Reactive-oxygen-species-mediated mechanism for photoinduced antibacterial and antiviral activities of Ag₃PO₄

Abstract

Cubic-shaped Ag₃PO₄ crystals with a mean size of 1 μm were synthesized by a precipitation method from a mixed solution of AgNO₃, Na₂HPO₄, and triethanolamine. The antibacterial activities against Escherichia coli, Listeria innocua, and Pseudomonas syringae DC3000 in both the absence and presence of Ag₃PO₄ under dark conditions and in the presence of Ag₃PO₄ under red-light (625 nm) and blue-light (460 nm) irradiation were examined. The concentrations of reactive oxygen species (ROS) were also measured in the antibacterial action of the Ag₃PO₄ against Escherichia coli. The photoinduced enhancement of the Ag₃PO₄ antibacterial activity under blue-light irradiation is explained by the formation of ROS during the antibacterial action of the Ag₃PO₄. Moreover, the antiviral activity of Ag₃PO₄ against amphotropic 10A1 murine leukemia virus enhanced under blue-light irradiation via ROS production. These results provide an insight into extended bio-applications of Ag₃PO₄.

Introduction

Photoenhanced catalytic and antibacterial materials have been extensively investigated in efforts to eliminate organic pollutants and microorganisms from wastewater (Chatterjee and Dasgupta 2005; Lapworth et al. 2012; Mouele et al. 2015; Schwarzenbach et al. 2006). In the process where electrons from the conduction band recombine with holes from the valence band of photocatalytic materials, reactive oxygen species (ROS) such as superoxide anions (O₂^•−), hydroxyl radicals (•OH), and singlet oxygen (¹O₂) are produced (Dickinson and Chang 2011; Li et al. 2012). These ROS play an important role in the photoenhanced catalytic activities. The ROS can also damage biomolecules and regulate cell death of microorganisms (Du and Gebicki 2004; Overmyer et al. 2003).

Silver phosphate (Ag₃PO₄), which has an indirect bandgap of 2.36 eV, exhibits excellent photoenhanced catalytic activity, with a quantum efficiency as high as 90% under irradiation at 420 nm (Bi et al. 2011; Chen et al. 2015). Ag₃PO₄ exhibits higher photocatalytic activity than TiO₂ in the degradation of organic dyes such as methylene blue and rhodamine B (Dong et al. 2014; Liang et al. 2012). The antibacterial activity and photoinduced antibacterial activity of Ag₃PO₄ have also been investigated (Buckley et al. 2010; Piccirillo et al. 2015; Seo et al. 2017; Suwanprateeb et al. 2012; Wu et al. 2013). However, to the best of our knowledge, there is no report that examines antibacterial or antiviral activities arising from the ROS photoinducibly generated by Ag₃PO₄. In this work, we assessed the role of ROS-mediated behavior in the photoinduced antibacterial and antiviral activities of Ag₃PO₄ crystals against various bacteria and amphotropic 10A1 murine leukemia virus (MLV), respectively.

Methods/experimental

Synthesis of Ag₃PO₄ microcrystal

AgNO₃ (99%, Aldrich), Na₂HPO₄ (99%, Aldrich), and triethanolamine (TEA; 98%, Aldrich) were used without further purification. Ag₃PO₄ was synthesized via a simple precipitation method at room temperature. Six milliliters of 1.0 M TEA aqueous solution was added to 30 mL of 0.01 M AgNO₃ aqueous solution with stirring at room temperature for 10 min. Then, 20 mL of 5 mM Na₂HPO₄ aqueous solution was added, and the resulting mixture was stirred for 1 min at room temperature. The product was collected by centrifugation at 4000 rpm for 5 min, washed several times with water and ethanol, and then dried for 24 h at room temperature.

Antibacterial and antiviral experimental conditions

A small fraction (10 μL) of Escherichia coli (E. coli) overnight culture was added evenly to fresh 5 mL Luria–Bertani (LB) medium containing 2 μg/mL of the Ag₃PO₄ product with or without 1 mM N-acetylcysteine (NAC, Aldrich) and then incubated in a 37 °C shaking incubator. Pseudomonas syringae (P. syringae) DC3000 was grown at 28 °C in NYGB medium (0.5% tryptone, 3% yeast extract, and 2% glycerol) containing rifampicin. Two-day grown P. syringae DC3000 culture was evenly aliquoted into 10 mL fresh NYGB medium containing rifampicin and further grown at 28 °C.

Antibacterial activity of Ag₃PO₄ to Listeria innocua (L. innocua), which was used as L. monocytogenes surrogate, was measured using the agar-overlay method. Bacterial culture incubated in tryptic soy broth (TSB) was inoculated to tryptic soy agar (TSA) or TSA containing Ag₃PO₄ (4 μg/mL). Oxford agar base (OAB; Difco, Sparks, MD) with antimicrobial supplement (Bacto Oxford antimicrobial supplement; Difco) was poured into 50-mm petri dish (bottom agar), overlaid with the inoculated TSA (top agar), and incubated at 37 °C with or without light treatment. After incubation at 37 °C for 22 h, OAB images were obtained and typical black colonies were enumerated.

For virus production, 293T human embryonic kidney cells (ATCC CRL-11268) were transiently transfected with a full-length molecular clone pMoMLV-10A1-EGFP using the CalPhos Mammalian Transfection Kit (TaKaRa Bio, Shiga, Japan). pMoMLV-10A1-EGFP is a replication-competent retroviral vector containing enhanced green fluorescent protein (EGFP). To determine the viral titer, 1 mL of virus-containing supernatants and 2 μg/mL Ag₃PO₄ were mixed at 37 °C for different irradiation time under the blue and red light sources. HT1080 human fibrosarcoma cells (ATCC CCL-121) were infected with 1 mL of viral supernatants at a multiplicity of infection (MOI) of 1 in the presence of 8 μg/mL polybrene. Two days after infection, green fluorescent protein (GFP)-positive cells were analyzed by a FACSCalibur^TM flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Intracellular amounts of ROS were analyzed by fluorescence spectroscopy after reaction with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, E. coli cells were treated with or without Ag₃PO₄ under light irradiation. The cells were then additionally incubated with phosphate-buffered saline (PBS) containing 500 μM DCFH-DA for 1 h at room temperature in the dark. Finally, the amounts of ROS were measured by fluorescence spectrophotometry (Synergy HTX multimode reader; λ_ex = 485 ± 20 nm, λ_em = 528 ± 20 nm). To obtain E. coli images, we placed DCFH-DA-stained cells on a slide glass, covered them with a cover slip, and then observed them by fluorescence microscopy (Axioplan 2 microscope) using a green filter.

Statistics

Data are presented as the mean ± SEM. Statistics were performed by Tukey’s post hoc test. A p < 0.05 is considered statistically significant.

Instrumentation

The structure and morphology of the Ag₃PO₄ product were examined by powder X-ray diffraction (XRD; PANalytical X'Pert-PRO MPD) with Cu K_α radiation and by scanning electron microscopy (SEM; Hitachi S-4300), respectively. To examine the antibacterial activities of the Ag₃PO₄ product, the growth rates of E. coli or P. syringae DC3000 in the absence or presence of Ag₃PO₄ without light and in the presence of Ag₃PO₄ under blue and red light were determined by measurement of the optical density at 600 nm with a UV–vis spectrophotometer (X-ma 1200V). A blue LED (NC LED, λ = 460 nm) and a red LED (NC LED, λ = 625 nm) with equivalent luminescence were used as the blue and red light sources, respectively.

Results and discussion

Figure 1a shows an SEM image of the Ag₃PO₄ crystals prepared by the precipitation method at room temperature. Most of the Ag₃PO₄ crystals exhibit a cubic shape with a size of 1 μm. Figure 1b shows the XRD pattern of the as-synthesized Ag₃PO₄ crystals. The Rietveld-refined cell parameters of the Ag₃PO₄ crystals in this work are consistent with those of body-centered cubic Ag₃PO₄ with a = 0.6013 nm (JCPDS 06-0505).

Fig. 1

a SEM images of the as-synthesized Ag₃PO₄ product prepared by the precipitation method. b (Top panel) XRD patterns of the Ag₃PO₄ product, along with the patterns calculated using the Rietveld refinement method; the solid line (black) and open circles (red) present the measured and calculated XRD data, respectively. The intensity differences (blue) between the measured and calculated patterns are shown. The vertical markers (black) indicate the Bragg reflections. (Bottom panel) The XRD pattern and Miller indices of the cubic crystal structure of Ag₃PO₄ (JCPDS 06-0505) are included for comparison

Figure 2a shows the growth rate of E. coli in the absence and presence of Ag₃PO₄ under dark conditions and in the presence of Ag₃PO₄ under red-light (625 nm) and blue-light (460 nm) irradiation. In the control experiment without Ag₃PO₄ crystals and under dark conditions, the growth rate of E. coli increases rapidly during the incubation period of 8 h and reaches a saturation plateau after 12–16 h. The incubation time for growth to 50% is known as the half-maximal growth time. The half-maximal growth time in the control experiment was 6.5 h for culturing the E. coli in the absence of Ag₃PO₄ and under dark conditions. When Ag₃PO₄ crystals were present under dark conditions, the growth rate of E. coli decreased compared with the growth rate in the control experiment and the half-maximal growth time increased to 11.0 h. These results indicate that the Ag₃PO₄ crystal exhibits antibacterial activity against E. coli.

Fig. 2

a The growth rate of E. coli in the absence (circles) or presence (squares) of Ag₃PO₄ under dark conditions and in the presence of Ag₃PO₄ under red-light (625 nm, triangles) or blue-light (460 nm, inverted triangles) irradiation. Line graphs represent mean ± SEM (n = 3). b Colony formation of L. innocua in the absence or presence of Ag₃PO₄ under dark conditions and in the presence of Ag₃PO₄ under red-light or blue-light irradiation. Quantified data are shown in bar graphs (mean ± SEM; n = 3). Scale bars are 10 μm in b. c The growth rate of P. syringae DC3000 in the absence (circles) or presence (squares) of Ag₃PO₄ under dark conditions and in the presence of Ag₃PO₄ under red-light (triangles) or blue-light irradiation (inverted triangles). Line graphs represent mean ± SEM (n = 3)

In the presence of Ag₃PO₄ crystals and under red-light (625 nm) irradiation, an E. coli growth curve very similar to that for Ag₃PO₄ crystals under dark conditions is observed, where the half-maximal growth time is 12.0 h. Because the indirect bandgap energy of crystalline Ag₃PO₄ is 2.36 eV (525 nm), the red light (625 nm, 1.98 eV) lacks sufficient energy to transfer the electron from the valance band to the conduction band of the Ag₃PO₄ crystal. This suggests that red light does not induce photoenhancement of the antibacterial activity of Ag₃PO₄ crystals. However, under blue-light irradiation (460 nm, 2.70 eV), the growth rate of E. coli is substantially decreased in the presence of Ag₃PO₄ crystals and the half-maximal growth time is increased to 15.5 h. Because the blue light has sufficient energy to transfer electrons from the valance band to the conduction band of the Ag₃PO₄ crystals, the photoinduced enhancement of antibacterial activity of Ag₃PO₄ is observed only under blue-light irradiation.

Similar trends were observed for L. innocua, which was used as a surrogate of representative Gram-positive foodborne pathogens, L. monocytogenes. Figure 2b shows colonies of L. innocua grown on the selective agar plates in the absence and presence of Ag₃PO₄ under dark conditions and red-light or blue-light irradiation. Ag₃PO₄ decreases the number of L. innocua colonies by twofold under dark conditions. The colony number is about 4/cm² under blue-light irradiation in the presence of Ag₃PO₄, when compared to 58/cm² and 55/cm² under dark and red-light conditions in the presence of Ag₃PO₄, respectively. This result indicates that blue-light irradiation remarkably and synergistically enhances the antibacterial activity of Ag₃PO₄. Photoinduced antibacterial activity on the agar plate gives an insight into applications of Ag₃PO₄ in anti-fouling and eco-friendly adhesive industry.

We then examined the photoinduced antibacterial activity of Ag₃PO₄ against the plant pathogenic P. syringae DC3000 bacterium. In Fig. 2c, the half-maximal growth rates of untreated control and Ag₃PO₄ under dark conditions are 6 h and 9 h, respectively. Comparatively, the half-maximal growth rates of Ag₃PO₄ under red-light and blue-light irradiations are 11 h and undetectable (caused by almost complete inhibition), respectively. Accordingly, Ag₃PO₄ under blue-light irradiation almost completely inhibits the growth of P. syringae DC3000, suggesting that Ag₃PO₄ under blue-light irradiation can be useful for crop protection from phytopathogenic bacteria.

To understand the mechanisms underlying the antibacterial activity of Ag₃PO₄ crystals, we examined whether Ag₃PO₄ crystals alter the levels of ROS in E. coli. Interestingly, Ag₃PO₄ crystals appeared to increase the level of ROS under blue-light irradiation, whereas Ag₃PO₄ crystals alone or under red light exhibited no effect, as shown in Fig. 3. Quantified amounts of ROS and ROS-stained E. coli cells are shown in Fig. 3a and b, respectively. In both panels, the level of ROS was highest in E. coli cells exposed to Ag₃PO₄ crystals in conjunction with blue-light irradiation. These data indicate that the antibacterial activity of Ag₃PO₄ under blue-light irradiation corresponds to the amount of ROS in E. coli. More convincingly, N-acetylcysteine (NAC) known as an ROS scavenger reverses the antibacterial activity of Ag₃PO₄ under blue-light irradiation as shown in Fig. 3c.

Fig. 3

a The ROS concentration produced in the process of antibacterial action against E. coli (mean ± SEM; n = 3) and b optic (left panels showing total cells) and fluorescent (right panels) images of E. coli in the absence and presence of Ag₃PO₄ under dark conditions and in the presence of Ag₃PO₄ under red-light (625 nm) and blue-light (460 nm) irradiation. DCFH-DA fluorescent dye was used for ROS detection. Scale bars are 10 μm in b. c The growth rate of E. coli in the absence or presence of 1 mM N-acetylcysteine (NAC) under dark conditions and in the presence of Ag₃PO₄ with or without 1 mM NAC under blue-light (460 nm, inverted triangles) irradiation. Bar graphs represent mean ± SEM (n = 3)

We furthermore examined the antiviral activity of Ag₃PO₄ under blue-light irradiation. Figure 4a shows that amphotropic 10A1 murine leukemia virus (MLV) was more severely inactivated by Ag₃PO₄ under blue-light irradiation, when compared to Ag₃PO₄ under dark conditions and Ag₃PO₄ under red-light irradiation. We assume that inactivation of the MLV by blue-light irradiated Ag₃PO₄ might be attributable to the peroxidation of the envelope membrane phospholipids, which is furthermore detrimental to DNA (Paiva and Bozza 2014). Given that the envelop membrane phospholids are damaged by blue-light irradiated Ag₃PO₄, other enveloped viruses including HIV-1, SARS-CoV, MERS-CoV, and SARS-CoV2 can be inactivated by blue-light irradiated Ag₃PO₄. To understand the antiviral activity of Ag₃PO₄ under blue-light irradiation, the possibility of the generation of ROS was examined when blue light irradiates on the Ag₃PO₄ solution. Figure 4b shows that ROS is substantially increased by photoinduction to the Ag₃PO₄ solution. This result supports that the ROS is detrimental to viral particles.

Fig. 4

a Antiviral activity of Ag₃PO₄ under red-light (625 nm) and blue-light (460 nm) irradiation. HT1080 cells were inoculated with MoMLV-10A1-EGFP at an MOI of 1. Viral supernatants were collected after mixing with Ag₃PO₄ under no light, red-light, and blue-light irradiation. Representative cells observed under optic and fluorescence microscopy are shown in the left panel. Forty-eight hours after infection, the GFP-expressed cells were analyzed by flow cytometry. Fluorescence-gated cell percents are shown in line graphs in the right panel (mean ± SEM; n = 3). b The ROS concentrations generated by Ag₃PO₄ suspended in PBS under dark conditions and under red-light or blue-light irradiation. Quantified data are shown in bar graphs (mean ± SEM; n = 3) *p < 0.0001

Conclusion

We synthesized cubic Ag₃PO₄ crystals with a mean size of 1 μm to investigate their antibacterial and antiviral activities. The Ag₃PO₄ crystals showed good antibacterial and antiviral activities against E. coli, L. innocua, P. syringae DC3000, and amphotropic 10A1 MLV. The photoinduced enhancement of the antibacterial and antiviral activities of Ag₃PO₄ under blue-light irradiation was observed. The ROS mediation process in the antibacterial and antiviral activities was confirmed through measurements of the concentrations of ROS. The formation of ROS plays an important role in the antibacterial and antiviral activities of Ag₃PO₄. These findings suggest that the photoinduced enhancement of antibacterial and antiviral activities of Ag₃PO₄ can be used for the biomedical application including anti-fouling, additives, and crop cultivations.