Piotr Orlowski

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

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.

Assessment of in vitro cellular responses of monocytes and keratinocytes to tannic acid modified silver nanoparticles

Hydrolyzable tannins are known to exhibit diverse biological effects, which can be used in combination with silver nanoparticles (AgNPs). In this study, we tested toxic and inflammatory properties of tannic-acid modified 13, 33, 46 nm and unmodified 10-65 nm AgNPs using murine 291.03C keratinocyte and RAW 264.7 monocyte cell lines. Both cell lines exposed for 24h to 1-10 μg/ml of 13 nm, 33 nm, 46 nm and unmodified AgNPs showed dose-dependent toxicity and decreased cell proliferation. Only small-sized AgNPs induced production of ROS by monocytes, but not keratinocytes. Monocytes internalized large aggregates of 33, 46 nm and 10-65 nm AgNPs in cytoplasmic vacuoles, whereas keratinocytes accumulated less particles. AgNPs of 13 nm were localized ubiquitously within both cell types. The tested AgNPs strongly down-regulated production of tumor necrosis factor-α (TNF-α) by monocytes, whereas keratinocytes exposed to AgNPs showed an opposite effect. Unmodified but not tannic acid-modified AgNPs increased production of the pro-inflammatory MCP-1 by monocytes and keratinocytes. In summary, low inflammatory potential and lack of ROS production by tannic-acid modified AgNPs sized above 30 nm suggests that tannic acid modification of large silver nanoparticles may help to increase AgNPs biosafety.

HSV-2 Regulates Monocyte Inflammatory Response via the Fas/FasL Pathway

Monocytic cells represent important cellular elements of the innate and adaptive immune responses in viral infections. We assessed the role of Fas/FasL in promoting monocyte apoptosis during HSV-2 infection by using an in vitro model based on the murine RAW 264.7 monocytic cell line and an in vivo murine model of HSV-2 infection applied to C57BL6, MRL-Faslpr/J (Fas−/−) and C3-Faslgld/J (FasL−/−) mice. HSV-2 infection of the monocytic cell line led to early induction of apoptosis, with no protective expression of anti-apoptotic Bcl-2. HSV-2 infected monocytes up-regulated Fas and FasL expression early during in vitro infection but were susceptible to Fas induced apoptosis. The vaginal monocytes in the HSV-2 murine model of infection up-regulated FasL expression and were susceptible to Fas induced apoptosis. HSV-2 infection of Fas and FasL- deficient mice led to decreased apoptosis of monocytes and impaired recruitment of NK, CD4+ and CD8+ T cells within the infection sites. The vaginal lavages of HSV-2 infected Fas and FasL- deficient showed decreased production of CXCL9, CXCL10 and TNF-α in comparison to HSV-2 infected wild-type mice strain. The decreased recruitment of immune competent cells was accompanied by delayed virus clearance from the infected tissue. Triggering of the Fas receptor on HSV-2 infected monocytes in vitro up-regulated the expression of CXCL9 chemokines and the cytokine TNF-α. Our study provides novel insights on the role of Fas/FasL pathway not only in apoptosis of monocytes but also in regulating local immune response by monocytes during HSV-2 infection.

The role of tannic acid and sodium citrate in the synthesis of silver nanoparticles

AbstractWe describe herein the significance of a sodium citrate and tannic acid mixture in the synthesis of spherical silver nanoparticles (AgNPs). Monodisperse AgNPs were synthesized via reduction of silver nitrate using a mixture of two chemical agents: sodium citrate and tannic acid. The shape, size and size distribution of silver particles were determined by UV–Vis spectroscopy, dynamic light scattering (DLS) and scanning transmission electron microscopy (STEM). Special attention is given to understanding and experimentally confirming the exact role of the reagents (sodium citrate and tannic acid present in the reaction mixture) in AgNP synthesis. The oxidation and reduction potentials of silver, tannic acid and sodium citrate in their mixtures were determined using cyclic voltammetry. Possible structures of tannic acid and its adducts with citric acid were investigated in aqueous solution by performing computer simulations in conjunction with the semi-empirical PM7 method. The lowest energy structures found from the preliminary conformational search are shown, and the strength of the interaction between the two molecules was calculated. The compounds present on the surface of the AgNPs were identified using FT-IR spectroscopy, and the results are compared with the IR spectrum of tannic acid theoretically calculated using PM6 and PM7 methods. The obtained results clearly indicate that the combined use of sodium citrate and tannic acid produces monodisperse spherical AgNPs, as it allows control of the nucleation, growth and stabilization of the synthesis process. Graphical abstractᅟ

Toxicity of tannic acid-modified silver nanoparticles in keratinocytes: potential for immunomodulatory applications

Hydrolyzable tannins are known to exhibit anti-inflammatory activity, which can be used in combination with silver nanoparticles (AgNPs) for dermal uses. In this study, we investigated the effects of tannic acid-modified 13, 33, 46nm and unmodified 10-65nm AgNPs using the human-derived keratinocyte HaCaT and VK2-E6/E7 cell lines in the form of stationary and spheroids cultures. After exposition to tannic acid-modified AgNPs, VK2-E6/E7 cells showed higher toxicity, increased production of reactive oxygen species (ROS) and activity of JNK stress kinase, while HaCaT cell line demonstrated less ROS production and activation of ERK kinase. AgNPs internalization was detected both in the superficial and internal layers of spheroids prepared from both cell lines. Tannic acid modified AgNPs sized above 30nm did not induce DNA breaks in comet assay performed in both cell lines. Tannic acid-modified but not unmodified AgNPs down-regulated TNF-α and LPS-triggered production of IL-8 in VK2-E6/E7 but not in HaCaT cells. In summary, tannic acid-modified AgNPs sized above 30nm show good toxicological profile both in vitro and possess immunomodulatory properties useful for potential dermal applications in humans.

Fas/FasL pathway participates in resolution of mucosal inflammatory response early during HSV-2 infection.

Apoptotic cell death is critical for maintaining integrity of the epithelia as well as for removal of the virus infected cells. We assessed the role of Fas/FasL-dependent pathway in apoptosis of genital epithelium during HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6, MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) mice and an in vitro model of HSV-2 infection in monocyte RAW 264.7 and keratinocyte 291.03C cell cultures and peritoneal macrophages. In contrast to keratinocyte in vitro cultures, HSV-2 infection of the monocytic cell cultures led to early induction of apoptosis. HSV-2 infection of peritoneal macrophages isolated from Fas- and FasL-deficient mice showed decreased activation of apoptosis, which could be further blocked by caspase-9 inhibitor. Infection of Fas and FasL-deficient mice increased the percentage of apoptotic cells and activation of caspase-9 in the vaginal tissue in comparison to C57BL6 wild type strain. Furthermore, Fas and FasL-deficient mice showed increased infiltration of neutrophiles in the vaginal mucosal epithelium at 3 and 7 day of infection in contrast to HSV-2 infected wild-type mice. Our results show that while the Fas/FasL pathway during HSV-2 infection of the vaginal epithelium plays an important role in controlling early local inflammatory response, mitochondrial apoptotic pathway also becomes activated by the inflammatory reaction.

Role of Fas/FasL signaling in regulation of anti-viral response during HSV-2 vaginal infection in mice

Fas receptor-Fas ligand (FasL) signaling is involved in apoptosis of virus-infected cells but increasing evidence accumulates on Fas receptor as a mediator of apoptosis-independent processes such as induction of activating and pro-inflammatory signals. In this study, we examined the role of Fas/FasL pathway in regulation of anti-viral response to genital HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6/J, B6. MRL-Faslpr/J and B6Smn.C3-Faslgld/J mice. HSV-2 infection of Fas- and FasL-deficient mice led to decreased migration of IFN-γ expressing NK cells and CD4+ T cells, but not of γδ T cells, into the vaginal tissue. The vaginal tissues of HSV-2 infected Fas- and FasL-deficient mice showed increased production of IL-10, followed by low expression of the early CD69 activation marker on CD4+ and CD8+ T cells and increased numbers of regulatory T cells (Tregs). Experiments in co-cultures of CD4+ T cells and bone marrow derived dendritic cells showed that lack of bilateral Fas-FasL signaling led to expansion of Tregs and increased production of IL-10 and TGF-β1. Our results demonstrate that Fas/FasL can regulate development of tolerogenic dendritic cells and expansion of Tregs early during HSV-2 infection, which further influences effective anti-viral response.

Involvement of Fas/FasL pathway in the murine model of atopic dermatitis

Objective and designThe aim of this study was to elucidate the role of apoptosis mediated through Fas/FasL pathway using the mouse model of atopic dermatitis (AD).Materials and treatment AD was induced by epicutaneous application of ovalbumin (OVA) in wild-type C57BL/6, B6. MRL-Faslpr/J (Fas−) and B6Smn.C3-Faslgld/J (FasL−) mouse strains.MethodsSkin samples were subjected to staining for Fas/FasL expression, M30 epitope and assessment of inflammatory response via immunohistochemical staining. Cytokine and chemokine production was assessed by real-time PCR.ResultsIn comparison to wild-type mice, OVA sensitization of Fas- and FasL-deficient mice led to increased epidermal and dermal thickness, collagen deposition and local inflammation consisting of macrophages, neutrophils and CD4+ T cells. Fas- and FasL-deficient mice showed increased total counts of regulatory T cells (Tregs) and IgE levels in blood as well as increased expression of IL-1β, IL-4, IL-5, IL-13 and TGF-1β mRNA in comparison to wild-type mice. On the other hand, expression of CXCL9 and CXCL10, IL-17 mRNAs in the skin samples in Fas- and FasL-deficient mice was decreased.ConclusionsOur results show that lack of the Fas-induced apoptosis leads to exacerbation of AD characteristics such as Th2 inflammation and dermal thickening. Therefore, Fas receptor can play an important role in AD pathogenesis by controlling development of the local inflammation.

Tannic acid-modified silver nanoparticles for wound healing: the importance of size

Introduction Silver nanoparticles (AgNPs) have been shown to promote wound healing and to exhibit antimicrobial properties against a broad range of bacteria. In our previous study, we prepared tannic acid (TA)-modified AgNPs showing a good toxicological profile and immunomodulatory properties useful for potential dermal applications. Methods In this study, in vitro scratch assay, antimicrobial tests, modified lymph node assay as well as a mouse splint wound model were used to access the wound healing potential of TA-modified and unmodified AgNPs. Results TA-modified but not unmodified AgNPs exhibited effective antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli and stimulated migration of keratinocytes in vitro. The tests using the mouse splint wound model showed that TA-modified 33 and 46 nm AgNPs promoted better wound closure, epithelialization, angiogenesis and formation of the granulation tissue. Additionally, AgNPs elicited expression of VEGF-α, PDGF-β and TGF-β1 cytokines involved in wound healing more efficiently in comparison to control and TA-treated wounds. However, both the lymph node assay and the wound model showed that TA-modified AgNPs sized 13 nm can elicit strong inflammatory response not only during wound healing but also when applied to the damaged skin. Conclusion TA-modified AgNPs sized >26 nm promote wound healing better than TA-modified or unmodified AgNPs. These findings suggest that TA-modified AgNPs sized >26 nm may have a promising application in wound management.

Multifunctional Tannic Acid/Silver Nanoparticle-Based Mucoadhesive Hydrogel for Improved Local Treatment of HSV Infection: In Vitro and In Vivo Studies

Mucoadhesive gelling systems with tannic acid modified silver nanoparticles were developed for effective treatment of herpes virus infections. To increase nanoparticle residence time after local application, semi solid formulations designed from generally regarded as safe (GRAS) excipients were investigated for their rheological and mechanical properties followed with ex vivo mucoadhesive behavior to the porcine vaginal mucosa. Particular effort was made to evaluate the activity of nanoparticle-based hydrogels toward herpes simplex virus (HSV) type 1 and 2 infection in vitro in immortal human keratinocyte cell line and in vivo using murine model of HSV-2 genital infection. The effect of infectivity was determined by real time quantitative polymerase chain reaction, plaque assay, inactivation, attachment, penetration and cell-to-cell assessments. All analyzed nanoparticle-based hydrogels exhibited pseudoplastic and thixotropic properties. Viscosity and mechanical measurements of hydrogels were found to correlate with the mucoadhesive properties. The results confirmed the ability of nanoparticle-based hydrogels to affect viral attachment, impede penetration and cell-to-cell transmission, although profound differences in the activity evoked by tested preparations toward HSV-1 and HSV-2 were noted. In addition, these findings demonstrated the in vivo potential of tannic acid modified silver nanoparticle-based hydrogels for vaginal treatment of HSV-2 genital infection.