Tannic-acid modified AgNPs reduce HSV-2 infection <i>in vivo</i>.

<p>(A) Schematics of <i>in vivo</i> 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 <i>p</i>≤0.05, while ** <i>p</i>≤0.001.</p

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

<p>(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.</p

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

<p>(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.</p

image_1_Tannic Acid-Modified Silver and Gold Nanoparticles as Novel Stimulators of Dendritic Cells Activation.PDF

<p>Silver nanoparticles (AgNPs) are promising new antimicrobial agents against a wide range of skin and mucosal pathogens. However, their interaction with the immune system is currently not fully understood. Dendritic cells (DCs) are crucial during development of T cell-specific responses against bacterial and viral pathogens. We have previously shown that tannic acid-modified silver nanoparticles (TA-AgNPs) consist of a promising microbicide against HSV-2. The aim of this study was to compare the ability of TA-AgNPs or TA-AuNPs of similar sizes (TA-Ag/AuNPs) to induce DCs maturation and activation in the presence of HSV-2 antigens when used at non-toxic doses. First, we used JAWS II DC line to test toxicity, ultrastructure as well as activation markers (MHC I and II, CD40, CD80, CD86, PD-L1) and cytokine production in the presence of TA-Ag/AuNPs. Preparations of HSV-2 treated with nanoparticles (TA-Ag/AuNPs-HSV-2) were further used to investigate HSV-2 antigen uptake, activation markers, TLR9 expression, and cytokine production. Additionally, we accessed proliferation and activation of HSV-2-specific T cells by DCs treated with TA-AgNP/AuNPs-HSV-2. We found that both TA-AgNPs and TA-AuNPs were efficiently internalized by DCs and induced activated ultrastructure. Although TA-AgNPs were more toxic than TA-AuNPs in corresponding sizes, they were also more potent stimulators of DCs maturation and TLR9 expression. TA-Ag/AuNPs-HSV-2 helped to overcome inhibition of DCs maturation by live or inactivated virus through up-regulation of MHC II and CD86 and down-regulation of CD80 expression. Down-regulation of CD40 expression in HSV-2-infected DCs was reversed when HSV-2 was treated with TA-NPs sized >30 nm. On the other hand, small-sized TA-AgNPs helped to better internalize HSV-2 antigens. HSV-2 treated with both types of NPs stimulated activation of JAWS II and memory CD8+ T cells, while TA-AgNPs treatment induced IFN-γ producing CD4+ and CD8+ T cells. Our study shows that TA-AgNPs or TA-AuNPs are good activators of DCs, albeit their final effect upon maturation and activation may be metal and size dependent. We conclude that TA-Ag/AuNPs consist of a novel class of nano-adjuvants, which can help to overcome virus-induced suppression of DCs activation.</p

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

<p>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.</p

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

<p>(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 <i>p</i>≤0.05, while ** <i>p</i>≤0.001.</p

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

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

Cytotoxicity and anti-HSV-2 activity of tannic acid-modified 13, 33, 46 nm AgNPs, unmodified 10–65 nm AgNPs or corresponding carriers in 291.03C cells.^*

<p>* The values shown are means from three independent experiments with each treatment performed in triplicate.</p>†<p>Cytotoxic effects were evaluated by neutral red assay to determine the concentration of 50% cellular cytotoxicity (CC₅₀) of the tested compounds.</p>‡<p>Antiviral effects were evaluated by plaque assay to determine the effective concentration that achieved 50% inhibition (EC₅₀) against HSV-2 infection.</p>§<p>SI. selectivity index.</p><p>CC₅₀/EC₅₀.</p

table_2_Tannic Acid-Modified Silver and Gold Nanoparticles as Novel Stimulators of Dendritic Cells Activation.PDF

<p>Silver nanoparticles (AgNPs) are promising new antimicrobial agents against a wide range of skin and mucosal pathogens. However, their interaction with the immune system is currently not fully understood. Dendritic cells (DCs) are crucial during development of T cell-specific responses against bacterial and viral pathogens. We have previously shown that tannic acid-modified silver nanoparticles (TA-AgNPs) consist of a promising microbicide against HSV-2. The aim of this study was to compare the ability of TA-AgNPs or TA-AuNPs of similar sizes (TA-Ag/AuNPs) to induce DCs maturation and activation in the presence of HSV-2 antigens when used at non-toxic doses. First, we used JAWS II DC line to test toxicity, ultrastructure as well as activation markers (MHC I and II, CD40, CD80, CD86, PD-L1) and cytokine production in the presence of TA-Ag/AuNPs. Preparations of HSV-2 treated with nanoparticles (TA-Ag/AuNPs-HSV-2) were further used to investigate HSV-2 antigen uptake, activation markers, TLR9 expression, and cytokine production. Additionally, we accessed proliferation and activation of HSV-2-specific T cells by DCs treated with TA-AgNP/AuNPs-HSV-2. We found that both TA-AgNPs and TA-AuNPs were efficiently internalized by DCs and induced activated ultrastructure. Although TA-AgNPs were more toxic than TA-AuNPs in corresponding sizes, they were also more potent stimulators of DCs maturation and TLR9 expression. TA-Ag/AuNPs-HSV-2 helped to overcome inhibition of DCs maturation by live or inactivated virus through up-regulation of MHC II and CD86 and down-regulation of CD80 expression. Down-regulation of CD40 expression in HSV-2-infected DCs was reversed when HSV-2 was treated with TA-NPs sized >30 nm. On the other hand, small-sized TA-AgNPs helped to better internalize HSV-2 antigens. HSV-2 treated with both types of NPs stimulated activation of JAWS II and memory CD8+ T cells, while TA-AgNPs treatment induced IFN-γ producing CD4+ and CD8+ T cells. Our study shows that TA-AgNPs or TA-AuNPs are good activators of DCs, albeit their final effect upon maturation and activation may be metal and size dependent. We conclude that TA-Ag/AuNPs consist of a novel class of nano-adjuvants, which can help to overcome virus-induced suppression of DCs activation.</p

table_1_Tannic Acid-Modified Silver and Gold Nanoparticles as Novel Stimulators of Dendritic Cells Activation.PDF

<p>Silver nanoparticles (AgNPs) are promising new antimicrobial agents against a wide range of skin and mucosal pathogens. However, their interaction with the immune system is currently not fully understood. Dendritic cells (DCs) are crucial during development of T cell-specific responses against bacterial and viral pathogens. We have previously shown that tannic acid-modified silver nanoparticles (TA-AgNPs) consist of a promising microbicide against HSV-2. The aim of this study was to compare the ability of TA-AgNPs or TA-AuNPs of similar sizes (TA-Ag/AuNPs) to induce DCs maturation and activation in the presence of HSV-2 antigens when used at non-toxic doses. First, we used JAWS II DC line to test toxicity, ultrastructure as well as activation markers (MHC I and II, CD40, CD80, CD86, PD-L1) and cytokine production in the presence of TA-Ag/AuNPs. Preparations of HSV-2 treated with nanoparticles (TA-Ag/AuNPs-HSV-2) were further used to investigate HSV-2 antigen uptake, activation markers, TLR9 expression, and cytokine production. Additionally, we accessed proliferation and activation of HSV-2-specific T cells by DCs treated with TA-AgNP/AuNPs-HSV-2. We found that both TA-AgNPs and TA-AuNPs were efficiently internalized by DCs and induced activated ultrastructure. Although TA-AgNPs were more toxic than TA-AuNPs in corresponding sizes, they were also more potent stimulators of DCs maturation and TLR9 expression. TA-Ag/AuNPs-HSV-2 helped to overcome inhibition of DCs maturation by live or inactivated virus through up-regulation of MHC II and CD86 and down-regulation of CD80 expression. Down-regulation of CD40 expression in HSV-2-infected DCs was reversed when HSV-2 was treated with TA-NPs sized >30 nm. On the other hand, small-sized TA-AgNPs helped to better internalize HSV-2 antigens. HSV-2 treated with both types of NPs stimulated activation of JAWS II and memory CD8+ T cells, while TA-AgNPs treatment induced IFN-γ producing CD4+ and CD8+ T cells. Our study shows that TA-AgNPs or TA-AuNPs are good activators of DCs, albeit their final effect upon maturation and activation may be metal and size dependent. We conclude that TA-Ag/AuNPs consist of a novel class of nano-adjuvants, which can help to overcome virus-induced suppression of DCs activation.</p