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

Abstract

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.

Keywords: Carbopol 974P; HSV 1/2; antiherpes activity; hydrogel; mucoadhesiveness; tannic acid modified silver nanoparticles.

Figure 1

Representative TEM micrographs of hydrogel H2/NP25 with 25 ppm TA-AgNPs concentration. The scale bar is: 500 nm (A); and 100 nm (B).

Figure 2

Plots of viscosity vs. shear rate of hydrogels with 25 ppm (H1/NP25–H4/NP25) or 50 ppm (H5/NP50–H8/NP50) TA-AgNPs concentration and commercially available vaginal gel Replens™ measured at: 25 °C (A); and 37 °C (B). Values are expressed as mean ± S.D (n = 3).

Figure 3

Hysteresis loops (expressed as shear stress vs. shear rate curves) of Carbopol 974P hydrogels with 25 ppm (H1–H4/NP25) or 50 ppm Ta-AgNPs concentration (H5–H8/NP50) and commercially available vaginal gel Replens™ measured at: 25 ± 1 °C (A); and 37 ± 1 °C (B). Values are expressed as mean ± S.D. (n = 3). Arrows indicate direction of the hysteresis loop.

Figure 4

Box-plot graphs displaying mucoadhesive properties: (A) maximum force of detachment; and (B) work of adhesion of formulations H1–H4/NP25 or H5–H8/NP50 with 25 or 50 ppm TA-AgNPs concentration; commercially available vaginal gel Replens™, 0.5% (w/w) Carbopol 974P base (Carb), and cellulose paper (CP) were used as controls. Values are expressed as median (n = 6). Box-plot extends from the first quartile to the third quartile. Whiskers represent minimum and maximum values.

Figure 5

(A) The scheme of HSV inactivation assay. (B) HSV-1 or HSV-2 inhibition (%) in HaCaT cells pre-incubated for 1 h with TA-AgNPs colloid in concentration of 5 ppm. At 24 h post infection h p.i., cells were collected and titrated to determine PFU/mL in comparison to HSV-1/2 infected cultures. DNA titers (copies/μg DNA) of: (C) HSV-1; and (D) HSV-2 in HaCaT cells infected with the virus incubated with placebo hydrogel (P₀) or TA-AgNPs-based hydrogels (H2/NP25 and H8/NP50 with 25 ppm or 50 ppm of TA-AgNPs, respectively) determined by RT-qPCR. Values are expressed as mean ± SD; * represents significant differences with p ≤ 0.05, while ** represents significant differences with p ≤ 0.01 in comparison to cells not exposed to hydrogels; †† symbolizes significant differences with p ≤ 0.01 in comparison to placebo.

Figure 6

(A) The scheme of HSV attachment and penetration experiments; (B) HSV-1 or HSV-2 attachment; and (C) penetration inhibition (expressed in %) in HaCaT cells in the presence TA-AgNPs-based hydrogels (H2/NP25 or H8/NP50 with 25 ppm or 50 ppm of TA-AgNPs, respectively) and the corresponding placebo hydrogel (P₀). At 24 hours post infection (h p.i.), cells and supernatants were collected and titrated to determine PFU/mL in comparison to HSV-1 or HSV-2 infected cultures. Values are expressed as mean ± SD; ** and *** represent significant differences with p ≤ 0.01 and p ≤ 0.001, respectively, in comparison to HSV-1 or HSV-2 infected untreated cultures, while † signifies substantial differences with p ≤ 0.05 and †† represents significant differences with p ≤ 0.01 in comparison to placebo.

Figure 7

(A) The scheme of cell-to-cell infection assay. The results of cell-to-cell infection assay expressed as DNA titers (copies/μg DNA) of: (B) HSV-1; or (C) HSV-2 in HaCaT cells, in which TA-AgNPs-based hydrogels (H2/NP25 and H8/NP50 with 25 ppm or 50 ppm of TA-AgNPs, respectively) or the corresponding placebo hydrogel (P₀) were added at 6 hours post infection (h p.i). Values are expressed as mean ± SD; ** represents significant differences with p ≤ 0.01.

Figure 7

(A) The scheme of cell-to-cell infection assay. The results of cell-to-cell infection assay expressed as DNA titers (copies/μg DNA) of: (B) HSV-1; or (C) HSV-2 in HaCaT cells, in which TA-AgNPs-based hydrogels (H2/NP25 and H8/NP50 with 25 ppm or 50 ppm of TA-AgNPs, respectively) or the corresponding placebo hydrogel (P₀) were added at 6 hours post infection (h p.i). Values are expressed as mean ± SD; ** represents significant differences with p ≤ 0.01.

Figure 8

The scheme of in vivo HSV challenge with TA-AgNPs-based hydrogels or the corresponding placebo.