Silver nanoparticles (AgNPs) are considered to be a potentially useful tool for controlling various pathogens. However, there are concerns about the release of AgNPs into environmental media, as they may generate adverse human health and ecological effects. In this study, we developed and evaluated a novel micrometer-sized magnetic hybrid colloid (MHC) decorated with variously sized AgNPs (AgNP-MHCs). After being applied for disinfection, these particles can be easily recovered from environmental media using their magnetic properties and remain effective for inactivating viral pathogens. We evaluated the efficacy of AgNP-MHCs for inactivating bacteriophage ϕX174, murine norovirus (MNV), and adenovirus serotype 2 (AdV2). These target viruses were exposed to AgNP-MHCs for 1, 3, and 6 h at 25°C and then analyzed by plaque assay and real-time TaqMan PCR. The AgNP-MHCs were exposed to a wide range of pH levels and to tap and surface water to assess their antiviral effects under different environmental conditions. Among the three types of AgNP-MHCs tested, Ag30-MHCs displayed the highest efficacy for inactivating the viruses. The ϕX174 and MNV were reduced by more than 2 log10 after exposure to 4.6 × 109 Ag30-MHCs/ml for 1 h. These results indicated that the AgNP-MHCs could be used to inactivate viral pathogens with minimum chance of potential release into environment.

With recent advances in nanotechnology, nanoparticles have been receiving increased attention worldwide in the fields of biotechnology, medicine, and public health (1, 2). Owing to their high surface-to-volume ratio, nano-sized materials, typically ranging from 10 to 500 nm, have unique physicochemical properties compared with those of larger materials (1). The shape and size of nanomaterials can be controlled, and specific functional groups can be conjugated on their surfaces to enable interactions with certain proteins or intracellular uptake (3,–5).

Silver nanoparticles (AgNPs) have been widely studied as an antimicrobial agent (6). Silver is used in the creation of fine cutlery, for ornamentation, and in therapeutic agents. Silver compounds such as silver sulfadiazine and certain salts have been used as wound care products and as treatments for infectious diseases due to their antimicrobial properties (6, 7). Recent studies have revealed that AgNPs are very effective for inactivating various types of bacteria and viruses (8,–11). AgNPs and Ag+ ions released from AgNPs interact directly with phosphorus- or sulfur-containing biomolecules, including DNA, RNA, and proteins (12,–14). They have also been shown to generate reactive oxygen species (ROS), causing membrane damage in microorganisms (15). The size, shape, and concentration of AgNPs are also important factors that affect their antimicrobial capabilities (8, 10, 13, 16, 17).

Previous studies have also highlighted several problems when AgNPs are used for controlling pathogens in a water environment. First, existing studies on the effectiveness of AgNPs for inactivating viral pathogens in water are limited. In addition, monodispersed AgNPs are typically subject to particle-particle aggregation because of their small size and large surface area, and these aggregates reduce the effectiveness of AgNPs against microbial pathogens (7). Finally, AgNPs have been shown to have various cytotoxic effects (5, 18,–20), and the release of AgNPs into a water environment could result in human health and ecological problems.

Recently, we developed a novel micrometer-sized magnetic hybrid colloid (MHC) decorated with AgNPs of various sizes (21, 22). The MHC core can be used to recover the AgNP composites from the environment. We evaluated the antiviral efficacy of these silver nanoparticles on MHCs (AgNP-MHCs) using bacteriophage ϕX174, murine norovirus (MNV), and adenovirus under different environmental conditions.

Antiviral effects of AgNP-MHCs at various concentrations against bacteriophage ϕX174 (a), MNV (b), and AdV2 (c). Target viruses were treated with different concentrations of AgNP-MHCs, and with OH-MHCs (4.6 × 109 particles/ml) as a control, in a shaking incubator (150 rpm, 1 h, 25°C). The plaque assay method was used to measure surviving viruses. Values are means ± standard deviations (SD) from three independent experiments. Asterisks indicate significantly different values (P < 0.05 by one-way ANOVA with Dunnett’s test).

This study demonstrated that AgNP-MHCs are effective for inactivating bacteriophages and MNV, a surrogate for human norovirus, in water. In addition, AgNP-MHCs can be easily recovered with a magnet, effectively preventing the release of potentially toxic AgNPs into the environment. A number of previous studies have shown that the concentration and particle size of AgNPs are critical factors for inactivating targeted microorganism (8, 16, 17). The antimicrobial effects of AgNPs also depend on the type of microorganism. The efficacy of AgNP-MHCs for inactivating ϕX174 followed a dose-response relationship. Among the AgNP-MHCs tested, Ag30-MHCs had a higher efficacy for inactivating ϕX174 and MNV. For MNV, only Ag30-MHCs displayed antiviral activity, with the other AgNP-MHCs not generating any significant inactivation of MNV. None of the AgNP-MHCs had any significant antiviral activity against AdV2.

In addition to particle size, the concentration of silver in the AgNP-MHCs was also important. The concentration of silver appeared to determine the efficacy of the antiviral effects of AgNP-MHCs. The silver concentrations in solutions of Ag07-MHCs and Ag30-MHCs at 4.6 × 109 particles/ml were 28.75 ppm and 200 ppm, respectively, and correlated with the level of antiviral activity. Table 2 summarizes the silver concentrations and surface areas of the AgNP-MHCs tested. Ag07-MHCs displayed the lowest antiviral activity and had the lowest silver concentration and surface area, suggesting that these properties are related to the antiviral activity of AgNP-MHCs.

Our previous study indicated that the major antimicrobial mechanisms of AgNP-MHCs are the chemical abstraction of Mg2+ or Ca2+ ions from microbial membranes, the creation of complexes with thiol groups located at the membranes, and the generation of reactive oxygen species (ROS) (21). Because AgNP-MHCs have a relatively large particle size (∼500 nm), it is unlikely that they can penetrate a viral capsid. Instead, AgNP-MHCs appear to interact with viral surface proteins. AgNPs on the composites tend to bind thiol group-containing biomolecules embedded in the coat proteins of viruses. Therefore, the biochemical properties of viral capsid proteins are important for determining their susceptibility to AgNP-MHCs. Figure 1 shows the different susceptibilities of the viruses to the effects of AgNP-MHCs. The bacteriophages ϕX174 and MNV were susceptible to AgNP-MHCs, but AdV2 was resistant. The high resistance level of AdV2 is likely to be associated with its size and structure. Adenoviruses range in size from 70 to 100 nm (30), making them much larger than ϕX174 (27 to 33 nm) and MNV (28 to 35 nm) (31, 32). In addition to their large size, adenoviruses have double-stranded DNA, unlike other viruses, and are resistant to various environmental stresses such as heat and UV radiation (33, 34). Our previous study reported that almost a 3-log10 reduction of MS2 occurred with Ag30-MHCs within 6 h (21). MS2 and ϕX174 have similar sizes with different types of nucleic acid (RNA or DNA) but have similar rates of inactivation by Ag30-MHCs. Therefore, the nature of the nucleic acid does not appear to be the major factor for resistance to AgNP-MHCs. Instead, the size and shape of viral particle appeared to be more important, because adenovirus is a much larger virus. The Ag30-MHCs achieved almost a 2-log10 reduction of M13 within 6 h (our unpublished data). M13 is single-stranded DNA virus (35) and is ∼880 nm in length and 6.6 nm in diameter (36). The rate of inactivation of the filamentous bacteriophage M13 was intermediate between those of small, round-structured viruses (MNV, ϕX174, and MS2) and a large virus (AdV2).

In the present study, the inactivation kinetics of MNV were significantly different in the plaque assay and the RT-PCR assay (Fig. 2b and ​andc).c). Molecular assays such as RT-PCR are known to significantly underestimate the inactivation rates of viruses (25, 28), as was found in our study. Because AgNP-MHCs interact primarily with the viral surface, they are more likely to damage viral coat proteins rather than viral nucleic acids. Therefore, an RT-PCR assay to measure viral nucleic acid may significantly underestimate the inactivation of viruses. The effect of Ag+ ions and the generation of reactive oxygen species (ROS) should be responsible for the inactivation of the tested viruses. However, many aspects of the antiviral mechanisms of AgNP-MHCs are still unclear, and further research using biotechnological approaches is required to elucidate the mechanism of the high resistance of AdV2.

Finally, we evaluated the robustness of the antiviral activity of Ag30-MHCs by exposing them to a wide range of pH values and to tap and surface water samples before measuring their antiviral activity (Fig. 3 and ​and4).4). Exposure to extremely low pH conditions resulted in the physical and/or functional loss of AgNPs from the MHC (unpublished data). In the presence of nonspecific particles, Ag30-MHCs consistently displayed antiviral activity, despite a decline in the antiviral activity against MS2. The antiviral activity was lowest in unfiltered surface water, as an interaction between Ag30-MHCs and nonspecific particles in the highly turbid surface water probably caused a reduction of antiviral activity (Table 3). Therefore, field evaluations of AgNP-MHCs in various types of water (e.g., with different salt concentrations or humic acid) should be performed in the future.

In conclusion, the new Ag composites, AgNP-MHCs, have excellent antiviral capabilities against several viruses, including ϕX174 and MNV. AgNP-MHCs maintain strong efficacy under different environmental conditions, and these particles can be easily recovered using a magnet, thus reducing their potential harmful effects on human health and the environment. This study showed that the AgNP composite can be an effective antiviral in various environmental settings, without significant ecological risks.