According to the Joint United Nations Programme on HIV/AIDS, an estimated 33 million people were living with HIV in 2007, 2.7 million fewer than in 2001 1 . Although the rate of new HIV infections has fallen in several countries, the HIV/AIDS pandemic still stands as a serious public health problem worldwide. The emergence of resistant strains is one of the principal challenges to containing the spread of the virus and its impact on human health. In different countries, studies have shown that 5%-78% of treated patients receiving antiretroviral therapy are infected with HIV-1 viruses that are resistant to at least one of the available drugs 2 . For these reasons, there is a need for new anti-HIV agents that function over viral stages other than retrotranscription or protease activity and that can be used for treatment and prevention of HIV/AIDS dissemination 3 .

Fusion or entry inhibitors are considered an attractive option, since blocking HIV entry into its target cell leads to suppression of viral infectivity, replication, and the cytotoxicity induced by the virus-cell interaction 4 . Since 2005, only two fusion inhibitors have been approved by the FDA (Enfurtivide and Maravirovic).

In addition to fusion inhibitors, virucidal agents are urgently needed for HIV/AIDS prevention because they directly inactivate the viral particle (virion), which prevents the completion of the viral replication cycle. Virucidal agents differ from virustatic drugs in that they act directly and rapidly by lysing viral membranes on contact or by binding to virus coat proteins 5 . These compounds would directly interact with HIV-1 virions to inactivate infectivity or prevent infection and could be used as an approach to provide a defense against sexual transmission of the virus 6 .

Previously, we explored the antiviral properties of silver nanoparticles against HIV-1 and found by in vitro assays that they are active against a laboratory-adapted HIV-1 strain at non-cytotoxic concentrations. Images obtained by high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) show gp120 as its possible molecular target. Using this technique, a regular spatial arrangement of the silver nanoparticles attached to HIV-1 virions was observed. The center-to-center distance between the silver nanoparticles (~28 nm) was similar to the spacing of gp120 spikes over the viral membrane (~22 nm). It was hypothesized that the exposed sulfur-bearing residues of the glycoprotein knobs would be attractive sites for nanoparticle interaction 7 . However, the mechanism underlying the HIV-inhibitory activity of silver nanoparticles was not fully elucidated.

Nanotechnology offers opportunities to re-explore biological properties of known antimicrobial compounds by manipulation of their sizes. Silver has long been known for its antimicrobial properties, but its medical applications declined with the development of antibiotics. Nonetheless, Credés prophylaxis of gonococcal ophthalmia neonatorum remained the standard of care in many countries until the end of the 20^th century 8 . Currently, silver sulfadiazine is listed by the World Health Organization as an essential anti-infective topical medicine 9 . Silver's mode of action is presumed to be dependent on Ag⁺ ions, which strongly inhibit bacterial growth through suppression of respiratory enzymes and electron transport components and through interference with DNA functions 10 . If silver as a bulk material works, would nano-size silver be appealing? In medicine, the potential of metal nanoparticles has been explored for early detection, diagnosis, and treatment of diseases, but their biological properties have largely remained unexplored 11 .

Silver nanoparticles have been studied for their antimicrobial potential and have proven to be antibacterial agents against both Gram-negative and Gram-positive bacteria 12 13 14 15 16 , and antiviral agents against the HIV-1 17 hepatitis B virus 18 respiratory syncytial virus 19 herpes simplex virus type 1 20 and monkeypox virus 21 . The development of silver nanoparticle products is expanding. They are now used as part of clothing, food containers, wound dressings, ointments, implant coatings, and other items 22 23 ; some silver nanoparticle applications have received approval from the US Food and Drug Administration 24 .

To better understand the mode of action by which silver nanoparticles inactivate HIV-1 and their potential as a virucidal agent, we used a panel of assays that included: (i) a challenge against a panel of various HIV-1 strains, (ii) virus adsorption assays, (iii) cell-based fusion assays, (iv) a gp120/CD4 capture ELISA, (v) time-of-addition experiments, (vi) virucidal activity assays with cell-free virus, and (vii) a challenge against cell-associated virus. The data from these experiments suggest that silver nanoparticles exerted anti-HIV activity at an early stage of viral replication, most likely as a virucidal agent or viral entry inhibitor.

Silver nanoparticles proved to be an antiviral agent against HIV-1, but its mode of action was not fully elucidated. Is gp120 its principal target? Do silver nanoparticles act as entry inhibitors? In this study, we investigated the mode of antiviral action of silver nanoparticles against HIV-1. Our results reveal, for the first time, that silver nanoparticles exert anti-HIV activity at an early stage of viral replication, most likely as a virucidal agent or viral entry inhibitor.

No significant difference was found in the antiviral activities of silver nanoparticles against the different drug-resistant strains (Table 1), so the mutations in antiretroviral HIV strains that confer resistance do not affect the efficacy of silver nanoparticles. These results further agree with previous findings, where it was proven that silver nanoparticles are broad-spectrum biocides 30 31 HIV-1 strains found in the human population can differ widely in their pathogenicity, virulence, and sensitivity to particular antiretroviral drugs 32 The fact that silver nanoparticles inhibit such a varied panel of strains makes them an effective broad-spectrum agent against HIV-1. This particular property can reduce the likelihood of the emergence of resistance and the subsequent spread of infection.

Silver nanoparticles inhibited a variety of HIV-1 strains regardless of their tropism (Table 1). Variation in gp120 among HIV strains is the major determinant of differing tropism among strains, with the V3 loop of gp120 recognizing the chemokine receptors CXCR4 (T-tropic virus), CCR5 (M-tropic virus), or both (dual-tropic virus) 33 The fact that silver nanoparticles inhibited all tested strains indicates that their mode of action does not depend on this determinant of cell tropism. Elechiguerra et al. postulated that silver nanoparticles undergo specific interaction with HIV-1 via preferential binding with gp120 7 If so, then our findings show that inhibition by silver nanoparticles is not dependent on the V3 loop, which has a net positive charge that contributes to its role in determining viral co-receptor tropism 34 Since silver particles have a positive surface charge, the V3 loop would not be their preferred site of interaction. Hence, the nanoparticles may possibly act as attachment inhibitors by impeding the gp120-CD4 interaction, rather than as co-receptor antagonists that interfere with the gp120-CXCR4/CCR5 contact 4

By means of a viral adsorption assay, it was shown that silver nanoparticles' mechanism of anti-HIV action is based on the inhibition of the initial stages of the HIV-1 cycle. In addition, the gp120-capture ELISA data (Figure 1B), combined with the results of the cell-based fusion assay (Figure 1A), supported the hypothesis that silver nanoparticles inhibit HIV-1 infection by blocking viral entry, particularly the gp120-CD4 interaction. The observations previously made by STEM analysis support this idea, since silver nanoparticles were seen to bind protein structures distributed over the viral membrane 7 If silver nanoparticles do not bind to the V3 loop, then they might preferentially interact with the negative cavity of gp120 that binds to CD4 35 The attraction between CD4 and gp120 is mostly electrostatic, with the primary end of CD4 binding in a recessed pocket on gp120, making extensive contacts over ~800 Ų of the gp120 surface 36

In addition, silver nanoparticles might interact with the two disulfide bonds located in the carboxyl half of the HIV-1 gp120 glycoprotein, an area that has been implicated in binding to the CD4 receptor 37 Silver ions bind to sulfhydryl groups, which lead to protein denaturation by the reduction of disulfide bonds 38 Therefore, we hypothesize that silver nanoparticles not only bind to gp120 but also modify this viral protein by denaturing its disulfide-bonded domain located in the CD4 binding region. This can be seen in our results of silver nanoparticles' capacity to more strongly diminish residual infectivity of viral particles after 60 minutes of incubation than after 5 minutes of incubation (Figure 3). Since the antiviral effect of silver nanoparticles increases with the incubation time, we can hypothesize that silver nanoparticles initially bind to gp120 knobs and then inhibit infection by irreversibly modifying these viral structures. However, further research is needed to define if silver nanoparticles interact with the negatively charged cavity and the two disulfide bonds located in gp120's CD4 binding region.

Resistance development may be an issue for compounds that target the envelope because of the high rate of substitutions in the variable regions of the Env protein. However, since the positions of the cysteine residues, the disulfide bonding pattern in gp120, and the ability of gpl20 to bind to the viral receptor CD4 are highly conserved between isolates 39 the development of resistance to silver nanoparticles would be complicated.

By comparing the antiviral effect (measured by the therapeutic index) of silver nanoparticles with two commonly used silver salts (AgSD and AgNO₃), it was observed that silver ions by themselves are less efficient than silver nanoparticles. Hence, if the observed anti-HIV-1 activity of silver nanoparticles would just have been due to silver ions present in the nanoparticles' solution, the therapeutic index would have been lower. High activity of silver nanoparticles is suggested to be due to species difference as they dissolve to release Ag⁰ (atomic) and Ag⁺ (ionic) clusters, whereas silver salts release Ag⁺ only 40

The time-of-addition experiments further confirmed silver nanoparticles as entry inhibitors (Figure 2). In addition, it was revealed that silver nanoparticles have other sites of intervention on the viral life cycle, besides fusion or entry. Since silver ions can complex with electron donor groups containing sulfur, oxygen, or nitrogen that are normally present as thiols or phosphates on amino acids and nucleic acids 41 they might inhibit post-entry stages of infection by blocking HIV-1 proteins other than gp120, or reducing reverse transcription or proviral transcription rates by directly binding to the RNA or DNA molecules. Besides, earlier studies have shown that silver nanoparticles suppress the expression of TNF-α 42 which is a cytokine that plays a pivotal role in HIV-1 pathogenesis by incrementing HIV-1 transcription 43 The inhibition of the TNF-α activated transcription might also be a target for the anti-HIV activity of silver nanoparticles. Having such a varied panel of targets in the HIV-1 replication cycle makes silver nanoparticles an agent that is not prone to contribute to the appearance of resistant strains.

Silver nanoparticles proved to be virucidal to cell-free and cell-associated HIV-1 as judged by viral infectivity assays (Figures 3 and 4). HIV infectivity is effectively eliminated following short exposure of isolated virus to silver nanoparticles. Silver nanoparticle treatment of chronically infected H9⁺ cells as well as human PBMC⁺ resulted in decreased infectivity.

A virucide must operate quickly and effectively in preventing infection of vulnerable target cells. According to Borkow et al. (1997), an ideal retrovirucidal agent should act directly on the virus, act at replication steps prior to integration of proviral DNA into the infected host cell genome, be absorbable by uninfected cells in order to provide a barrier to infection by residual active virus, and be effective at non-cytotoxic concentrations readily attainable in vivo 44 Silver nanoparticles act directly on the virus at steps that prevent integration inside the host cell, but further pharmacokinetic, pharmacodynamic, and toxicological studies in animal models are needed to define safety parameters for the use of silver nanoparticles as preventive tools for HIV-1 transmission.

Finally, we propose that the antiviral activity of silver nanoparticles results from their inhibition of the interaction between gp120 and the target cell membrane receptors. According to our results, this mode of antiviral action allows silver nanoparticles to inhibit HIV-1 infection regardless of viral tropism or resistance profile, to bind to gp120 in a manner that prevents CD4-dependent virion binding, fusion, and infectivity, and to block HIV-1 cell-free and cell-associated infection, acting as a virucidal agent. In conclusion, silver nanoparticles are effective virucides as they inactivate HIV particles in a short period of time, exerting their activity at an early stage of viral replication (entry or fusion) and at post-entry stages. The data presented here contribute to a new and still largely unexplored area; the use of nanomaterials against specific targets of viral particles.

The authors declare that they have no competing interests.

All authors read and approved the final manuscript. HHL participated in the conception and experimental design of the in vitro HIV-1 manipulation and infectivity assays, in analysis and interpretation of the data, and in writing and revision of this report. NVAN. participated in the conception and design of the in vitro HIV-1 manipulation and infectivity assays, in analysis and interpretation of the data, and in writing and revision of this report. LIT participated in collection of in vitro HIV-1 manipulation and infectivity assays. C.R-P. participated in the experimental design of this research.