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

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 (O 2 •− ), hydroxyl radicals (•OH), and singlet oxygen ( 1 O 2 ) are produced (Dickinson and Chang 2011;. 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 (Ag 3 PO 4 ), 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). Ag 3 PO 4 exhibits higher photocatalytic activity than TiO 2 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 Ag 3 PO 4 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 Ag 3 PO 4 . In this work, we assessed the role of ROSmediated behavior in the photoinduced antibacterial and antiviral activities of Ag 3 PO 4 crystals against various bacteria and amphotropic 10A1 murine leukemia virus (MLV), respectively.

Synthesis of Ag 3 PO 4 microcrystal
AgNO 3 (99%, Aldrich), Na 2 HPO 4 (99%, Aldrich), and triethanolamine (TEA; 98%, Aldrich) were used without further purification. Ag 3 PO 4 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 AgNO 3 aqueous solution with stirring at room temperature for 10 min. Then, 20 mL of 5 mM Na 2 HPO 4 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 Ag 3 PO 4 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 Ag 3 PO 4 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 Ag 3 PO 4 (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 Ag 3 PO 4 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 Ag 3 PO 4 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 Ag 3 PO 4 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 Ag 3 PO 4 product, the growth rates of E. coli or P. syringae DC3000 in the absence or presence of Ag 3 PO 4 without light and in the presence of Ag 3 PO 4 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 Ag 3 PO 4 crystals prepared by the precipitation method at room temperature. Most of the Ag 3 PO 4 crystals exhibit a cubic shape with a size of 1 μm. Figure 1b shows the XRD pattern of the as-synthesized Ag 3 PO 4 crystals. The Rietveldrefined cell parameters of the Ag 3 PO 4 crystals in this work are consistent with those of body-centered cubic Ag 3 PO 4 with a = 0.6013 nm (JCPDS 06-0505). Figure 2a shows the growth rate of E. coli in the absence and presence of Ag 3 PO 4 under dark conditions and in the presence of Ag 3 PO 4 under red-light (625 nm) and blue-light (460 nm) irradiation. In the control experiment without Ag 3 PO 4 crystals and under dark conditions, the growth rate of E. coli increases rapidly during the incubation period of 8 h and reaches a Fig. 2 a The growth rate of E. coli in the absence (circles) or presence (squares) of Ag 3 PO 4 under dark conditions and in the presence of Ag 3 PO 4 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 Ag 3 PO 4 under dark conditions and in the presence of Ag 3 PO 4 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 Ag 3 PO 4 under dark conditions and in the presence of Ag 3 PO 4 under red-light (triangles) or bluelight irradiation (inverted triangles). Line graphs represent mean ± SEM (n = 3) 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 Ag 3 PO 4 and under dark conditions. When Ag 3 PO 4 crystals were present under dark conditions, the growth rate of E. coli decreased compared with the growth rate in the control experiment and the halfmaximal growth time increased to 11.0 h. These results indicate that the Ag 3 PO 4 crystal exhibits antibacterial activity against E. coli.
In the presence of Ag 3 PO 4 crystals and under red-light (625 nm) irradiation, an E. coli growth curve very similar to that for Ag 3 PO 4 crystals under dark conditions is observed, where the half-maximal growth time is 12.0 h. Because the indirect bandgap energy of crystalline Ag 3 PO 4 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 Ag 3 PO 4 crystal. This suggests that red light does not induce photoenhancement of the antibacterial activity of Ag 3 PO 4 crystals. However, under blue-light irradiation (460 nm, 2.70 eV), the growth rate of E. coli is substantially decreased in the presence of Ag 3 PO 4 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 Ag 3 PO 4 crystals, the photoinduced enhancement of antibacterial activity of Ag 3 PO 4 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 Ag 3 PO 4 under dark conditions and red-light or blue-light irradiation. Ag 3 PO 4 decreases the number of L. innocua colonies by twofold under dark conditions. The colony number is about 4/ cm 2 under blue-light irradiation in the presence of Ag 3 PO 4 , when compared to 58/cm 2 and 55/cm 2 under dark and red-light conditions in the presence of Ag 3 PO 4 , respectively. This result indicates that blue-light irradiation remarkably and synergistically enhances the antibacterial activity of Ag 3 PO 4 . Photoinduced antibacterial activity on the agar plate gives an insight into applications of Ag 3 PO 4 in anti-fouling and eco-friendly adhesive industry. We then examined the photoinduced antibacterial activity of Ag 3 PO 4 against the plant pathogenic P. syringae DC3000 bacterium. In Fig. 2c, the half-maximal growth rates of untreated control and Ag 3 PO 4 under dark conditions are 6 h and 9 h, respectively. Comparatively, the half-maximal growth rates of Ag 3 PO 4 under red-light and blue-light irradiations are 11 h and undetectable (caused by almost complete inhibition), respectively. Accordingly, Ag 3 PO 4 under blue-light irradiation almost completely inhibits the growth of P. syringae DC3000, suggesting that Ag 3 PO 4 under blue-light irradiation can be useful for crop protection from phytopathogenic bacteria.
To understand the mechanisms underlying the antibacterial activity of Ag 3 PO 4 crystals, we examined whether Ag 3 PO 4 crystals alter the levels of ROS in E. coli. Interestingly, Ag 3 PO 4 crystals appeared to increase the level of ROS under blue-light irradiation, whereas Ag 3 PO 4 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 Ag 3 PO 4 crystals in conjunction with blue-light irradiation. These data indicate that the antibacterial activity of Ag 3 PO 4 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 Ag 3 PO 4 under blue-light irradiation as shown in Fig. 3c.
We furthermore examined the antiviral activity of Ag 3 PO 4 under blue-light irradiation. Figure 4a shows that amphotropic 10A1 murine leukemia virus (MLV) was more severely inactivated by Ag 3 PO 4 under bluelight irradiation, when compared to Ag 3 PO 4 under dark conditions and Ag 3 PO 4 under red-light irradiation. We assume that inactivation of the MLV by blue-light irradiated Ag 3 PO 4 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 Ag 3 PO 4 , other enveloped viruses including HIV-1, SARS-CoV, MERS-CoV, and SARS-CoV2 can be inactivated by blue-light irradiated Ag 3 PO 4 . To understand the antiviral activity of Ag 3 PO 4 under blue-light irradiation, the possibility of the generation of ROS was examined when blue light irradiates on the Ag 3 PO 4 solution. Figure 4b shows that ROS is substantially increased by photoinduction to the Ag 3 PO 4 solution. This result supports that the ROS is detrimental to viral particles.

Conclusion
We synthesized cubic Ag 3 PO 4 crystals with a mean size of 1 μm to investigate their antibacterial and antiviral activities. The Ag 3 PO 4 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 Ag 3 PO 4 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 Ag 3 PO 4 . These findings suggest that the photoinduced enhancement of antibacterial and antiviral activities of Ag 3 PO 4 can be used for the biomedical application including anti-fouling, additives, and crop cultivations.