic acid showed inferior antiviral effects in vitro and were ineffective in blocking in vivo infection. Our results show that tannic acid modified silver nanoparticles are good candidates for microbicides used in treatment of herpesvirus infections.

Figure 1. Transmission electron microscopy (TEM) images of silver nanoparticles and DLS histograms.

(A) 13±5 (13) nm, (B) 33±7 (33) nm, (C) 46±9 (46) nm and (D) 10±1–65±10 nm AgNPs.

Figure 2. Inactivation of HSV-2 infection by tannic acid modified AgNPs is dose and size related.

(A) Schematics of dose response experiments. Viral inhibition (%) in 291.03C cells infected with HSV-2 pre-incubated for 1 h with 13 nm (B), 33 nm (C), 46 nm (D) AgNPs and unmodified 10–65 nm (E) AgNPs or respective carrier buffers at 0.5, 1, 2.5 and 5 µg/ml. At 24 h p.i. cells and supernatants were collected and titrated to determine PFU/ml in comparison to HSV-2 infected cultures. The data are expressed as means from three independent experiments ± SEM. * represents significant differences with p≤0.001.

Figure 3. Tannic acid-modified AgNPs block HSV-2 attachment and penetration.

(A) Schematics of attachment and penetration experiments. (B) Viral inhibition (%) for virus attachment and penetration experiments in 291.03C cell cultures with the use of 33 nm and 46 nm AgNPs (5 µg/ml) and 13 nm AgNPs (2.5 µg/ml) and corresponding carriers. At 24 h p.i. cells and supernatants were collected and titrated to determine PFU/ml in comparison to HSV-2 infected cultures. (C) SEM images in EDS mode of HSV-2 incubated with 13 nm, 33 nm and 46 nm AgNPs, white arrows indicate nanoparticles on the viron's surface. White bars indicate 100 nm. (D) Kinetics of AgNPs and HSV-2 interaction expressed as % of HSV-2 infected positive controls. HSV-2 aliquots were mixed with 2.5 µg/ml of 13, 33 or 46 nm AgNPs or corresponding carriers, incubated for indicated time points, then used to infect GMK-AH1 cells and determine PFU/ml in comparison to HSV-2 infected cultures. The data are shown as means from three independent experiments ± SEM. * represents significant differences with p≤0.05, while ** p≤0.001.

Figure 4. Antiviral effects of tannic-acid modified AgNPs require direct interaction.

(A) Schematics and results for pre-treatment experiments. The results are expressed as % of HSV-2 infected control in 291.03C cells pre-treated with 2.5 µg/ml of tannic acid-modified 13 nm, 33 nm and 46 nm AgNPs or respective carriers for 2 h, then infected with HSV-2. (B) Schematics for post-treatment experiments. The results are expressed as percentage of viral inhibition in HSV-2 infected 291.03C cell cultures, in which complete medium containing 33 nm and 46 nm AgNPs (5 µg/ml) and 13 nm AgNPs (2.5 µg/ml) or respective carriers were added at the indicated time points for up to 24 h. The data are shown as means from three independent experiments ± SEM. * represents significant differences with p≤0.05.

Figure 5. Tannic-acid modified AgNPs reduce HSV-2 infection in vivo.

(A) Schematics of in vivo experiments. C57BL/6 mice were infected with HSV-2 pre-incubated or not with 13, 33 and 46 nm AgNPs or corresponding carriers (5 µg/ml). (B) HSV-2 DNA titers (copies/µg DNA) in the whole vaginal tissues determined by real-time PCR at 48 h p.i. (N = 6). (C) Sizes of infected sites determined by immunohistochemistry of HSV-2 antigens. (D) Schematics of post-infection treatment at 3 and 18 h p.i. with 33 AgNPs or carrier buffer (3×100 µl at 5 µg/ml) (N = 5). (E) HSV-2 DNA titers (copies/µg DNA) in the whole vaginal tissues determined by real-time PCR at 48 h p.i. (N = 6). (F) Sizes of infected sites determined by immunohistochemistry of HSV-2 antigens (N = 5). The bars represent means from three independent experiments ± SEM. * represents significant differences with p≤0.05, while ** p≤0.001.

Figure 6. Tannic-acid modified AgNPs reduce inflammation during HSV-2 infection of C57BL/6 mice.

(A) Immunohistochemical staining for Gr-1+ cells in the vaginal tissue of control mice, mice 48 h after treatment with 13, 33 and 46 nm AgNPs or corresponding carriers (5 µg/mice); mice at 48 h p.i. with HSV-2 pre-incubated with 13, 33 and 46 nm AgNPs or corresponding carriers. (B) Numbers of Gr-1+ cells/infectious site in the vaginal tissue at 48 h p.i., HSV-2 was pretreated, or not, with 13, 33 and 46 nm AgNPs or corresponding carriers. (C) Numbers of Gr-1+ cells/infectious site in the vaginal tissue at 48 h p.i. after treatment at 3 and 18 h p.i. with 33 AgNPs or the corresponding carrier buffer. The bars represent means from three independent experiments ± SEM (N = 5). * p≤0.01.

Figure 7. Tannic-acid modified AgNPs regulate cytokine and chemokine production in a size-related manner.

TNF-α (A), IFN-γ (B), IL-6 (C), IL-10 (D) and CCL2 (E) production in the vaginal lavages of uninfected or HSV-2 infected C57BL/6 mice at 48 h with the virus dose pre-incubated or not with 13, 33 and 46 nm AgNPs or corresponding carriers. (F) INF-γ, IL-10 and CCL-2 production in the vaginal lavages from mice treated at 3 and 18 h p.i. with 33 nm AgNPs or the corresponding carrier buffer (3×100 µl of 5 µg/ml). The bars represent means from 3 separate experiments ± SEM (N = 5). * represents significant differences with p≤0.05, ** p≤0.01. n.d. - not detected.