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Preparation of sonoactivated TiO2-DVDMS nanocomposite for enhanced antibacterial activity
Journal Pre-proofs Preparation of sonoactivated TiO2-DVDMS nanocomposite for enhanced antibacterial activity Yihui Wang, Yue Sun, Shupei Liu, Lijuan Zhi, Xiaobing Wang PII: DOI: Reference:
S1350-4177(19)31664-5 https://doi.org/10.1016/j.ultsonch.2020.104968 ULTSON 104968
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Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
22 October 2019 30 December 2019 9 January 2020
Please cite this article as: Y. Wang, Y. Sun, S. Liu, L. Zhi, X. Wang, Preparation of sonoactivated TiO2-DVDMS nanocomposite for enhanced antibacterial activity, Ultrasonics Sonochemistry (2020), doi: https://doi.org/10.1016/ j.ultsonch.2020.104968
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Title：Preparation of sonoactivated TiO2-DVDMS nanocomposite for enhanced antibacterial activity Authors：Yihui Wang, Yue Sun, Shupei Liu, Lijuan Zhi, Xiaobing Wang* Affiliation: National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China; Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education; College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi 710119, China. Corresponding Author E-mail: [email protected] (Xiaobing Wang) Tel: +86-29-85310275
Abstract Titanium dioxide (TiO2) nanoparticle has good photo-/sono-catalytic features, the reunion of this particle in solution-phase generally limits the extensive biomedical application. In the present study, the aggregation of TiO2 nanoparticles was alleviated by facile fabrication under different pH conditions. A novel TiO2 nanocomposite was further synthesized by properly conjugation with trace amount of DVDMS sensitizer (named DFT). The characterization, sonoactivity, as well as the antibacterial efficiency were specially evaluated. The results showed that the sonochemical activity of DFT was greatly improved as compared with the simple surface modification of TiO2 (F-TiO2) and free DVDMS, regarding to the hydroxyl radicals and singlet oxygen yields using the same ultrasound exposure. Moreover, ultrasonic stimulation of DFT exhibited excellent bacterial eradication, with up to 92.41% of killing efficiency in S. aureus. The flow cytometry analysis indicated an increased intracellular ROS and membrane disturbance by combination of DFT and ultrasound. The findings suggest that the proper fabrication and DVDMS incorporation greatly improved the sonocatalytic process of TiO2, and the ultrasound based biomedical applications of DFT deserve future deep investigation.
Key word: TiO2 nanoparticles, Ultrasound, DVDMS, Sonochemical reaction, Antibacterial effect 1. Introduction As an inorganic semiconductor material, titanium dioxide (TiO2) has made dramatic breakthroughs in nanomaterial due to its advantages of easy fabrication, abundant composition and versatile structures [1, 2]. In the past decades, the photocatalytic activity of TiO2 nanoparticles (NPs) has attracted much attention. TiO2 NPs can convert the natural light energy into the chemical reaction energy to initiate catalytic effect, and cause the surrounding O2 and H2O to be excited into free anions with oxidation power [3]. Nevertheless, the single TiO2 products have some flaws like in stability and energy transfer efficiency. Based on this, Geng et al. prepared Fe3O4/TiO2-N-GO nanocomposites to utilize the photocatalysis of TiO2 to degrade humic acid [4]. Hafeeza et al. developed a ternary TiO2 nanocomposite loaded on rGO plates (InVO4-TiO2) to produce efficient hydrogen energy by converting solar energy [5]. These diverse findings have inspired researchers’ great interests to develop various functional TiO2 nanomaterials for conquering the structural defects of semiconductor particles themselves and improving the photo-/sono-catalysis [6]. In recent years, along with the rapid development of TiO2 nanomaterials, not only the potential photocatalytic activities have been explored, but also the possible ultrasound (US)-responsiveness has been continuously confirmed [1]. US has been generally used as an auxiliary method for TiO2 multi-composites synthesis, and the
stimulation of different US frequency and power would affect the sonochemical efficiency [7, 8]. Sonochemical reaction is widely used because of its cavitation effect, especially in sonodynamic therapy (SDT). Upon activation by US, the sonosensitizers would transfer energy to oxygen molecules, leading to the production of reactive oxygen species (ROS), further causing serious cellular toxicity of SDT [9]. TiO2 has shown good sonoactivity in SDT. Harada et al. investigated the therapeutic effects of US stimulating TiO2 NPs on melanoma cells in vitro and in vivo [10]. Since TiO2 NPs strongly absorb US energy and generate a large amount of ROS such as hydroxyl radicals and super oxides, that can be applied in biomedicine fields [11]. Deepagan et al. prepared a long-circulating Au-TiO2 nanocomposite as sonosensitizer and optimized the pore size of mesoporous TiO2 for SDT [12]. Dai et al. reported on augmenting the sonocatalytic efficiency of TiO2-based nanosonosensitizer for highly efficient SDT by the integration of two-dimensional ultrathin graphene with TiO2 [13]. Chen et al. synthesized B-TiO2 by high-temperature reduction method, broadening the second near-infrared region imaging and TiO2-based drug delivery [14]. To some extent, these diverse designs facilitated the separation of electron-hole (e--h+) pairs from TiO2 and improved the sonocatalytic activity. As one of the representative paradigm of inorganic nanosonosensitizers, the sonodynamic efficacy of TiO2 is limited by the particle agglomeration which hinders the segregation of electron and hole pairs from the energy band post US trigger, thus yields low ROS [9]. Through tremendous effort on modification of TiO2 NPs, the problems of TiO2 aggregation have not been solved exhaustively [15]. Because of the
high surface tension of TiO2 NPs in liquid, they are easy to agglomerate into powder blocks which tremendous difficulties to solution dispersion and nanomaterial synthesis, and engender barriers in the exertion of their functions [16, 17]. Studies have found that the size of TiO2 NPs and surrounding pH condition impacted their photocatalytic and biomedical properties. Pettibone et al showed that the natural pH condition exacerbate the aggregation of TiO2 nanoparticles [18]. The decrease of the particle size elicits the increase of the specific surface area as well as the catalytic activity [19]. The proper modification of nanoparticles, on one hand, enables the material to disperse well in the liquid, and on the other hand, strengthens the active center of particles [20]. Therefore, the usage of some suitable modifications to overcome TiO2 NPs agglomeration would be a good choice to improve the sonotherapy efficiency. We hope to enhance the deprotonation of the functional groups of TiO2 by adjusting the pH value, thus affecting the agglomeration behavior of the particles themselves. In brief, two different pH systems (8 and 4) were used to influence the ion distribution in the solution for evaluating the stability of nanoparticles. At the same time, the addition of different modifiers exhibited more excellent dispersion of TiO2.We also introduced a porphyrin sensitizer-DVDMS, to develop new nanocomposite for augmenting the sonosensitivity of TiO2. DVDMS (Sinoporphyrin sodium) has been previously confirmed with fabulous water solubility and high photo-/sono-activity [21]. Herein, a new hybrid nanocomposite (designated as DFT) was developed by incorporation of a small amount of DVDMS on surfactant modified TiO2 NPs.
Firstly, the surface fabrication of TiO2 NPs was optimized using different modifiers (APTMS, F127, PEG2000) under distinct pH values. A novel TiO2-DVDMS nanocomposite was then prepared and evaluated for sonochemical effects. Antimicrobial sonodynamic therapy has shown great potentials in eradicating bacterial infections [22]. Therefore, the enhanced sonodynamic antibacterial activity was specially examined in Gram-positive bacteria. The results showed that the surface modifications partially alleviated the aggregation of TiO2 NPs, and the DVDMS incorporation further improved the dispersion and biocompatibility of the nanocomposite. The designed hybrid DFT exhibit excellent sonochemical responsiveness and bacterial eradication efficiency. Such constructs can be expanded to many other disease treatment using sono/phototherapy, and the design strategy may derive several therapeutic nanoplatforms. 2. Experimental 2.1. Materials Titanium dioxide (TiO2, 60 nm, anatase phase) and terephthalic acid (TA) were obtained from Aladdin. DVDMS (purity> 98%) was the property of Qinglong Hi-tech Co. Ltd. (Jiangxi, China). Singlet oxygen sensor green (SOSG) probe was supplied by Molecular Probes Inc. (Invitrogen, USA). All other chemicals were analytical grade. Deionized water was used through out the study. 2.2. Surface fabrication of TiO2 In detail, 0.01 g TiO2 was completely dissolved in 10 mL water and stirred at
room temperature for 1 h, then adjusted the pH of the solution (4 or 8) and subjected to ultrasonic dispersion (400 w, 40 kHz) for 15 min. 0.5 wt% of modifiers (APTMS, F127, PEG2000) were individually added to the dispersion and constantly stirred for 2 h, and the samples were named A-TiO2, F-TiO2, P-TiO2, respectively. The final dispersion was washed and centrifuged (3000 rpm, 5 min) three times to remove impurities, then re-suspended in deionized water and stored at 4°C. 2.3. Preparation of DFT dispersion An appropriate amount of DVDMS was dissolved in 5 mL of physiological saline to prepare a 2 mg/mL pure liquid. 1 mL of 200 μg/mL DVDMS dilution was added to 1 mL of F-TiO2 dispersion (pH = 8) and stirred for 12 h in the dark. The DFT dispersion was washed and centrifuged three times (6000 rpm, 5 min), and re-suspended in water at 4°C in the dark. 2.4. Characterization The optical characteristics of TiO2 and DFT were measured by UV − vis spectrophotometer (SpectraMax M5, Molecular Devices, US). IR-spectra were collected and recorded with a Fourier infrared spectrometer (Nicolet iS10, Thermo Scientific, UK) with a wavenumber range of 4000–650 cm-1, scanned with the resolution of 4 cm-1. Dynamic light scattering (DLS, BI-90 Plus, Brookhaven, USA) and Zetasizer Nano Instrument (ZS90, Malvern, UK) were used for characterizing particle size and the average potential. Fluorescence quantification was performed using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) and a flow cytometer
(NovoCyte, ACEA Biosciences Inc., USA). 2.5. Sonocatalytic efficiency The sonocatalytic efficiency was quantified by labeling different fluorescent substances. For extracellular, the production of hydroxyl radicals and singlet oxygen was used to reflect the ultrasound response of TiO2 NPs. Terephthalic acid (TA) has been used to reflect the sonochemical sensitivity, especially for hydroxyl radicals. Non-fluorescent TA could combine with hydroxyl radical to form a fluorescent substance-2-ethyl terephthalate ion (HTA). The fluorescence intensity of HTA could indicate the ultrasonic cavitation level. In brief, the above TiO2 samples were added to the prepared TA dispersion (1×10-3 M). Then the treatment groups were subjected to ultrasonic stimulation in the dark, and the HTA fluorescence intensity was immediately detected by a fluorescence spectrophotometer (λex = 310 nm, λem = 425 nm). In addition, singlet oxygen is another indicator for evaluating the sonoactivated nanoparticles in this experimental system. SOSG (Single Oxygen Sensor Green) is a single oxygen specific capture agent. Because of the impermeability through the cell membrane, SOSG is generally used to detect extracellular molecular oxygen. The SOSG itself emits weak blue fluorescence, but emits strong green fluorescence when captures singlet oxygen.The probe was diluted to an appropriate concentration and incubated with the nanoparticles for 15 min. After the ultrasound treatment, the green fluorescence was detected (λex = 504 nm, λem = 525 nm).
2.6. Sonodynamic antibacterial tests Gram-positive Staphylococcus aureus (S. aureus) (CMCC 260003, Shaanxi Provincial Institute of Microbiology) was diluted with PBS and suspended at 1×106 colony forming units (CFU) per mL. After 1 h co-culture of bacteria and DFT, the bacteria were then irradiated by US (3 W, 60 s). The treated bacteria were diluted into 1 × 103 (CFU/mL) and inoculated onto the plate. After incubation for another 24 h, bacteria were counted with three repeats. The antibacterial efficiency was reported as percentage of bacteria reduction calculated from the average number of surviving bacteria
before
(A)
and
after
(B)
treatment.
Bacteria
reduction(%)
=
[(A-B)/A]×100%. Fluorescence probes play important roles in intracellular tests. DCFH-DA (2’, 7’ -dichlorofluorescent yellow diacetate) has no fluorescence, it could freely cross the cell membrane and be hydrolyzed by intracellular esterase to form DCFH. DCFH can not penetrate the cell membrane, making the probe easily entrapped into the cell. Bacteria were incubated with DFT in the diluent of DCFH-DA (2 × 10-3 M) for 0.5 h, then subjected to US treatment. The reactive ROS in the bacteria could oxidize DCFH to form DCF (with green fluorescence). The fluorescence intensity of DCF was detected to evaluate the level of ROS in bacteria. Besides, after ultrasonic treatment, the membrane impermeable dye propidium iodide (PI) was also used to examine the cell membrane disturbance. The bacterial suspensions (1×106 CFU/mL) were incubated with DFT (40 μg/mL) and exposed to US (3 W, 60 s), after that, PI (1 × 10-3 M) was added and the red fluorescence was recorded by flow cytometry.
3. Results and discussion Compared with traditional organic sonosensitizers, the physiochemical property of inorganic nanomaterials makes them good candidates. The most representative paradigm of inorganic nanosonosensitizers is TiO2 NP, which has been reported as a good sensitizer to respond to ultrasound exposure, and generate ROS such as singlet oxygen (1O2), hydroxyl radical (OH-), etc [23]. The mechanical US wave can affect the energy band of TiO2 and induce electronic transition occurrence. The electron (e-)-hole (h+) pairs are separated and migrate to the surface of the particle, thus inducing a series of redox reactions [14]. The active oxygens generated by sonocatalysis attack the H atoms in the unsaturated bond structure of organisms [24], eventually causing biological effects [25]. The easy-aggregation of bare TiO2 NPs, however, hinders the separation of e--h+ pairs from the energy band upon US exposure. The ROS yields and sonosensitizing effects are therefore low [26]. It is a big challenge to prevent the recombination of US-triggered e--h+ pairs from TiO2 NPs. Accordingly, the combination of TiO2 NPs with other noble metals such as Pt, Au, and Ag has been hybridized to improve the ROS yields [12]. In order to overcome the agglomeration of inorganic nanoparticles and enhance their biocompatibility, we expect to design an organic/inorganic hybrid nanosystem to solve the above problems. In this experimental system, TiO2 NPs were first modified by different modifiers, and the admirable F-TiO2 was chosen for further incorporation of DVDMS. The developed DFT nanocomposite could convert US energy into chemical energy, significantly initiated sonodynamic response to combat bacteria.
The sonocatalysis of TiO2 could generate hydroxyl radicals and single oxygen in aqueous solution. In this work, TA was used to verify the hydroxyl radicals, and the produced fluorescent HTA gradually increased as the concentration of TiO2 NPs increased, which showed a dose-dependent trend and reached peak at 50 μg/mL TiO2, then gradually decreased after that (Fig. 1b). Similarly, SOSG probe was used to trap singlet oxygen generated by ultrasonic stimulation of TiO2. Under 50 μg/mL TiO2, the SOSG fluorescence intensity increased with the increase of ultrasound power, and reached the maximum at 3 W US (1 MHz) (Fig. 1c). For inorganic nanoparticles, the appropriate concentration could ensure the ultrasonic response of each particle. When the concentration is too high, many particles accumulation will affect the uniformity of solute dispersion and cannot produce more ROS [27]. From another point of view, ultrasonic intensity is also important. The lower power is not enough to initiate the sonodynamic process of TiO2. While, the sono-energy generated by too high-power US couldn’t be efficiently converted into active radicals, because the e--h+ pairs provided by NPs are limited. Therefore, the additionally increased US dose (5 W) did not further elevate the SOSG fluorescence intensity when compared with 3 W US. The results suggest that the proper concentration of TiO2 combined with optimal ultrasonic dose could stimulate the biochemical advantage of a sonosensitive agent. It is worth noting that the agglomeration of TiO2 NPs is a big issue which would not only affect the particle size but also hinder the catalytic reaction [28, 29]. When the particles were refined to nanometer level, a large amount of charges can accumulate on the surface, thereby increasing the surface nanoaction and leading to
particle agglomeration [30]. The orientation of proper pH condition also affect the charge distribution of the solution. Lin et al. revealed the correlation between different pH conditions and the particle agglomeration [31]. With the increase of pH, the functional groups of nanoparticles gradually became negatively charged due to deprotonation of carboxyl (pH 2.0-6.0), phospholipid (pH 2.4-7.2), phosphodiester (pH 3.2-3.5), hydroxyl (pH 9.0-10.0) and amino (pH 9.0-11.0) groups. While, the neutral pH condition will amplify the aggregation of nanoparticles. In our studies, we characterized the NPs by particle size (DLS), zeta potential and electron microscopy, and found that TiO2 NPs dispersed hard under neutral pH, which would lead to much larger particle size (1800 nm) and positive zeta potential (53 mV) of the product. Therefore, we chose pH 4 or 8 to provide a suitable deprotonation environment for the modification of nanoparticles, which could help the modifier to relieve the agglomeration of TiO2. As shown in Fig. 2a, three common modifiers, APTMS, F127 and PEG2000 were added respectively to synthesize A-TiO2, F-TiO2 and P-TiO2. The particle sizes of the modifier combinations at pH=4 were all much higher than those of samples at pH=8 (Fig. 2a). The pH condition also changed the potential of particles, in which the modified TiO2 potential decreased sharply because of deprotonation [32] (A-TiO2 = - 23 mV, F-TiO2= - 33.13 mV, P-TiO2 = - 30.8 mV) under pH 8 (Fig. 2b). As a tri-block amphiphilic polymer, F127 has been used as a non-ionic surfactant in material synthesis, which can not only increase the water-solubility of nanoparticles but also increase the permeability of cells [33, 34]. After TiO2 NPs were packed by F127, the original agglomerated blocks dispersed to
some extent under the guidance of hydrophilic chain and pH charge values. The modified F-TiO2 showed largely reduced particle size (212 nm versus 1800 nm) and zeta potential (-33.13 mV versus 53 mV) as compared with the naked TiO2 (Fig. 2a&b). As shown in Fig. 3, the morphologic images of TiO2 and F-TiO2 were observed through field emission scanning electron microscope (HRSEM). The addition of F127 indeed greatly alleviated the agglomeration of TiO2. Based on the comparisons of particle size and stability, we selected F-TiO2 sample (pH = 8) for subsequent tests. Moreover, we also confirmed that F-TiO2 has no optical characteristic peaks and retained the tensile vibration (650 cm-1) of Ti-O (Fig. 2c&d). One hand, the use of our modification method has greatly improved the stability of TiO2 colloids, which presented a stable negative potential. On the other hand, the modified TiO2 was more favorable for the sono/photocatalysis based on its good dispersion. Therefore, we further evaluated the ultrasound sensitivity of F-TiO2. The results in Fig. 2e&f showed that the addition of F127 increased the acoustic efficiency of TiO2. The facial modification with F127 might provide more redox reaction sites, even improved the generation of hydroxyl radicals and singlet oxygen. However, we are not satisfied with the sonochemical efficiency of F-TiO2, which may be due to the fact that the particles has not been sufficiently dispersed and the active sites cannot be exposed to the maximum. Therefore, we hope to further improve the sonocatalytic performance by optimizing the material system. In previous studies, TiO2 NPs have been developed as nanocomposites by combination with other inorganic components such as Au and MnO2, showing
improved sonodynamic efficiency [12, 13]. The TiO2-organic elements nanohybrids have been rarely reported. Porphyrins are good organic sonosensitizer in SDT, with high biocompatibility. We suppose the TiO2-porphyrin nanocomposites would augment the sonochemical effects of either alone. DVDMS is a newly invented sensitizer, which has been assembled with other NPs (MnO2, GO) to produce enhanced photocatalytic effects [35, 36]. In this study, we introduced a small amount of DVDMS to F-TiO2 to obtain the novel nanohybrid-DFT. As shown in Fig. 4a, DFT presented the characteristic porphyrin peaks with slight red-shift compared with free DVDMS, suggesting the successful loading of DVDMS on F-TiO2. The infrared spectrum in Fig. 4b showed that the stretching vibration of Ti-O was shifted from 650 cm-1 to 725 cm-1, when DVDMS adsorbed on the surface of F-TiO2. The asymmetric stretching vibration caused by COO- on DVDMS was at 1566 cm-1 and the vibration at 1067 cm-1 was probably due to the stretching of porphyrin rings. As a small water-soluble molecule, the adjunction of DVDMS not only optimized the nano-system of F-TiO2, but also improved the dispersibility of nano-composites. As shown by DLS in Fig. 4c, the particle size of DFT further decreased to 181.6 nm. As expected, the sonochemical efficiency of DFT nanocomposite was significantly heightened. Under 3 W US stimulation, DFT showed the highest yields of hydroxyl radical and singlet oxygen, as compared with the other groups (Fig. 4d&e). Moreover, the sonochemical efficiency of DFT also showed drug concentration- dependent manners. DFT with high sonoactivity further decreased the drug dosage for cavitational effect and free radicals production. Fig. 4f showed the hydroxyl radicals
reached peak at a low concentration of 20 μg/mL DFT (versus 50 μg/mL TiO2 in Fig. 1b), under 3 W US stimulation. In addition, the free radicals induced by US plus DFT (20 μg/mL) was significantly inhibited by ROS scavengers like catalase (CAT) and histidine (his) (Fig. 5a&b), which confirmed the cavitational events in the process. The results suggest a synergistic combination between DVDMS and F-TiO2. Under US stimulation, both TiO2 and DVDMS in a close affinity in the same particle were fully activated and boosted ROS production. Recently, the combination of TiO2 NPs with other particles has shown improved quantum yields. Such hybrid modification not only prevents the e--h+ recombination by trapping the excited electron but also increases the absorption spectrum via surface plasmon resonance [37-38]. The enhanced catalytic property of TiO2 nanocomposites has been confirmed in various industrial and biomedical fields [39]. At present, pathogenic bacteria are a formidable public health enemy that causes many diseases ranging from minor skin infections to life-threatening abscesses [40-41]. Sonocatalytic antibacteria is a newly developed strategy with high efficiency and easy application [42]. In this work, we move forward to explore the potential antibacterial efficiency of DFT+US. S. aureus was chosen as the research model because it is the representative strain of Gram-positive bacteria with high toxicity. As shown in Fig. 6, US stimulating DFT significantly inhibited the survival and proliferation of S. aureus, with as high as 92.41% of inactivation efficiency. The temperature increase in the stimulated samples during the whole process was below 37°C, so the main reasons for bacterial eradication would be sonodynamic effects. Simultaneously, the increased
intracellular ROS level and membrane disturbance were shown by fluorescence dyes and flow cytometry (Fig. 6b). In the past reports, the photocatalytic efficiency of TiO2 suffered from the surficial aggregation and insufficient electron-hole pairs, and led to unsatisfied antibacterial efficacy [43]. Accordingly, the sonodynamic action in this study achieved more acceptable results, because US has deep penetration and DFT shows reformative features. Besides, the negative potential property of DFT was conducive to interaction with Gram-positive bacteria. Therefore, the developed combination of DFT and US would be helpful in combating Gram-positive bacteria and related infections. 4. Conclusions In summary, we successfully developed an US-responsive nanocomposite DFT which consisted of an organic porphyrin sensitizer DVDMS, and an inorganic semiconductor material TiO2 with proper surface modification. Considering that TiO2 NPs with usual aggregation compromised their performance in applications, this work optimized the nanoparticle system by surface modification with F127, under alkaline condition (pH = 8). The additional incorporation of DVDMS into F-TiO2 showed much better dispersion and stability, as well as the decreased usage for sonochemical effects. Ultrasound stimulating DFT showed improved ROS generation and highly efficient bacterial eradication. The designed nanohybrid in this study is promising in future sono-/photo-catalytic applications and antibacterial studies. Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 81972900, 81872497), the Innovative Talents Promotion Plan in Shaanxi Province (2017KJXX-78), the Natural Science Foundation of Shaanxi province (2019JZ-13), the Academic Leaders and Academic Backbones, Shaanxi Normal University (16QNGG012), the Fundamental Research Funds for the Central Universities (Grant Nos. 2019CSLY011, GK201802002). References [1] X. Qian, X. Han, Y. Chen, Insights into the unique functionality of inorganic micro/nanoparticlesforversatile ultrasound theranostics, Biomaterials. 142 (2017) 13-30.
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Figure legends Fig. 1. (a) Ultrasonic treatment of TiO2 NPs. (b) The produced hydroxyl radicals by US stimulation (1 MHz, 3 W) of TiO2 NPs using TA method. (c) The generated single oxygen of TiO2 NPs (50 μg/mL) under different US conditions (1 MHz, 1-5 W, 60 s) using SOSG probe. #p<0.05, ##p<0.01, ###p<0.001,
compared with control; *p<0.05, **p<0.01, ***p<0.001, between groups.
Fig. 2. (a) Changes in the size of TiO2, A-TiO2, F-TiO2, and P-TiO2 as a function of pH conditions (pH 4 and pH 8). (b) The zeta potential of A-TiO2, F-TiO2 and P-TiO2 under pH 8. (c, d) UV-vis spectra and FTIR patterns of F-TiO2. (e) The evaluation of hydroxyl radicals between TiO2 and F-TiO2 (50 μg/mL) with or without 3 W US stimulation. (f) The evaluation of single oxygen between TiO2 and F-TiO2 (50 μg/mL) with or without 3 W US stimulation. *p<0.05, **p<0.01, ***p<0.001, between groups.
Fig. 3. HRSEM images of different particles (a, b: TiO2; c, d: F- TiO2) (bar=5 μm, 1 μm).
Fig. 4. (a) UV-vis spectra of DVDMS and DFT. (b) FTIR pattern of DFT. (c) The size
distributions of F-TiO2 and DFT. (d) The hydroxyl radical production of DVDMS, F-TiO2 and DFT under the same US exposure (3 W, 60 s). (e) The single oxygen production of DVDMS, F-TiO2 and DFT under the same US exposure (3 W, 60 s). (f) The generation of hydroxyl radicals using 3 W US stimulating different concentration of DFT. #p<0.05,
##p<0.01, ###p<0.001,
compared with control; *p<0.05, **p<0.01, ***p<0.001, between groups.
Fig. 5. (a) The hydroxyl radicals of US+DFT (20 μg/mL) with or without CAT, under distinct US exposure (1 W, 3 W, 5 W). (b) The single oxygen of US+DFT (20 μg/mL) with or without his, under distinct US exposure (1 W, 3 W, 5 W). ***p<0.001, between groups.
Fig. 6. (a) The antibacterial mechanism of DFT nanoparticles. (b) The optical photographs of petri dish after different treatment (upper channel); Flow cytometric analysis of intracellular ROS generation (middle channel) and membrane disruption (lower channel) after ultrasonic treatment of S. aureus. (c) The antimicrobial rate of DFT plus US in S. aureus (3 W US; 40 μg/mL DFT). **p<0.01, ***p<0.001, compared with control.
Conflict of Interest This manuscript has not been published or presented elsewhere and is not under consideration by another journal. We have read and understood your journal’s policies, and we believe that the manuscript complies with these policies. There are no conflicts of interest to declare.
Highlights: Under different pH systems, a series of modifiers were added to disperse the TiO2 even better. The new porphyrin sensitizer-DVDMS was introduced for augmenting the sonosensitivity of TiO2. The designed novel nanohybrid DFT exhibit excellent sonochemical responsiveness and bacterial eradication efficiency.
S1350-4177(19)31664-5 https://doi.org/10.1016/j.ultsonch.2020.104968 ULTSON 104968
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
22 October 2019 30 December 2019 9 January 2020
Please cite this article as: Y. Wang, Y. Sun, S. Liu, L. Zhi, X. Wang, Preparation of sonoactivated TiO2-DVDMS nanocomposite for enhanced antibacterial activity, Ultrasonics Sonochemistry (2020), doi: https://doi.org/10.1016/ j.ultsonch.2020.104968
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Title：Preparation of sonoactivated TiO2-DVDMS nanocomposite for enhanced antibacterial activity Authors：Yihui Wang, Yue Sun, Shupei Liu, Lijuan Zhi, Xiaobing Wang* Affiliation: National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China; Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education; College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi 710119, China. Corresponding Author E-mail: [email protected] (Xiaobing Wang) Tel: +86-29-85310275
Abstract Titanium dioxide (TiO2) nanoparticle has good photo-/sono-catalytic features, the reunion of this particle in solution-phase generally limits the extensive biomedical application. In the present study, the aggregation of TiO2 nanoparticles was alleviated by facile fabrication under different pH conditions. A novel TiO2 nanocomposite was further synthesized by properly conjugation with trace amount of DVDMS sensitizer (named DFT). The characterization, sonoactivity, as well as the antibacterial efficiency were specially evaluated. The results showed that the sonochemical activity of DFT was greatly improved as compared with the simple surface modification of TiO2 (F-TiO2) and free DVDMS, regarding to the hydroxyl radicals and singlet oxygen yields using the same ultrasound exposure. Moreover, ultrasonic stimulation of DFT exhibited excellent bacterial eradication, with up to 92.41% of killing efficiency in S. aureus. The flow cytometry analysis indicated an increased intracellular ROS and membrane disturbance by combination of DFT and ultrasound. The findings suggest that the proper fabrication and DVDMS incorporation greatly improved the sonocatalytic process of TiO2, and the ultrasound based biomedical applications of DFT deserve future deep investigation.
Key word: TiO2 nanoparticles, Ultrasound, DVDMS, Sonochemical reaction, Antibacterial effect 1. Introduction As an inorganic semiconductor material, titanium dioxide (TiO2) has made dramatic breakthroughs in nanomaterial due to its advantages of easy fabrication, abundant composition and versatile structures [1, 2]. In the past decades, the photocatalytic activity of TiO2 nanoparticles (NPs) has attracted much attention. TiO2 NPs can convert the natural light energy into the chemical reaction energy to initiate catalytic effect, and cause the surrounding O2 and H2O to be excited into free anions with oxidation power [3]. Nevertheless, the single TiO2 products have some flaws like in stability and energy transfer efficiency. Based on this, Geng et al. prepared Fe3O4/TiO2-N-GO nanocomposites to utilize the photocatalysis of TiO2 to degrade humic acid [4]. Hafeeza et al. developed a ternary TiO2 nanocomposite loaded on rGO plates (InVO4-TiO2) to produce efficient hydrogen energy by converting solar energy [5]. These diverse findings have inspired researchers’ great interests to develop various functional TiO2 nanomaterials for conquering the structural defects of semiconductor particles themselves and improving the photo-/sono-catalysis [6]. In recent years, along with the rapid development of TiO2 nanomaterials, not only the potential photocatalytic activities have been explored, but also the possible ultrasound (US)-responsiveness has been continuously confirmed [1]. US has been generally used as an auxiliary method for TiO2 multi-composites synthesis, and the
stimulation of different US frequency and power would affect the sonochemical efficiency [7, 8]. Sonochemical reaction is widely used because of its cavitation effect, especially in sonodynamic therapy (SDT). Upon activation by US, the sonosensitizers would transfer energy to oxygen molecules, leading to the production of reactive oxygen species (ROS), further causing serious cellular toxicity of SDT [9]. TiO2 has shown good sonoactivity in SDT. Harada et al. investigated the therapeutic effects of US stimulating TiO2 NPs on melanoma cells in vitro and in vivo [10]. Since TiO2 NPs strongly absorb US energy and generate a large amount of ROS such as hydroxyl radicals and super oxides, that can be applied in biomedicine fields [11]. Deepagan et al. prepared a long-circulating Au-TiO2 nanocomposite as sonosensitizer and optimized the pore size of mesoporous TiO2 for SDT [12]. Dai et al. reported on augmenting the sonocatalytic efficiency of TiO2-based nanosonosensitizer for highly efficient SDT by the integration of two-dimensional ultrathin graphene with TiO2 [13]. Chen et al. synthesized B-TiO2 by high-temperature reduction method, broadening the second near-infrared region imaging and TiO2-based drug delivery [14]. To some extent, these diverse designs facilitated the separation of electron-hole (e--h+) pairs from TiO2 and improved the sonocatalytic activity. As one of the representative paradigm of inorganic nanosonosensitizers, the sonodynamic efficacy of TiO2 is limited by the particle agglomeration which hinders the segregation of electron and hole pairs from the energy band post US trigger, thus yields low ROS [9]. Through tremendous effort on modification of TiO2 NPs, the problems of TiO2 aggregation have not been solved exhaustively [15]. Because of the
high surface tension of TiO2 NPs in liquid, they are easy to agglomerate into powder blocks which tremendous difficulties to solution dispersion and nanomaterial synthesis, and engender barriers in the exertion of their functions [16, 17]. Studies have found that the size of TiO2 NPs and surrounding pH condition impacted their photocatalytic and biomedical properties. Pettibone et al showed that the natural pH condition exacerbate the aggregation of TiO2 nanoparticles [18]. The decrease of the particle size elicits the increase of the specific surface area as well as the catalytic activity [19]. The proper modification of nanoparticles, on one hand, enables the material to disperse well in the liquid, and on the other hand, strengthens the active center of particles [20]. Therefore, the usage of some suitable modifications to overcome TiO2 NPs agglomeration would be a good choice to improve the sonotherapy efficiency. We hope to enhance the deprotonation of the functional groups of TiO2 by adjusting the pH value, thus affecting the agglomeration behavior of the particles themselves. In brief, two different pH systems (8 and 4) were used to influence the ion distribution in the solution for evaluating the stability of nanoparticles. At the same time, the addition of different modifiers exhibited more excellent dispersion of TiO2.We also introduced a porphyrin sensitizer-DVDMS, to develop new nanocomposite for augmenting the sonosensitivity of TiO2. DVDMS (Sinoporphyrin sodium) has been previously confirmed with fabulous water solubility and high photo-/sono-activity [21]. Herein, a new hybrid nanocomposite (designated as DFT) was developed by incorporation of a small amount of DVDMS on surfactant modified TiO2 NPs.
Firstly, the surface fabrication of TiO2 NPs was optimized using different modifiers (APTMS, F127, PEG2000) under distinct pH values. A novel TiO2-DVDMS nanocomposite was then prepared and evaluated for sonochemical effects. Antimicrobial sonodynamic therapy has shown great potentials in eradicating bacterial infections [22]. Therefore, the enhanced sonodynamic antibacterial activity was specially examined in Gram-positive bacteria. The results showed that the surface modifications partially alleviated the aggregation of TiO2 NPs, and the DVDMS incorporation further improved the dispersion and biocompatibility of the nanocomposite. The designed hybrid DFT exhibit excellent sonochemical responsiveness and bacterial eradication efficiency. Such constructs can be expanded to many other disease treatment using sono/phototherapy, and the design strategy may derive several therapeutic nanoplatforms. 2. Experimental 2.1. Materials Titanium dioxide (TiO2, 60 nm, anatase phase) and terephthalic acid (TA) were obtained from Aladdin. DVDMS (purity> 98%) was the property of Qinglong Hi-tech Co. Ltd. (Jiangxi, China). Singlet oxygen sensor green (SOSG) probe was supplied by Molecular Probes Inc. (Invitrogen, USA). All other chemicals were analytical grade. Deionized water was used through out the study. 2.2. Surface fabrication of TiO2 In detail, 0.01 g TiO2 was completely dissolved in 10 mL water and stirred at
room temperature for 1 h, then adjusted the pH of the solution (4 or 8) and subjected to ultrasonic dispersion (400 w, 40 kHz) for 15 min. 0.5 wt% of modifiers (APTMS, F127, PEG2000) were individually added to the dispersion and constantly stirred for 2 h, and the samples were named A-TiO2, F-TiO2, P-TiO2, respectively. The final dispersion was washed and centrifuged (3000 rpm, 5 min) three times to remove impurities, then re-suspended in deionized water and stored at 4°C. 2.3. Preparation of DFT dispersion An appropriate amount of DVDMS was dissolved in 5 mL of physiological saline to prepare a 2 mg/mL pure liquid. 1 mL of 200 μg/mL DVDMS dilution was added to 1 mL of F-TiO2 dispersion (pH = 8) and stirred for 12 h in the dark. The DFT dispersion was washed and centrifuged three times (6000 rpm, 5 min), and re-suspended in water at 4°C in the dark. 2.4. Characterization The optical characteristics of TiO2 and DFT were measured by UV − vis spectrophotometer (SpectraMax M5, Molecular Devices, US). IR-spectra were collected and recorded with a Fourier infrared spectrometer (Nicolet iS10, Thermo Scientific, UK) with a wavenumber range of 4000–650 cm-1, scanned with the resolution of 4 cm-1. Dynamic light scattering (DLS, BI-90 Plus, Brookhaven, USA) and Zetasizer Nano Instrument (ZS90, Malvern, UK) were used for characterizing particle size and the average potential. Fluorescence quantification was performed using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) and a flow cytometer
(NovoCyte, ACEA Biosciences Inc., USA). 2.5. Sonocatalytic efficiency The sonocatalytic efficiency was quantified by labeling different fluorescent substances. For extracellular, the production of hydroxyl radicals and singlet oxygen was used to reflect the ultrasound response of TiO2 NPs. Terephthalic acid (TA) has been used to reflect the sonochemical sensitivity, especially for hydroxyl radicals. Non-fluorescent TA could combine with hydroxyl radical to form a fluorescent substance-2-ethyl terephthalate ion (HTA). The fluorescence intensity of HTA could indicate the ultrasonic cavitation level. In brief, the above TiO2 samples were added to the prepared TA dispersion (1×10-3 M). Then the treatment groups were subjected to ultrasonic stimulation in the dark, and the HTA fluorescence intensity was immediately detected by a fluorescence spectrophotometer (λex = 310 nm, λem = 425 nm). In addition, singlet oxygen is another indicator for evaluating the sonoactivated nanoparticles in this experimental system. SOSG (Single Oxygen Sensor Green) is a single oxygen specific capture agent. Because of the impermeability through the cell membrane, SOSG is generally used to detect extracellular molecular oxygen. The SOSG itself emits weak blue fluorescence, but emits strong green fluorescence when captures singlet oxygen.The probe was diluted to an appropriate concentration and incubated with the nanoparticles for 15 min. After the ultrasound treatment, the green fluorescence was detected (λex = 504 nm, λem = 525 nm).
2.6. Sonodynamic antibacterial tests Gram-positive Staphylococcus aureus (S. aureus) (CMCC 260003, Shaanxi Provincial Institute of Microbiology) was diluted with PBS and suspended at 1×106 colony forming units (CFU) per mL. After 1 h co-culture of bacteria and DFT, the bacteria were then irradiated by US (3 W, 60 s). The treated bacteria were diluted into 1 × 103 (CFU/mL) and inoculated onto the plate. After incubation for another 24 h, bacteria were counted with three repeats. The antibacterial efficiency was reported as percentage of bacteria reduction calculated from the average number of surviving bacteria
before
(A)
and
after
(B)
treatment.
Bacteria
reduction(%)
=
[(A-B)/A]×100%. Fluorescence probes play important roles in intracellular tests. DCFH-DA (2’, 7’ -dichlorofluorescent yellow diacetate) has no fluorescence, it could freely cross the cell membrane and be hydrolyzed by intracellular esterase to form DCFH. DCFH can not penetrate the cell membrane, making the probe easily entrapped into the cell. Bacteria were incubated with DFT in the diluent of DCFH-DA (2 × 10-3 M) for 0.5 h, then subjected to US treatment. The reactive ROS in the bacteria could oxidize DCFH to form DCF (with green fluorescence). The fluorescence intensity of DCF was detected to evaluate the level of ROS in bacteria. Besides, after ultrasonic treatment, the membrane impermeable dye propidium iodide (PI) was also used to examine the cell membrane disturbance. The bacterial suspensions (1×106 CFU/mL) were incubated with DFT (40 μg/mL) and exposed to US (3 W, 60 s), after that, PI (1 × 10-3 M) was added and the red fluorescence was recorded by flow cytometry.
3. Results and discussion Compared with traditional organic sonosensitizers, the physiochemical property of inorganic nanomaterials makes them good candidates. The most representative paradigm of inorganic nanosonosensitizers is TiO2 NP, which has been reported as a good sensitizer to respond to ultrasound exposure, and generate ROS such as singlet oxygen (1O2), hydroxyl radical (OH-), etc [23]. The mechanical US wave can affect the energy band of TiO2 and induce electronic transition occurrence. The electron (e-)-hole (h+) pairs are separated and migrate to the surface of the particle, thus inducing a series of redox reactions [14]. The active oxygens generated by sonocatalysis attack the H atoms in the unsaturated bond structure of organisms [24], eventually causing biological effects [25]. The easy-aggregation of bare TiO2 NPs, however, hinders the separation of e--h+ pairs from the energy band upon US exposure. The ROS yields and sonosensitizing effects are therefore low [26]. It is a big challenge to prevent the recombination of US-triggered e--h+ pairs from TiO2 NPs. Accordingly, the combination of TiO2 NPs with other noble metals such as Pt, Au, and Ag has been hybridized to improve the ROS yields [12]. In order to overcome the agglomeration of inorganic nanoparticles and enhance their biocompatibility, we expect to design an organic/inorganic hybrid nanosystem to solve the above problems. In this experimental system, TiO2 NPs were first modified by different modifiers, and the admirable F-TiO2 was chosen for further incorporation of DVDMS. The developed DFT nanocomposite could convert US energy into chemical energy, significantly initiated sonodynamic response to combat bacteria.
The sonocatalysis of TiO2 could generate hydroxyl radicals and single oxygen in aqueous solution. In this work, TA was used to verify the hydroxyl radicals, and the produced fluorescent HTA gradually increased as the concentration of TiO2 NPs increased, which showed a dose-dependent trend and reached peak at 50 μg/mL TiO2, then gradually decreased after that (Fig. 1b). Similarly, SOSG probe was used to trap singlet oxygen generated by ultrasonic stimulation of TiO2. Under 50 μg/mL TiO2, the SOSG fluorescence intensity increased with the increase of ultrasound power, and reached the maximum at 3 W US (1 MHz) (Fig. 1c). For inorganic nanoparticles, the appropriate concentration could ensure the ultrasonic response of each particle. When the concentration is too high, many particles accumulation will affect the uniformity of solute dispersion and cannot produce more ROS [27]. From another point of view, ultrasonic intensity is also important. The lower power is not enough to initiate the sonodynamic process of TiO2. While, the sono-energy generated by too high-power US couldn’t be efficiently converted into active radicals, because the e--h+ pairs provided by NPs are limited. Therefore, the additionally increased US dose (5 W) did not further elevate the SOSG fluorescence intensity when compared with 3 W US. The results suggest that the proper concentration of TiO2 combined with optimal ultrasonic dose could stimulate the biochemical advantage of a sonosensitive agent. It is worth noting that the agglomeration of TiO2 NPs is a big issue which would not only affect the particle size but also hinder the catalytic reaction [28, 29]. When the particles were refined to nanometer level, a large amount of charges can accumulate on the surface, thereby increasing the surface nanoaction and leading to
particle agglomeration [30]. The orientation of proper pH condition also affect the charge distribution of the solution. Lin et al. revealed the correlation between different pH conditions and the particle agglomeration [31]. With the increase of pH, the functional groups of nanoparticles gradually became negatively charged due to deprotonation of carboxyl (pH 2.0-6.0), phospholipid (pH 2.4-7.2), phosphodiester (pH 3.2-3.5), hydroxyl (pH 9.0-10.0) and amino (pH 9.0-11.0) groups. While, the neutral pH condition will amplify the aggregation of nanoparticles. In our studies, we characterized the NPs by particle size (DLS), zeta potential and electron microscopy, and found that TiO2 NPs dispersed hard under neutral pH, which would lead to much larger particle size (1800 nm) and positive zeta potential (53 mV) of the product. Therefore, we chose pH 4 or 8 to provide a suitable deprotonation environment for the modification of nanoparticles, which could help the modifier to relieve the agglomeration of TiO2. As shown in Fig. 2a, three common modifiers, APTMS, F127 and PEG2000 were added respectively to synthesize A-TiO2, F-TiO2 and P-TiO2. The particle sizes of the modifier combinations at pH=4 were all much higher than those of samples at pH=8 (Fig. 2a). The pH condition also changed the potential of particles, in which the modified TiO2 potential decreased sharply because of deprotonation [32] (A-TiO2 = - 23 mV, F-TiO2= - 33.13 mV, P-TiO2 = - 30.8 mV) under pH 8 (Fig. 2b). As a tri-block amphiphilic polymer, F127 has been used as a non-ionic surfactant in material synthesis, which can not only increase the water-solubility of nanoparticles but also increase the permeability of cells [33, 34]. After TiO2 NPs were packed by F127, the original agglomerated blocks dispersed to
some extent under the guidance of hydrophilic chain and pH charge values. The modified F-TiO2 showed largely reduced particle size (212 nm versus 1800 nm) and zeta potential (-33.13 mV versus 53 mV) as compared with the naked TiO2 (Fig. 2a&b). As shown in Fig. 3, the morphologic images of TiO2 and F-TiO2 were observed through field emission scanning electron microscope (HRSEM). The addition of F127 indeed greatly alleviated the agglomeration of TiO2. Based on the comparisons of particle size and stability, we selected F-TiO2 sample (pH = 8) for subsequent tests. Moreover, we also confirmed that F-TiO2 has no optical characteristic peaks and retained the tensile vibration (650 cm-1) of Ti-O (Fig. 2c&d). One hand, the use of our modification method has greatly improved the stability of TiO2 colloids, which presented a stable negative potential. On the other hand, the modified TiO2 was more favorable for the sono/photocatalysis based on its good dispersion. Therefore, we further evaluated the ultrasound sensitivity of F-TiO2. The results in Fig. 2e&f showed that the addition of F127 increased the acoustic efficiency of TiO2. The facial modification with F127 might provide more redox reaction sites, even improved the generation of hydroxyl radicals and singlet oxygen. However, we are not satisfied with the sonochemical efficiency of F-TiO2, which may be due to the fact that the particles has not been sufficiently dispersed and the active sites cannot be exposed to the maximum. Therefore, we hope to further improve the sonocatalytic performance by optimizing the material system. In previous studies, TiO2 NPs have been developed as nanocomposites by combination with other inorganic components such as Au and MnO2, showing
improved sonodynamic efficiency [12, 13]. The TiO2-organic elements nanohybrids have been rarely reported. Porphyrins are good organic sonosensitizer in SDT, with high biocompatibility. We suppose the TiO2-porphyrin nanocomposites would augment the sonochemical effects of either alone. DVDMS is a newly invented sensitizer, which has been assembled with other NPs (MnO2, GO) to produce enhanced photocatalytic effects [35, 36]. In this study, we introduced a small amount of DVDMS to F-TiO2 to obtain the novel nanohybrid-DFT. As shown in Fig. 4a, DFT presented the characteristic porphyrin peaks with slight red-shift compared with free DVDMS, suggesting the successful loading of DVDMS on F-TiO2. The infrared spectrum in Fig. 4b showed that the stretching vibration of Ti-O was shifted from 650 cm-1 to 725 cm-1, when DVDMS adsorbed on the surface of F-TiO2. The asymmetric stretching vibration caused by COO- on DVDMS was at 1566 cm-1 and the vibration at 1067 cm-1 was probably due to the stretching of porphyrin rings. As a small water-soluble molecule, the adjunction of DVDMS not only optimized the nano-system of F-TiO2, but also improved the dispersibility of nano-composites. As shown by DLS in Fig. 4c, the particle size of DFT further decreased to 181.6 nm. As expected, the sonochemical efficiency of DFT nanocomposite was significantly heightened. Under 3 W US stimulation, DFT showed the highest yields of hydroxyl radical and singlet oxygen, as compared with the other groups (Fig. 4d&e). Moreover, the sonochemical efficiency of DFT also showed drug concentration- dependent manners. DFT with high sonoactivity further decreased the drug dosage for cavitational effect and free radicals production. Fig. 4f showed the hydroxyl radicals
reached peak at a low concentration of 20 μg/mL DFT (versus 50 μg/mL TiO2 in Fig. 1b), under 3 W US stimulation. In addition, the free radicals induced by US plus DFT (20 μg/mL) was significantly inhibited by ROS scavengers like catalase (CAT) and histidine (his) (Fig. 5a&b), which confirmed the cavitational events in the process. The results suggest a synergistic combination between DVDMS and F-TiO2. Under US stimulation, both TiO2 and DVDMS in a close affinity in the same particle were fully activated and boosted ROS production. Recently, the combination of TiO2 NPs with other particles has shown improved quantum yields. Such hybrid modification not only prevents the e--h+ recombination by trapping the excited electron but also increases the absorption spectrum via surface plasmon resonance [37-38]. The enhanced catalytic property of TiO2 nanocomposites has been confirmed in various industrial and biomedical fields [39]. At present, pathogenic bacteria are a formidable public health enemy that causes many diseases ranging from minor skin infections to life-threatening abscesses [40-41]. Sonocatalytic antibacteria is a newly developed strategy with high efficiency and easy application [42]. In this work, we move forward to explore the potential antibacterial efficiency of DFT+US. S. aureus was chosen as the research model because it is the representative strain of Gram-positive bacteria with high toxicity. As shown in Fig. 6, US stimulating DFT significantly inhibited the survival and proliferation of S. aureus, with as high as 92.41% of inactivation efficiency. The temperature increase in the stimulated samples during the whole process was below 37°C, so the main reasons for bacterial eradication would be sonodynamic effects. Simultaneously, the increased
intracellular ROS level and membrane disturbance were shown by fluorescence dyes and flow cytometry (Fig. 6b). In the past reports, the photocatalytic efficiency of TiO2 suffered from the surficial aggregation and insufficient electron-hole pairs, and led to unsatisfied antibacterial efficacy [43]. Accordingly, the sonodynamic action in this study achieved more acceptable results, because US has deep penetration and DFT shows reformative features. Besides, the negative potential property of DFT was conducive to interaction with Gram-positive bacteria. Therefore, the developed combination of DFT and US would be helpful in combating Gram-positive bacteria and related infections. 4. Conclusions In summary, we successfully developed an US-responsive nanocomposite DFT which consisted of an organic porphyrin sensitizer DVDMS, and an inorganic semiconductor material TiO2 with proper surface modification. Considering that TiO2 NPs with usual aggregation compromised their performance in applications, this work optimized the nanoparticle system by surface modification with F127, under alkaline condition (pH = 8). The additional incorporation of DVDMS into F-TiO2 showed much better dispersion and stability, as well as the decreased usage for sonochemical effects. Ultrasound stimulating DFT showed improved ROS generation and highly efficient bacterial eradication. The designed nanohybrid in this study is promising in future sono-/photo-catalytic applications and antibacterial studies. Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 81972900, 81872497), the Innovative Talents Promotion Plan in Shaanxi Province (2017KJXX-78), the Natural Science Foundation of Shaanxi province (2019JZ-13), the Academic Leaders and Academic Backbones, Shaanxi Normal University (16QNGG012), the Fundamental Research Funds for the Central Universities (Grant Nos. 2019CSLY011, GK201802002). References [1] X. Qian, X. Han, Y. Chen, Insights into the unique functionality of inorganic micro/nanoparticlesforversatile ultrasound theranostics, Biomaterials. 142 (2017) 13-30.
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Figure legends Fig. 1. (a) Ultrasonic treatment of TiO2 NPs. (b) The produced hydroxyl radicals by US stimulation (1 MHz, 3 W) of TiO2 NPs using TA method. (c) The generated single oxygen of TiO2 NPs (50 μg/mL) under different US conditions (1 MHz, 1-5 W, 60 s) using SOSG probe. #p<0.05, ##p<0.01, ###p<0.001,
compared with control; *p<0.05, **p<0.01, ***p<0.001, between groups.
Fig. 2. (a) Changes in the size of TiO2, A-TiO2, F-TiO2, and P-TiO2 as a function of pH conditions (pH 4 and pH 8). (b) The zeta potential of A-TiO2, F-TiO2 and P-TiO2 under pH 8. (c, d) UV-vis spectra and FTIR patterns of F-TiO2. (e) The evaluation of hydroxyl radicals between TiO2 and F-TiO2 (50 μg/mL) with or without 3 W US stimulation. (f) The evaluation of single oxygen between TiO2 and F-TiO2 (50 μg/mL) with or without 3 W US stimulation. *p<0.05, **p<0.01, ***p<0.001, between groups.
Fig. 3. HRSEM images of different particles (a, b: TiO2; c, d: F- TiO2) (bar=5 μm, 1 μm).
Fig. 4. (a) UV-vis spectra of DVDMS and DFT. (b) FTIR pattern of DFT. (c) The size
distributions of F-TiO2 and DFT. (d) The hydroxyl radical production of DVDMS, F-TiO2 and DFT under the same US exposure (3 W, 60 s). (e) The single oxygen production of DVDMS, F-TiO2 and DFT under the same US exposure (3 W, 60 s). (f) The generation of hydroxyl radicals using 3 W US stimulating different concentration of DFT. #p<0.05,
##p<0.01, ###p<0.001,
compared with control; *p<0.05, **p<0.01, ***p<0.001, between groups.
Fig. 5. (a) The hydroxyl radicals of US+DFT (20 μg/mL) with or without CAT, under distinct US exposure (1 W, 3 W, 5 W). (b) The single oxygen of US+DFT (20 μg/mL) with or without his, under distinct US exposure (1 W, 3 W, 5 W). ***p<0.001, between groups.
Fig. 6. (a) The antibacterial mechanism of DFT nanoparticles. (b) The optical photographs of petri dish after different treatment (upper channel); Flow cytometric analysis of intracellular ROS generation (middle channel) and membrane disruption (lower channel) after ultrasonic treatment of S. aureus. (c) The antimicrobial rate of DFT plus US in S. aureus (3 W US; 40 μg/mL DFT). **p<0.01, ***p<0.001, compared with control.
Conflict of Interest This manuscript has not been published or presented elsewhere and is not under consideration by another journal. We have read and understood your journal’s policies, and we believe that the manuscript complies with these policies. There are no conflicts of interest to declare.
Highlights: Under different pH systems, a series of modifiers were added to disperse the TiO2 even better. The new porphyrin sensitizer-DVDMS was introduced for augmenting the sonosensitivity of TiO2. The designed novel nanohybrid DFT exhibit excellent sonochemical responsiveness and bacterial eradication efficiency.