Mode of antiviral action of silver nanoparticles against HIV-1

Abstract

Background: Silver nanoparticles have proven to exert antiviral activity against HIV-1 at non-cytotoxic concentrations, but the mechanism underlying their HIV-inhibitory activity has not been not fully elucidated. In this study, silver nanoparticles are evaluated to elucidate their mode of antiviral action against HIV-1 using a panel of different in vitro assays.

Results: Our data suggest that silver nanoparticles exert anti-HIV activity at an early stage of viral replication, most likely as a virucidal agent or as an inhibitor of viral entry. Silver nanoparticles bind to gp120 in a manner that prevents CD4-dependent virion binding, fusion, and infectivity, acting as an effective virucidal agent against cell-free virus (laboratory strains, clinical isolates, T and M tropic strains, and resistant strains) and cell-associated virus. Besides, silver nanoparticles inhibit post-entry stages of the HIV-1 life cycle.

Conclusions: These properties make them a broad-spectrum agent not prone to inducing resistance that could be used preventively against a wide variety of circulating HIV-1 strains.

Figure 1

Inhibition of the gp120-CD4 interaction. (A) A cell-based fusion assay was used to mimic the gp120-CD4 mediated fusion of the viral and host cell membranes. HL2/3 and HeLa-CD4-LTR-β-gal cells were incubated with a two-fold serial dilution of silver nanoparticles and known antiretrovirals. The assay was performed in triplicate; the data points represent the mean ± s.e.m. (B) The degree of inhibition of the gp120-CD4 protein binding was assessed with a gp120/CD4 ELISA capture in the presence or absence of silver nanoparticles. Gp120 protein was pretreated for 10 min with a two-fold serial dilution of silver nanoparticles, then added to a CD4-coated plate. The assay was done twice; the error bars indicate the s.e.m.

Figure 2

Time-of-addition experiment. HeLa-CD4-LTR-β-gal cells were infected with HIV- 1_IIIB, and silver nanoparticles (1 mg/mL) and different antiretrovirals were added at different times post infection. Activity of silver nanoparticles was compared with (A) Fusion inhibitors (Tak-779, 2 μM), (B) RT inhibitors (AZT, 20 μM), (C) Protease inhibitors (Indinavir, 0.25 μM), and (D) Integrase inhibitors (118-D-24, 100 μM). Dashed lines indicate the moment when the activity of the silver nanoparticles and the antiretroviral differ. The assay was performed in triplicate; the data points represent the mean and the colored lines are nonlinear regression curves done with SigmaPlot 10.0 software.

Figure 3

Virucidal activity of silver nanoparticles against M and T tropic HIV-1. Serial two-fold dilutions of silver nanoparticles were added to 10⁵ TCID₅₀ of HIV-1_Bal (A) and HIV-1_IIIB (B) cell-free virus with a 0.2-0.5 m.o.i. After incubation for 5 min and 60 min, the mixtures were centrifuged three times at 10,000 rpm, the supernatant fluids removed, and the pellets washed three times. The final pellets were placed into 96-well plates with HeLa-CD4-LTR-β-gal cells. Assessment of HIV-1 infection was made with a luciferase-based assay. The percentage of residual infectivity after silver nanoparticle treatment was calculated with respect to the positive control of untreated virus. The assay was performed in triplicate; the data points represent the mean, and the solid lines are nonlinear regression curves done with SigmaPlot 10.0 software.

Figure 4

Treatment of HIV-1 cell-associated virus. Chronically HIV-1-infected H9 (A) and PBMC (B) cells were incubated with serial two-fold dilutions of silver nanoparticles for 1 min and 60 min. Treated cells were centrifuged, washed three times with cell culture media, and then added to TZM-bl cells. Assessment of HIV-1 infection was made with a luciferase-based assay after 48 h. The assay was performed in triplicate; the error bars indicate the s.e.m.