Inactivation of microbial infectiousness by silver nanoparticles-coated condom: a new approach to inhibit HIV- and HSV-transmitted infection

Abstract

Recent research suggests that today's condoms are only 85% effective in preventing human immunodeficiency virus (HIV) and other sexually transmitted diseases. In response, there has been a push to develop more effective ways of decreasing the spread of the disease. The new nanotechnology-based condom holds the promise of being more potent than the first-generation products. The preliminary goal of this study was to develop a silver nanoparticles (Ag-NPs)-coated polyurethane condom (PUC) and to investigate its antimicrobial potential including the inactivation of HIV and herpes simplex virus (HSV) infectiousness. The Ag-NPs-coated PUC was characterized by using ultraviolet-visible spectrophotometry, Fourier transform-infrared spectroscopy, high-resolution scanning electron microscopy, and energy-dispersive analysis of X-ray spectroscopy. Nanoparticles were stable on the PUC and not washed away by water. Morphology of the PUC was retained after coating. The NP binding is due to its interaction with the nitrogen atom of the PUC. No significant toxic effects was observed when human HeLa cells, 293T and C8166 T cells were contacted to Ag-NPs-coated PUC for three hours. Interestingly, our results demonstrated that the contact of the Ag-NPs-coated PUC with HIV-1 and HSV-1/2 was able to efficiently inactivate their infectiousness. In an attempt to elucidate the antiviral action of the Ag-NPs, we have demonstrated that the anti-HIV activity was primarily mediated by the Ag-NPs, which are associated with the PUC. In addition, the data showed that both macrophage (M)-tropic and T lymphocyte (T)-tropic strains of HIV-1 were highly sensitive to the Ag-NPs-coated PUC. Furthermore, we also showed that the Ag-NPs-coated PUC was able to inhibit the growth of bacteria and fungi. These results demonstrated that the Ag-NPs-coated PUC is able to directly inactivate the microbe's infectious ability and provides another defense line against these sexually transmitted microbial infections.

Keywords: HIV-1; HSV-1/2; antimicrobial; condom; silver nanoparticles.

Figure 1

Photographs of a PUC (A, left) and an Ag-NPs-coated PUC (A, right). Yellow color in (B) is due to the saturation coverage of Ag-NPs. Spot EDAX analysis (B) confirmed the presence of elemental silver on the surface of the PUC. SEM of PUC (C) and PU coated with Ag-NPs (D). Abbreviations: Ag-NPs, silver nanoparticles; EDAX, energy-dispersive analysis of X-ray; PUC, polyurethane condom; SEM, scanning electron microscopy.

Figure 2

(A) Diffused reflectance spectra of PUC before (control) and after coating with Ag-NPs. (B) FTIR spectra of noncoated (control) and coated PUC. Note: The slight shift in N–H peak after coating with Ag-NPs clearly indicates binding is due to the interaction between the nitrogen of the N(H) bond of polyurethane and Ag-NPs. Abbreviations: Ag-NPs, silver nanoparticles; FTIR, Fourier transform infrared; PUC, polyurethane condom.

Figure 3

Toxicity of Ag-NPs-coated PUC. HeLa cells (A) were incubated with Ag-NPs-coated or noncoated condom for 10, 30, and 180 minutes. A WST assay was performed to determine the growth of cell populations at day 1, day 2, and day 4 after incubation with Ag-NPs-coated or noncoated condom. To test the toxic effect of the Ag-NPs-coated PUC in human 293 T cells and C8166 T line, 0.5 × 10⁶ cells of 293 T (B) or C8166 T cells (C) were incubated with an Ag-NPs-coated PUC or noncoated PUC for 180 minutes. Then, the PUC was removed and the cell growth was monitored using the cell proliferation (WST-1) assay. Abbreviations: Ag-NPs, silver nanoparticles; PUC, polyurethane condom; WST, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate.

Figure 4

The inhibitory effect of the Ag-NPs-coated PUC on T-tropic HIV-1 infection in C8166 T cells. pNL4.3 GFP + virus was first incubated with the Ag-NPs-coated or noncoated condom for 10, 30, 60, or 120 minutes, in RPMI with consistent shaking. Then, the RPMI-containing viruses were used to infect CD4 + C8166 cells. The same amount of pNL4.3-GFP + virus without incubation with condom was used as positive control. At different days postinfection, HIV-1-infected GFP positive cells were observed under fluorescence microscopy (B) and the levels of HIV-1 Gag P24 in supernatants were measured by ELISA assay (A). Abbreviations: Ag-NPs, silver nanoparticles; GFP, green fluorescent protein; HIV, human immunodeficiency virus; PUC, polyurethane condom.

Figure 5

The inhibitory effect of Ag-NPs-coated PUC on M-tropic HIV-1 infection in HeLa β-gal-CD4 + -CCR + cells. Notes: pNL4.3-Bal virus were first incubated with Ag-NPs-coated or noncoated condom for 10, 30, or 60 minutes, in DMEM with consistent shaking. Then, the DMEM-containing viruses were used to infect HeLa β-gal-CD4 + -CCR + cells. The same amount of pNL4.3-Bal virus lacking incubation with condom was used as positive control. After 48 hours of infection, MAGI assay was used to detect HIV-1 infected cells. Abbreviations: Ag-NPs, silver nanoparticles; DMEM, Dulbecco’s modified Eagle’s medium; M, macrophage; MAGI, multinuclear activation of a galactosidase indicator; HIV, human immunodeficiency virus; PUC, polyurethane condom.

Figure 6

Ag-NPs that tightly bind to a polyurethane condom play the major role in inactivating HIV-1 infectivity. pNL4.3 GFP + virus (5 ng of GagP24) was added to wells in a 24-well plate that contains RPMI (medium) after being incubated with an Ag-NPs-coated or noncoated PUC for 30 minutes, or wells containing both RPMI and a piece of Ag-NPs-coated or noncoated PUC (1 cm²). After incubating for 30 minutes with continuous shaking, the virus-containing mediums were collected and used to infect 0.2 × 10⁶ CD4 + C8166 T cells. At day 3 postinfection, HIV-1 infectivity was examined by the measurement of the HIV-1 Gag antigen levels present in the supernatants (A) and by the observation of the virus-mediated syncytium formation and HIV-1-infected GFP + cells (B). Note: *RPMI medium was incubated with Ag-NPs-coated or noncoated PUC for 30 minutes, and then the PUC was removed. Abbreviations: Ag-Nps, silver nanoparticles; HIV, human immunodeficiency virus; GFP, green fluorescent protein; RPMI medium, Roswell Park Memorial Institute medium; PUC, polyurethane condom; cm, centimeters.

Figure 7

The inhibitory effect of the Ag-NPs-coated PUC on HSV infection. Notes: Different titers (pfu) of HSV-1 or HSV-2 virus in 200 μL of DMEM were first incubated with the Ag-NPs-coated PUC or noncoated condom for 30 minutes. Then, the virus-containing medium was used to infect Vero-E6 cells in 24-well plates. After 48 hours, the virus-induced cytopathic effects (lesion) were observed under microscope and the cytopathic lesions in five views were counted and calculated as lesions per well. (−) no HSV-induced cytopathic lesion; (+) average two cytopathic lesions/view; (++) two cytopathic lesions/view; (+++) three cytopathic lesions/view; (++++) four cytopathic lesions/view. Abbreviations: Ag-NPs, silver nanoparticles; UC, polyurethane condom; HSV, herpes simplex virus; DMEM, Dulbecco’s modified Eagle’s medium; PFU, plaque-forming units.

Figure 8

The inhibitory effect of the Ag-NPs-coated PUC on (A) bacteria (B) Candida spp. Ag-NPs-coated PUC (2 cm²) or noncoated PUC (2 cm²) material was inoculated in 3-mL Muller Hilton broth containing fresh E. coli, S. aureus, M. luteus, K. pneumoniae, or Candida spp. at a concentration of 10⁵ colony-forming units per mL (cfu/mL). Notes: After 10 minutes incubation in shaking condition, Ag-NPs-coated or noncoated PUC samples were removed from the test tubes and the bacteria cultures were incubated at 37°C for 24 hours. The sample without condom material inoculated was treated as positive control (PC). The growth inhibition of bacteria was determined by measuring optical density at 600 nm using the ELISA plate reader. Abbreviations: Ag-NPs, silver nanoparticles; PUC, polyurethane condom; ELISA, enzyme-linked immunosorbent assay; cm, centimeter; nm, nanometer.