key: cord-0957765-d0rm7r68 authors: Matsumoto, Takumi; Sunada, Kayano; Nagai, Takeshi; Isobe, Toshihiro; Matsushita, Sachiko; Ishiguro, Hitoshi; Nakajima, Akira title: Effects of cerium and tungsten substitution on antiviral and antibacterial properties of lanthanum molybdate date: 2020-08-04 journal: Mater Sci Eng C Mater Biol Appl DOI: 10.1016/j.msec.2020.111323 sha: 0e0bc0a3a1d2354786c3554e9bf61938701c9841 doc_id: 957765 cord_uid: d0rm7r68 Abstract Powders of cerium (Ce)-substituted and tungsten (W)-substituted La2Mo2O9 (LMO) were prepared using polymerizable complex method. Their antiviral and antibacterial performances were then evaluated using bacteriophage Qβ, bacteriophage Φ6, Escherichia coli, and Staphylococcus aureus. The obtained powders, which were almost single-phase, exhibited both antiviral and antibacterial properties. Effects of dissolved ions on their antiviral activity against bacteriophage Qβ were remarkable. A certain contribution of direct contact to the powder surface was also inferred along with the dissolved ion effect for antiviral activity against bacteriophage Φ6. Dissolved ion effects and pH values suggest that both Mo and W are in the form of polyacids. Antiviral activity against bacteriophage Φ6 was improved by substituting Ce for La in LMO. Similarly to LMO, Ce-substituted LMO exhibited hydrophobicity. Inactivation of alkaline phosphatase enzyme proteins was inferred as one mechanism of the antiviral and antibacterial activities of the obtained powders. The history of inorganic antiviral materials is not as long as that of organic antiviral materials. However, studies of inorganic antiviral materials are increasing gradually because one such material can affect various viruses under a wide temperature range with only a small probability of resistance development by a virus. Various materials have been investigated. Antiviral properties have been reported for metals [1-3] (e.g. Ag and Cu), photocatalysts (e.g. TiO 2 [4, 5] ), and other materials (e.g. ZnO and CaO [6, 7]). In fact, some of these materials have already been applied for practical use. Nevertheless, these materials entail several difficulties such as coloration or inactivation because of oxidation, and usage environment restrictions (requirement of light illumination, or alkalization). Development of new inorganic antiviral materials has been demanded to overcome these and other difficulties. We specifically examined the hydrophobicity of rare-earth oxides [8] [9] [10] [11] [12] and antibacterial effects of molybdenum [13] [14] [15] [16] . Then we developed the complex oxide La 2 Mo 2 O 9 (hereinafter LMO) using polymerizable complex method [17] . This material exhibited hydrophobicity and simultaneous antibacterial effects against gram-negative (Escherichia coli, E. coli) and gram-positive (Staphylococcus aureus, S. J o u r n a l P r e -p r o o f 6 Emmett-Teller (BET) method with N 2 (BEL SORP mini; MicrotracBEL Corp., Japan). The crystalline phase of the powders was evaluated using X-ray diffraction (XRD, XRD-6100; Shimadzu Corp., Japan) with a Cu Kα radiation source. The UV-vis absorption spectra of the samples were obtained using a UV-vis scanning spectrophotometer (V-660; Jasco Corp., Tokyo, Japan). Barium sulfate powder was used as a reference for this measurement. The chemical composition of the powders was evaluated using inductively coupled plasma analysis (ICP-OES, 5100 VDV; Agilent Technologies Japan Ltd., Tokyo, Japan) and an X-ray photoelectron spectroscope (XPS, Quantera SXM; Ulvac-Phi Inc., Japan) with an Al K X-ray line (1486.6 eV). For this study, antiviral and antibacterial activity measurements were taken not only for prepared powders but also for La 2 O 3 (99.99%; Fujifilm Wako Pure Chemical Corp.), CeO 2 (99.5%; Fujifilm Wako J o u r n a l P r e -p r o o f 8 Then the mixture was spread out on 1.5% LB agar plate to form a double agar layer. The plate was incubated at 37°C for 48 h to form the plaques. The titer after incubation (N) was calculated by counting the plaques. Control data were obtained using the same experimental procedure with a pristine glass plate instead of glass plates with sample powders. The plaque assay was performed twice for each point. Hereinafter, we designate this method as the film adhesion method. As described also for an earlier study, we evaluated the ion concentration when dissolved into 1/500 NB. To elucidate the contribution of dissolved ions to the overall antiviral activity, we took antiviral activity measurements using the solution after filtering the powder samples. Each prepared powder was mixed with 1/500 NB by the same solid-liquid ratio as that used for the film adhesion method. Then the mixture was shaken at 100 times/min for 2 h. Each suspension was filtered to exclude the powder. The pH value of the filtrate was measured. In addition, the amount of the dissolved ion (La, Ce, W, and Mo) was measured using ICP-OES. The filtrate solution was used for antiviral activity measurements against Qand 6. The filtrate solution, the suspensions of Qor 6 (2.2 × 10 7 PFU/ml), and the distilled water were mixed at a ratio of 8:1:1. This mixture was stirred and incubated at room temperature (ca. 25°C) in the dark for 2, 4, and 6 h. After incubation, the reaction was stopped with SCDLP medium. The medium was diluted with 0.01 M PBS. Then the antiviral activity was evaluated using the same procedure as that used for the film adhesion method. The initial virus concentration before contact with the filtrate solution including dissolved ions was set as the 0 h value. The control data were obtained using 1/500NB instead of the filtrate solution. Therefore, the ratio J o u r n a l P r e -p r o o f 9 of 1/500NB, the virus suspensions, and the distilled water was 8:1:1; then, the same measurements were conducted. Hereinafter, this method is described as the dissolved ion contact method. Evaluation of the antibacterial activity was conducted according to a film adhesion method described for ISO 17094, with minor modifications. For this study, we used E. coli (NBRC 3972) and S. aureus (NBRC 12732). Each had been precultured on nutrient agar (NA; Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) at 37°C for 18 h and had been suspended in 1/500 NB. The concentrations of these bacteria were fixed to ca. 2.0 × 10 6 colony-forming units (CFU)/ml. A 50 μl (= 10 5 CFU) bacteria suspension was pipetted onto a substrate loaded with the sample powder. After the substrate was covered with a transparent film to contact the bacteria suspension with the particles, it was incubated under a humid condition at room temperature (ca. 25°C) in a dark room. After a certain period (0, 2, 4, and 6 h), the bacteria were harvested by shaking with 5 ml SCDLP for 2 min to halt the incubation. The bacteria in SCDLP were diluted with 0.01 M PBS. Each 1 ml of the diluted bacteria suspension was mixed in NA and was incubated at 37°C for 48 h to produce bacterial colonies. The concentration of viable bacterial cells at each time point (N) was calculated by multiplying the number of colonies and the dilution ratio. The control cell concentrations were calculated using the same procedure as that used for a pristine glass substrate. The initial cell concentration for each sample was presented as the cell concentration at 0 h. The colony assay was conducted twice for each point. Journal Pre-proof J o u r n a l P r e -p r o o f CCL-34) purchased from American Type Culture Collection (Manassas, VA, U.S.A.). Cells were maintained using modified Eagle's medium containing 10% fetal bovine serum and were incubated at 35°C under a humidified 5% CO 2 atmosphere in an incubator. The prepared sample glass was put in a Petri dish into which was poured 5 ml of PBS. The extraction solutions of sample powders were prepared by shaking 100 times/min for 2 h. The extraction solution (50 µl) and MDCK cell solution (4.0 × 10 5 cells/ml, 150 l) were mixed and incubated at 35°C under a humidified 5% CO 2 atmosphere in an incubator for 4 days. After incubation, an adenosine triphosphate detection reagent (Viral ToxGlo™ Assay; Promega Corp., U.S.A.) was added (100 l). A spectrophotometer (infiniteM200; Tecan Group Ltd., Austria) was used to measure the emitted light intensity. The number of living cells was calculated. Control data were obtained using the same experimental procedure with a pristine glass plate used instead of a glass plate with a sample powder. The contributions of dissolved ions, especially those of ions of La, W, and Mo, are large for antivirus activity against Qbecause the activity achieved using dissolved ion contact method was higher than that achieved using film adhesion method. Before this study, we conducted a preliminary study of LaVO 4 , for which we replaced Mo by V in LMO. This material exhibited a small pH value (4.71) under the same experiment conditions. Because the dissolved amount of V is much greater than that of La, ion exchange between VO 4 3and OHwere expected for charge compensation. Nevertheless, this material exhibited little antiviral or antibacterial activity (see Table SI The slight antiviral activity shown by CeO 2 might be attributable to the major Ce ion valence (not III but IV). Based on the relation between the dissolved ion amount and antiviral activity against Q, we can infer that the activity order of prepared powders depends on the dissolved ion amount, and especially on the contact method is higher than that by film adhesion method, other oxide reagents exhibited similar antiviral activity from these two methods. Dissolved Mo and W ion also contribute to the overall antiviral activity against 6 to some degree, although their contributions are not as strong as that of Q. The antiviral activity by dissolved ion contact method was lower than that by film adhesion method for prepared powders. Therefore, we can infer that the effects of direct contact of the virus to the powder surface also exist for these powders. This trend differs from Q. The ion concentration is expected to be high around the powder surface. Therefore, this difference might be attributable to the difference of virus characteristics such as the resistance against dissolved ions. Figures 6(a) and 6(b) present results of antibacterial activity against E. coli for prepared powders ( Fig. 6(a) ) and for oxide reagents (Fig. 6(b) ). (Fig. 9) . However, photocatalytic decomposition activity was not obtained from this material. Therefore, this material might be applicable to UV shielding coatings for windows or cosmetics. 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