The Influence of the AgNPs Ligand on the Antiviral Activity Against HSV-2

Abstract

Introduction: In this paper, we discuss the influence of the ligand type present on the surface of silver nanoparticles (AgNPs) on its affinity to the virus surface and its virucidal activity against herpes simplex virus type 2 (HSV-2). We selected four different ligands, which potentially exhibit different affinity to the HSV-2 virus surface and used them for functionalization of AgNPs: i) sodium citrate: ii) tannic acid; iii) 1-mercaptoundecane-1-sulfonate (MUS); iv) and poly(ethylene glycol) (PEG).

Methods: The antiviral activity was performed by in vitro Vero cell culture. Anti- inflammatory activity was performed by measurement of NF-κB activity. The antiviral potential of functional NPs in vivo was tested with HSV-2 model of genital infection. Cryo- transmission electron microscopy (cryo-TEM) was used to directly visualize the interactions or lack of interactions of functional NPs with the surface of the HSV-2 virus and to assess their affinity for the virus surface.

Results: It was found that the surface chemistry of NPs plays a key role in modulation of its interaction with the HSV-2 virus. Two of the selected ligands (sodium citrate and PEG) were inert and show no affinity to the virus surface. AgNPs functionalized with heparan sulfate-mimic ligand (MUS) showed high affinity to the virus surface, and the appearance of these interactions resulted in virus deactivation in about 50%. In the case of silver nanoparticles functionalized with tannic acid, the assessment of the affinity is difficult to be resolved, mainly because TA-AgNPs exhibit very strong virucidal effect (~100%) and immediately after the contact of the HSV-2 virus with those NPs the virus structure is being destroyed.

Discussion: The obtained results indicate that the high affinity of functional nanoparticles to the virus surface does not provide the high virucidal effectiveness. The most effective revealed to be TA-AgNPs which exhibit very strong virucidal effect against HSV-2 virus.

Keywords: antiviral functional nanoparticles; cryo-TEM; interactions of nanoparticles with HSV-2; surface chemistry of antiviral nanoparticles.

Graphical abstract

Figure 1

The DLS size distribution histograms and HR-STEM images of functional nanoparticles: SC-AgNPs-SC (A); PEG- AgNPs (B); MUS-AgNPs (C) TA-AgNPs-TA (D).

Figure 2

Functional-AgNPs inhibit HSV-2 infection in vitro. HSV titers (PFU/mL) in Vero cells infected with HSV-2 pre-incubated for 1 h with SC-AgNPs, PEG-AgNPs, MUS-AgNPs and TA-AgNPs functional (7.5 μg/mL or 2.5 μg/mL of functional-AgNPs). The data are expressed as means from three independent experiments ± SEM. ***represents significant differences with p ≤ 0.001, **with p ≤ 0.01, and *with p ≤ 0.05 in comparison to infected control.

Figure 3

Anti- inflammatory activity upon AgNPs treatment measured in RAW-Dual™ (IRF-Lucia/KI-[MIP-2] SEAP) cell. Two-way ANOVA test. *Represents p ≤ 0.05, and **p ≤ 0.01 in comparison to untreated control, either uninfected, HSV-1/2-infected or poly (I:C)-treated.

Figure 4

HSV-2 titers in vaginal lavages collected after 3 treatments with SC-AgNPs, TA-AgNPs, PEG-AgNPs or MUS-AgNPs or NaCl (HSV-2) at 72h post infection. HSV-2 gB titers (copies/ng DNA) were measured by qPCR (N = 6). The bars represent means ± SEM. *represents significant differences with p ≤ 0.05, **p ≤ 0.01 in comparison to untreated infected tissues.

Figure 5

Cryo-TEM images (A and B) of HSV-2 virus (unstained sample). The measured diameter of the HSV-2 virus is equal 120 nm (B).

Figure 6

Cryo-TEM images of unstained samples: HSV-2 virus after incubation with: SC-AgNPs (A), PEG-AgNPs (B) MUS-AgNPs (C) and TA-AgNPs (D). Samples imaged after about 30s of incubation of virus with functional nanoparticles. Images presenting the interaction of functional nanoparticles with HSV-2 surface.

Figure 7

Cryo-TEM images of damaged HSV-2 structure after exposition to TA-AgNPs (A and B) (30s of exposition, samples unstained; arrows indicate the separated strands of the DNA helix from destroyed structure of HSV-2 viruses).