nanomaterials Article
Antibacterial Properties of Visible-Light-Responsive Carbon-Containing Titanium Dioxide Photocatalytic Nanoparticles against Anthrax Der-Shan Sun 1 , Jyh-Hwa Kau 2,3 , Hsin-Hsien Huang 3 , Yao-Hsuan Tseng 4 , Wen-Shiang Wu 1 and Hsin-Hou Chang 1, * 1 2 3 4
*
Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien 97004, Taiwan;
[email protected] (D.-S.S.);
[email protected] (W.-S.W.) Institute of Microbiology and Immunology, National Defense Medical Center, Taipei 11490, Taiwan;
[email protected] Institute of Preventive Medicine, National Defense Medical Center, Taipei 23742, Taiwan;
[email protected] Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan;
[email protected] Correspondence:
[email protected]; Tel.: +886-3-8565301 (ext. 2667)
Academic Editor: Thomas Nann Received: 31 August 2016; Accepted: 6 December 2016; Published: 9 December 2016
Abstract: The bactericidal activity of conventional titanium dioxide (TiO2 ) photocatalyst is effective only on irradiation by ultraviolet light, which restricts the applications of TiO2 for use in living environments. Recently, carbon-containing TiO2 nanoparticles [TiO2 (C) NP] were found to be a visible-light-responsive photocatalyst (VLRP), which displayed significantly enhanced antibacterial properties under visible light illumination. However, whether TiO2 (C) NPs exert antibacterial properties against Bacillus anthracis remains elusive. Here, we evaluated these VLRP NPs in the reduction of anthrax-induced pathogenesis. Bacteria-killing experiments indicated that a significantly higher proportion (40%–60%) of all tested Bacillus species, including B. subtilis, B. cereus, B. thuringiensis, and B. anthracis, were considerably eliminated by TiO2 (C) NPs. Toxin inactivation analysis further suggested that the TiO2 (C) NPs efficiently detoxify approximately 90% of tested anthrax lethal toxin, a major virulence factor of anthrax. Notably, macrophage clearance experiments further suggested that, even under suboptimal conditions without considerable bacterial killing, the TiO2 (C) NP-mediated photocatalysis still exhibited antibacterial properties through the reduction of bacterial resistance against macrophage killing. Our results collectively suggested that TiO2 (C) NP is a conceptually feasible anti-anthrax material, and the relevant technologies described herein may be useful in the development of new strategies against anthrax. Keywords: anthrax spore; antibacterial agents; TiO2 ; carbon-containing TiO2 ; visible light responsive photocatalyst
1. Introduction Anthrax is a life-threatening infectious disease that spreads through contact with spores of the Gram-positive bacterium Bacillus anthracis through skin contact (generally with infected animal products), inhalation, or ingestion [1]. Approximately 2000 to 20,000 cases occur worldwide annually [2], mostly in Africa and central and south Asia [3]. Anthrax spores have been developed as a biological weapon by several countries [4–6]. The 2001 US anthrax letter attacks further evidenced an emerging terrorist threat, leading to renewed attention to the importance of prophylaxis, prevention, and handling procedures for anthrax [7]. Agents commonly cited to inactivate anthrax spores Nanomaterials 2016, 6, 237; doi:10.3390/nano6120237
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include formaldehyde, hypochlorite solutions, chlorine dioxide, and radiation [8]. However, most of these agents are harmful to humans, limiting their use in public environments. Therefore, a safer disinfection technique that can exert a sustainable antimicrobial effect in human living environments is highly desirable. Photocatalytic titanium dioxide (TiO2 ) substrates have been demonstrated to eliminate organic compounds and to function as disinfectants [9]. On stimulation by ultraviolet (UV) light irradiation, the photon energy excites valance electrons and generates pairs of electrons and holes (electron vacancy in the valence band) that diffuse and become trapped on the TiO2 surfaces. These excited electrons and holes have strong reducing and oxidizing activities and react with atmospheric water and oxygen to yield reactive oxygen species (ROS) such as hydrogen peroxide (H2 O2 ), hydroxyl radicals (•OH), and superoxide anions (O2 − ) [10], which are extremely reactive on contact with organic compounds, and have been shown to operate in concert to attack polyunsaturated phospholipids and DNA in bacteria [9,11]. The oxidation of bacterial cell components such as lipids and DNA might therefore result in subsequent bacterial cell death [9]. Consequently, the TiO2 photocatalytic process is a conceptually feasible disinfectant technology. The TiO2 photocatalyst, however, is effective only on irradiation with UV light at the necessary levels, which can induce severe damage to human eyes and skin [12–15]. This greatly restricts the potential applications of the photocatalyst for use in human living environments. To solve this problem, impurity doping of TiO2 with different elements has been used, including carbon, sulfur, nitrogen, and silver, resulting in excitation wavelength shifts from the UV to visible-light [16–25]. Simultaneously, the proper amount of impurity doping of TiO2 may also reduce the recombination rates of electron and hole pairs. Previously, we reported visible-light-responsive photocatalyst (VLRP) films, which offered a complementary and possibly alternative approach for meeting this need to control the spread of anthrax [24]. However, these VLRP films must be precoated on the surfaces of particular objects, whereas photocatalytic NPs do not, and as such may have broader applications. To solve this problem, the anti-anthrax properties of VLRP carbon-containing titanium dioxide [TiO2 (C)] nanoparticles [TiO2 (C) NPs; C200 NPs] [17] were evaluated in this study. The visible-light-responsive photocatalytic activity of C200 NPs has been respectively validated by degradation of methylene blue in liquid phase, oxidation of NO in gas phase, and sterilization in these works under visible light illumination [17,19,26–28]. The existence of carbonaceous species on TiO2 surface was analyzed by X-ray photoelectron spectroscopy (XPS) and diffuse reflectance infrared Fourier transform spectra. The effect of carbonaceous species on physical properties was observed on UV-visible absorption spectra, photoluminescence spectroscopy, and Raman spectroscopy as shown in our previous works [26–28]. In addition, we have further demonstrated that C200 NPs exert superior Escherichia coli killing properties under visible light illumination when compared to anatase TiO2 NPs [17,19]. These results collectively suggested that the C200 NPs exhibit a photocatalytic property under visible light illumination. However, whether C200 NPs can eliminate spore-forming bacteria such as Bacillus species has remained uncertain. Therefore, the visible-light-responsive C200 NP-mediated anti-anthrax property was evaluated. The potential applications are discussed herein. 2. Results 2.1. Analyses of TiO2 NPs Detailed physical properties of UV-responsive pure TiO2 (TiO2 ; UV100 TiO2 ) and carboncontaining TiO2 (C200) NPs have been characterized in our previous work [17,26,27]. In the present study, scanning electron microscopy and UV-Vis absorption analyses of the newly prepared C200 NPs were performed (Figure 1). We found that both TiO2 and C200 displayed nanoscale structures (Figure 1A,B), and that an increased content of carbon (Figure 1C) and C200 displayed considerable redshift absorbance compared with TiO2 NPs (Figure 1D), indicating absorbance in the visible light range (wavelength > 380 nm). The UV-Visible diffuse reflectance spectra were converted by instrument
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Nanomaterials 2016, 6,values, 237 3 ofa12sharp software to absorbance F(R), based on the Kubelka-Munk theory. In the C200 sample, edge extending to approximately 438 nm and corresponding to a band gap of approximately 2.83 eV sample, a sharp edge extending to approximately 438 nm and corresponding to a band gap of was observed, as indicated in one of our previous reports [27].
approximately 2.83 Nanomaterials 2016, 6, 237
eV was observed, as indicated in one of our previous reports [27].
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sample, a sharp edge extending to approximately 438 nm and corresponding to a band gap of approximately 2.83 eV was observed, as indicated in one of our previous reports [27].
Figure 1. Scanning electron microscopy and ultraviolet-visible (UV-Vis) absorption spectrum analyses. Figure 1. Scanning electron microscopy and ultraviolet-visible (UV-Vis) absorption spectrum Scanning analyses. electron Scanning microscopy (A,B), X-ray photoelectron spectroscopyspectroscopy (XPS) analysis for the 1s for atomic electron microscopy (A, B), X-ray photoelectron (XPS) analysis orbital of the carbon (C) and UV-Vis absorption spectra (D) of spectra UV100(D) TiO and C200 NPsC200 usedNPs in this 1s atomic orbital of carbon (C) and UV-Vis absorption of2 UV100 TiO2 and Figure 1. Scanning electron and ultraviolet-visible (UV-Vis) used in this study. The C200microscopy sample absorbed light into the visible (>380 nm) spectrum region. study. The C200 sample absorbed light extending into extending the visible (>380 nm)absorption region. analyses. Scanning electron microscopy (A, B), X-ray photoelectron spectroscopy (XPS) analysis for
2.2. and Kinetic Analyses of Photocatalytic Inactivation B. theDose-Dependent 1s atomic orbital of carbon (C) and absorptionInactivation spectra (D) ofof TiO2 and C200 NPs 2.2. Dose-Dependent and Kinetic Analyses of UV-Vis Photocatalytic ofUV100 B.Subtilis Subtilis used in this study. The C200 sample absorbed light extending into the visible (>380 nm) region.
The antibacterial properties of C200 NPs have been demonstrated [17]; however, whether C200
The antibacterial properties of C200 NPs have been demonstrated [17]; however, whether C200 can also functionally eliminate spore-forming bacteria such as B. anthracis and B. subtilis remains to 2.2. Dose-Dependent and Kinetic Analyses of Photocatalytic B. Subtilis and B. subtilis remains to can also functionally eliminate spore-forming bacteriaInactivation such as B.ofanthracis be further elucidated. Because B. anthracis is hazardous to humans, before analysis using B. anthracis, be furtherwe elucidated. Because B. anthracis is hazardous to humans, before analysis using B. with anthracis, The antibacterial properties of C200 NPs have been demonstrated [17]; however, whether C200 employed B. subtilis as a surrogate. To obtain dose-dependent and kinetic data for B. subtilis canC200 also NPs, functionally spore-forming such as anthracis and B. data subtilis remains toand with we employed B. subtilis aseliminate a surrogate. obtain dose-dependent and kinetic for B. subtilis we further analyzed theTo effects ofbacteria illumination byB.visible light at various time points be at further elucidated. Because B.cm, anthracis isillumination hazardous humans, before B. anthracis, various distances (5 cm,the 15 andof with different to illumination intensities of 3using × 104time and 5points × 102 lux C200 NPs, we further analyzed effects by visible lightanalysis at various and at employed2(5 subtilis as aThe surrogate. To obtain that dose-dependent andcan kinetic data subtilis 4 and (lumen/m );B.Figure results C200 substrates inactivate B. B. subtilis inwith an 2 lux variouswe distances cm, 152A). cm, and withindicated different illumination intensities of 3for × 10 5half × 10 C200 NPs, we exposed further analyzed effectsofofillumination illuminationbybyvisible visible light at various timebacteria-killing points and hour when to variousthe light (Figure 2B). The 2 ); (lumen/m Figure 2A). The resultsdegrees indicated that C200 substrates can inactivate B. subtilis in half an 4 and 5 × 102 lux at various distances (5 cm, 15 cm, and with different illumination intensities of 3 × 10 efficiency in the C200 groups was significantly higher than in the respective UV100 TiO2 groups hour when exposed to**various of illumination visible light (Figure B. 2B). The in bacteria-killing (lumen/m Figure Thedegrees indicated that C200by substrates can inactivate subtilis half an (Figure2); 2A,B; P2A). < 0.01, * results P < 0.05). efficiency the exposed C200 groups was significantly higher than light in the respective TiO2 groups hourin when to various degrees of illumination by visible (Figure 2B). The UV100 bacteria-killing C200 was significantly higher than in the respective UV100 TiO2 groups (Figureefficiency 2A,B; ** in P
