Enhanced antibacterial activity of silica nanorattles with ZnO combination nanoparticles against methicillin-resistant Staphylococcus aureus

Accepted Manuscript Article Enhanced antibacterial activity of silica nanorattles with ZnO combination nanoparticles against methicillin-resistant Staphylococcus aureus Qianqian Chai, Qiong Wu, Tianlong Liu, Longfei Tan, Changhui Fu, Xiangling Ren, Yue Yang, Xianwei Meng PII: DOI: Reference:

S2095-9273(17)30414-0 http://dx.doi.org/10.1016/j.scib.2017.08.016 SCIB 201

To appear in:

Science Bulletin

Received Date: Revised Date: Accepted Date:

16 March 2017 2 June 2017 12 June 2017

Please cite this article as: Q. Chai, Q. Wu, T. Liu, L. Tan, C. Fu, X. Ren, Y. Yang, X. Meng, Enhanced antibacterial activity of silica nanorattles with ZnO combination nanoparticles against methicillin-resistant Staphylococcus aureus, Science Bulletin (2017), doi: http://dx.doi.org/10.1016/j.scib.2017.08.016

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Article Received 16 March 2017; Revised 2 June, 2017; Accepted 12 June, 2017

Enhanced antibacterial activity of silica nanorattles with ZnO combination nanoparticles against methicillin-resistant Staphylococcus aureus Qianqian Chai1,2, Qiong Wu1,2, Tianlong Liu*1, Longfei Tan1, Changhui Fu1, Xiangling Ren1, Yue Yang1, Xianwei Meng*1

1 Laboratory of Controllable Preparation and Application of Nanomaterials, CAS Key Laboratory of Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China 2 University of Chinese Academy of Sciences, Beijing 100049, China

*Corresponding Author: E-mail addresses: [email protected]; [email protected]

Abstract Silica nanorattles (SNs) with zinc oxide (ZnO) combination nanoparticles are reported to inhibit methicillin-resistant Staphylococcus aureus (MRSA) for the first time. SNs loaded with ZnO nanoparticles, which can produce free radicals, can cause severe damage to bacteria. ZnO nanoparticles not only provide free radicals in the combined nanostructures, which can inhibit the growth of bacteria, but also form nanorough surfaces with an irregular distribution of spikes on the SNs, which can enhance their adhesion to bacteria. Nanorough silica shell surfaces maintain the high activity and stability of small-sized ZnO nanoparticles and gather ZnO nanoparticles together to enhance production, which improves the efficiency of free radicals against the cytomembranes of bacterial cells. The enhanced adhesion of ZnO@SN nanoparticles to MRSA cells shortens the effective touching distance between free radicals and MRSA, which also improves antibacterial activity. As we expected, the ZnO@SN nanoparticles exhibit a better antibacterial effect than free ZnO nanoparticles against MRSA in vitro and in vivo. We also demonstrate that SNs loaded with ZnO nanoparticles can accelerate wound healing in MRSA skin inflammation models. This method of multilevel functionalization will be potentially applicable to the antibacterial field.

Keywords: Silica nanorattles, ZnO nanoparticles, methicillin-resistant Staphylococcus aureus (MRSA), rough surface, reactive oxygen species

1

1. Introduction In recent decades, methicillin-resistant Staphylococcus aureus (MRSA) has attracted increasing attention because of its ability to cause widespread infections all over the world and its high prevalence in clinics. Due to these facts and the decrease in new antibiotics reaching the market, the development of new and effective therapeutic approaches for MRSA treatment is needed. Nanomaterials, as novel and effective agents to inhibit, reduce or even kill numerous microorganisms, have been extensively applied in biomedical fields [1-4]. Some nanoparticles, such as zinc oxide, silver, copper and iron oxide nanoparticles, have been successfully introduced in anti-infecting agents applied in wound dressings [5,6], protective clothing [7], nanomedicines, water treatment [8], food preservation and disinfecting agents [9-12]. ZnO nanoparticles are among the most heavily researched antibacterial materials due to their broad spectrum antibacterial properties, variety of preparation methods, easy modification of physico-chemical properties, and their low toxicity. It should be noted that the physicochemical parameters of ZnO nanoparticles, such as size, shape, and zeta potential, can directly influence their antibacterial activity [13-15]. For example, small ZnO nanoparticles can exhibit outstanding antibacterial properties in terms of their highly efficient production of reactive oxygen species. However, it is still challenge to obtain monodispersed ZnO nanoparticles [16,17]. Recently, novel mesoporous core-shell nanostructures have received increasing attention [18-20]. By introducing gold and silver nanoparticles into the inner cavities of rattle nanospheres, the catalytic properties and stability of encapsulated nanomaterials have been extensively improved. Thus, we hypothesize that the integration of ZnO nanoparticles in well-defined cavities of silica nanorattles is a powerful stabilizing strategy to obtain good monodispersion, enhancing the antibacterial properties of ZnO nanoparticles [21-23]. In the present study, ZnO nanoparticles were successfully prepared in the cavities of silica nanorattles by impregnating Zn ions in a water solution into silica nanorattles under a negative pressure environment, followed by a simple heating process. This kind of nanoarchitecture, in which ZnO was hybridized to rattle-type silica nanoparticles (ZnO@SN), was successfully applied in the treatment of MRSA infection. The inhibition zone test and the minimum inhibitory concentration (MIC) test against MRSA indicated that the as-prepared ZnO@SN nanoparticles showed a significant antibacterial effect compared to ZnO nanoparticles. More importantly, ZnO@SN nanoparticles showed excellent antibacterial and healing properties in a mouse skin infection model caused by MRSA strains. Furthermore, the antibacterial mechanism of ZnO@SN nanoparticles against MRSA was also discussed based on the augmented generation of ROS from ZnO particles after their combination with silica nanorattles as a nanobomb full of ROS.

2. Materials and methods 2.1 Materials

2

Zn(Ac)2•2H2O and KOH were purchased from Lanyi Reagent Co., Ltd. Methyl sulfoxide was obtained from Sigma Aldrich. Methanol solution was purchased from Beijing Chemical Reagent Company. The ultrapure water was treated by a Millipore-Q water system. The chemical

regents utilized in following procedures were analytic-reagent grade. 2.2 Synthesis of ZnO@SN nanoparticles The synthesis of SNs followed the method in previous reports [24]. After preparing the SNs, ZnO nanoparticles were formed by the following procedures. First, Zn(Ac)2•2H2O was mixed with SNs in an aqueous solution. Then, the system was vacuumed with the help of a vacuum pump for 30 min. Subsequently, the system was centrifuged at 9,000 r/min for 10 min in order to obtain the precipitate, and the precipitate was dissolved in a methanol solution. At the same time, some methyl sulfoxide was also added. Then, the system was maintained at 65 ℃ for 1 h, and the system was homothermal and uniform. Afterwards, KOH was added to the reaction at a rate of 1 mL min-1. The reaction lasted for another 3 h. Finally, the products were washed several times in ethanol and water to remove redundant impurities with the help of centrifugation at 10,000 r/min for 10 min. Then, the clean ZnO@SN nanoparticles were stored in glass bottles. 2.3 Characterization of nanoparticles The sizes and morphologies of the ZnO nanoparticles, SNs and ZnO@SN nanoparticles were investigated with transmission electron microscopy (TEM, JEOL2100F) and scanning electron microscopy (SEM, S-4800). The crystal structure was investigated by powder X-ray diffraction (XRD). XRD was measured using a Rigaku Ulima IV diffractometer operated at 40 kV and 44 mA using Nifiltered Cu Kа radiation with a wavelength of 1.5408 Å in the wide-angle region from 20° to 70° on the 2θ scale. The data were analyzed with the help of MDI Jade6 software. Element analysis was conducted using energy dispersive spectrometry (EDS), which was connected to an S-4800 SEM. 2.4 Bacterial growth The gram-negative bacterium MRSA was used to investigate the antibacterial activity of the ZnO@SN nanoparticles and ZnO nanoparticles. All supplies used in the experiments were sterilized with an autoclave before use. Nutrient agar and Luria Bertani (LB) broth were adapted in the experiments as a nutrition source. The number of bacteria in the liquid cultures was counted by optical density (OD) measurements at a 600-nm wavelength. The ideal density of the bacterial liquid was reached by maintaining the OD at 0.8–1.0. The ideal cell suspensions adapted for the antibacterial tests were approximately 105 CFU/mL (CFU, colony forming units). 2.5 Agarose diffusion assay The antibacterial effects of the ZnO@SN nanoparticles and ZnO nanoparticles were investigated by inhibitory zone tests. The bacterial strain adapted in these tests was MRSA. The detailed procedure was described previously. Briefly, MRSA was first added into a bottle of Luria Bertani (LB) broth and incubated overnight with shaking at 37 ℃. This procedure lasted for 12 h. Then, the bacterial liquid was harvested, and its density was confirmed with OD measurements at a 600-nm wavelength. Next,

3

the bacterial liquid was diluted to 105 CFU/mL. At the same time, a 10-mL mixed solution of agarose and LB broth (1% agarose (low EEO; Sigma, St. Louis, MO), 0.03% tryptic soy broth (TSB), 0.02% Tween 20, and 10 mmol/L sodium phosphate, pH 7.4) was prepared by sterilization with an autoclave. After the mixed solution cooled to 50 ℃, 1 mL of the bacterial liquid was added. Then, the mixture was poured into Petri dishes and allowed to freeze. After the dishes hardened, sample wells were made with an agar punch. A volume of 20 μL of material was added into each well after 1 h. The dishes were placed in a sterile environment at room temperature for 30 min in order to ensure that the materials in the wells spread into the agarose. In addition, the dishes were placed into an incubator at 37 ℃ upside-down for overnight growth. The antibacterial effect was observed directly from the clear zones. In addition, the antibacterial activity was quantitatively analyzed by measuring the diameter of each clear zone. 2.6 Minimum inhibitory concentration test The modified resazurin method was used. Briefly, a sterile 96-well plate was used in the test. Each material was tested in 3 parallel groups in 3 rows. Materials were pipetted into wells from high to low concentrations with a volume of 200 μL into 1–6 columns of the plate. The wells of column 7 and 8 were pipetted with 200 μL of LB broth. A volume of 20 μL of MRSA was added into the wells of columns 1 through 7. Vancomycin was used as a positive control for the minimum inhibitory concentration (MIC) tests. After the above procedures, the plate was placed into an incubator at 37 ℃ for 16 h. The resazurin was dissolved in sterile water at a concentration of 0.675 mg/mL. The solution was well dissolved with the help of a vortex mixer. The plate continued to incubate at 37 ℃ for 4 h after adding 10 μL of resazurin solution as an indicator. The color changes directly reflected the antibacterial effect. 2.7 Detection of the integrity of the cell membranes The ZnO nanoparticles and ZnO@SN nanoparticles were diluted in serial concentrations before testing. Then, K+ kits were adapted in the experiments. The reagents utilized in these procedures were added by following the kit instructions step by step, and the plates were incubated at 37 ℃ for 15 min under guidance. After all the preparations were finished, the absorption of the samples was detected using an enzyme-labeling instrument (Perkin Elmer). 2.8 Electron paramagnetic resonance spectrometer (ESR) measurements The ZnO nanoparticles and ZnO@SN nanoparticles were dispersed in an aqueous solution for the hydroxyl radical tests. The detailed procedure was performed according to the method described previously. The trapping agent utilized in this test was DMPO. The trapping agent was pipetted into the solution and mixed homogeneously for preparation, and then, capillaries were introduced to load the samples. Then, the capillaries were placed into an electron paramagnetic resonance machine (JEOL, JES-FA200) to collect information about the radicals. 2.9 SEM of bacteria samples

4

MRSA was incubated with ZnO@SN nanoparticles at 37 ℃ for 6 h and washed three times with phosphate-buffered saline (PBS). The cells were suspended in a PBS solution and dropped on 9-mm cover glass. The specimens were fixed in 3% glutaraldehyde for 3 h then submerged in acetone solutions at concentrations of 10%, 20%, 40%, 60% and 80% for 15 min and later in 100% acetone for 1 h. After 24 h of desiccation at 37 °C, they were mounted on aluminum stubs with copper tape. Afterwards, they were coated with gold in a low-pressure atmosphere using an ion sputter coater. The surface topographies of the bacterial cells were visualized and photographed using the SEM. 2.10 HO• (hydroxyl radical) trapping in Fenton system 3,3',5,5'-Tetramethylbenzidine (TMB) oxidation reactions were conducted in this work. In the experiments, several groups of different concentrations were tested. The concentrations ranged from 8 to 250 μg/mL. At first, 1 mL of SNs or H2O was added in the system. Then, 1 mL of Fe3O4 nanoparticles, 25 μL of H2O2 and 25 μL of TMB were added afterwards. After 30 s, both groups were examined using a UV spectrophotometer (JASCO V-570) at 652 nm. 2.11 Animals and treatment The experimentation with animals was governed by the Regulations of Experimental Animals of Beijing Authority and approved by the Animal Ethics Committee of Peking University. Institute of Cancer Research (ICR) mice (provided by Vital River Laboratory Animal Technology Co., Ltd., Beijing) aged 6–8 weeks were used in the experiments. The mice were raised in independent ventilated cages and received pathogen-free food and water. 2.12 Animal anti-infection model assay Five mice per group were housed in stainless steel cages containing sterile paddy husks as bedding in ventilated animal rooms. Mice were anaesthetized with isoflurane, disinfected with ethanol (70%) and shaved in the middle of the back (approximately a one-inch by one-inch square region around the injection site) one day prior to infection. The skin on the back was punctured with a sterile syringe needle and inoculated with 5×107 CFU/mL MRSA in 100 μL of sterile PBS. MRSA was transferred to fresh TSB and shaken at 37 °C until an OD600 value of 1.0 was achieved. The cells were centrifuged, washed once with PBS, recentrifuged, and then resuspended in PBS. Clinical examination, bacterial culture washing and biochemical identification were performed to verify the mouse skin infection model. Mice that did not receive treatment were used as the blank control. ZnO@SN nanoparticle and ZnO nanoparticle solutions (100 μL) in PBS with final levels of 2 mg/mL were inoculated at the wound site at 8 h after infection. All animals were treated a single time and given 10 d of recovery. Mice receiving a particular treatment regimen were housed separately in a ventilated cage with appropriate bedding, food, and water. Mice were checked twice per day during infection and treatment to ensure that no adverse reactions were observed. Infected animals were divided into three groups. At 24 h, 3 d, 6 d and the last day, clinical examination, bacterial culture and biochemical identification were performed. The colonies were then enumerated and reported as recovered CFU per mL of washing fluid. The skin tissue from each mouse was fixed in 10% buffered formalin phosphate at

5

room temperature followed by paraffin embedding. Histological slide preparation and H&E staining were performed. The H&E stained mouse histology sections collected above were visualized with an optical microscope (Olympus X71). All identification and analyses of the pathology slides were blinded to the pathologist. 2.13 Statistical analysis Results were expressed as the mean ± standard deviation (S.D). Multigroup comparisons of the means were carried out by one-way analysis of variance (ANOVA) tests using SPSS 16.0 (SPSS Inc., Chicago, IL). The statistical significance for all tests was set at P< 0.05. 3 Results 3.1 Material synthesis and characterization The typical structural characterizations of the ZnO nanoparticles, SNs and ZnO@SN nanoparticles are shown in Fig. 1. Fig.1a presents the TEM images of the ZnO nanoparticles produced according to the previous method [25]. The sizes of the prepared ZnO nanoparticles measured by TEM were between 4 and 6 nm. TEM images of the 150-nm size SNs are shown in Fig. 1b. At different magnifications (Fig. 1c and d), it is clear that each ZnO@SN nanoparticle has an inner hollow cavity and a rough shell studded with many 4-6 nm ZnO nanoparticles (white arrows) on the surface of the shells. The sizes of the ZnO@SN nanoparticles were approximately 150 nm, which is similar to those of the SNs. SEM images further indicated that the surface topography of the ZnO@SN nanoparticles was similar to that of the envelope-spike structure observed in some viruses, showing a surface roughness with an irregular distribution of spikes on the surface (Fig. 1e). In addition, XRD patterns are displayed in Fig. 1f and g, which represent the ZnO nanoparticles and ZnO@SN nanoparticles, respectively. Comparing the two patterns, they all had three strong peaks, and the values of were 2.8143, 2.603 and 2.475 Ǻ, respectively. They were attributed to the lattice constants of 3.346, 1.4072 and 5.205 Ǻ, and they were indexed to the (100), (200) and (101) reflections, respectively. The other diffraction peaks in Fig. 1 were consistent with the JCPDS reference (PDF NO. 36-1415). They are typical peaks of ZnO nanoparticles. Furthermore, energy dispersive X-ray spectrometric microanalyses with the SEM utilized in this investigation are shown in Fig. S1. Silica and zinc were detected in the ZnO nanoparticles and ZnO@SN nanoparticles, respectively, and the insert image is an SEM result of the ZnO@SN nanoparticles (Fig. S1a and b online). These results indicate that the ZnO nanoparticles were successfully introduced into SNs after a series of reactions. When ZnO

nanoparticles are loaded into hollow silica nanoparticles, the ZnO nanoparticles are fixed on the surface of silica nanoparticles. The ZnO nanoparticles are relative stationary, and there is no chance to interact with each other. To confirm this opinion, we measured the zeta potential of both ZnO nanoparticles and ZnO@SN nanoparticles. The results are given in Figs. S2 and S3 (online). The zate potential of the ZnO nanoparticles is +21.5, whereas that of the

6

ZnO@SN nanoparticles is 53.8. The zeta potential is more than 30, which demonstrates an enhancement of dispersity.

Fig. 1 TEM images of the ZnO nanoparticles (a), SNs (b) and ZnO@SN nanoparticles (c, d). SEM image (e) and X-ray diffraction spectra (f, g) of the ZnO nanoparticles and ZnO@SN nanoparticles.

3.2 Hypothesized mechanism We measured the hydroxyl radicals produced by the free ZnO nanoparticles and ZnO@SN nanoparticles using EPR. The generation of hydroxyl radicals in the particle suspensions was studied in the presence of spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). As shown in Fig. 2a, ZnO nanoparticle and ZnO@SN nanoparticle suspensions were incubated with DMPO and subjected to 5 min of UV light irradiation, and both samples exhibited similar EPR spectra. ZnO@SN nanoparticles exhibited increasing peak intensities. Prominent 1:2:2:1 quartet EPR spectra were observed, confirming the DMPO-OH adduct with a split center at 3400 Gauss, and this result confirmed the formation of hydroxyl radicals. Without UV light irradiation, no signal was observed. It is well known that aqueous suspensions of quartz silica dust release free radicals, which has been implied by the pathogenicity of silica; however, the peaks for hydroxyl radicals were not detected by adding SNs alone with or without UV light irradiation in our present work (Fig. S4 online).

7

Fig. 2 (Color online) (a) ESR spectra of hydroxyl radicals produced by ZnO nanoparticles and ZnO@SN nanoparticles. 1 and 3 refer to ZnO nanoparticles and ZnO@SN nanoparticles before UV irradiation; 2 and 4 refer to ZnO nanoparticles and ZnO@SN nanoparticles after UV light irradiation. (c) The absorbance of TMB oxidation using different SN concentrations (b) and the values of the peaks at 650 nm.

As seen in Fig. 2b, the TMB oxidized by the Fe3O4 nanoparticles produced a blue color with a major absorbance peak at 652 nm. Hydroxyl radicals played an important role in the catalytic oxidation reaction. We found that the addition of SNs into the reaction system accelerated the catalytic rate compared to Fe3O4 nanoparticles alone in the same reaction conditions, including the concentration, pH, time and temperature. We also found that the SNs could accelerate the catalytic reaction in a controlled concentration-dependent manner from 8 to 250 μg/ml (Fig. 2c). However, solid silica nanoparticles at the same concentrations with similar surface charges were unable to accelerate the catalysis (Fig. S5 online). In addition, the Fe3O4 nanoparticles used in our work were approximately 100 nm (Fig. S6 online).

3.3 Antibacterial activity in vitro The sensitivity of the MRSA isolates to commonly used antibiotics was tested by the disc diffusion method, and the results are shown in the supporting information (Table S1 online). For the antibacterial examination in vitro, 4-nm ZnO nanoparticles and 150-nm ZnO@SN nanoparticles were used in the study. Concentration in this work refers to the concentration of ZnO nanoparticles. To be specific, the concentration of ZnO@SN nanoparticles was defined by the concentration of ZnO nanoparticles contained in the SNs, and it was measured using an inductively coupled plasma mass spectrometer (ICP-MS, NexION 300X). The results from ICP-MS indicated that the ratio between ZnO:SN in the composite was 1. The agarose diffusion assay results are shown in Fig. 3a and b, representing the ZnO nanoparticles and ZnO@SN nanoparticles, respectively. Well numbers 1 to 5 of ZnO nanoparticles and ZnO@SN nanoparticles all had clear rings around the wells, in which materials with different concentrations from 1,000 to 50 μg/mL were added. These results indicate the concentration-dependent effect of both kinds of nanoparticles against MRSA in vitro. The diameters of the rings that directly reflect the antibacterial effect are shown in Fig. 3c. At the same concentration, each inhibition zone diameter in the ZnO@SN nanoparticles group was higher than that in the ZnO nanoparticle group. These results indicate that ZnO@SN nanoparticles had better antibacterial activity than ZnO nanoparticles against MRSA in vitro. Minimum inhibitory concentration tests (MIC) were consistent with the results of the inhibition zone tests, and the results are shown in Fig. 3d. Numbers 1 to 6 refer to the materials under different concentrations of 250, 125, 50, 25, 12.5, and 6.25 μg/mL in sequence. Then, numbers 7 and 8 refer to negative and positive control groups, respectively. The top three rows are the ZnO@SN nanoparticle-treated groups while the other three rows are the ZnO

8

nanoparticle groups. The blue wells represent no bacterial growth, and the light wells represent the failure of inhibition. ZnO@SN nanoparticles showed much stronger antibacterial effects against MRSA than ZnO nanoparticles at a concentration of 12.5 μg/mL because the ZnO@SN nanoparticles still remained blue, whereas the ZnO nanoparticles turned into a light color. The MIC of the ZnO@SN nanoparticles was 6.25 μg/mL, whereas the MIC of the ZnO nanoparticles was 12.5 μg/mL.

Fig. 3 (Color online) Images of the inhibitory zones tests of ZnO nanoparticles (a) and ZnO@SN nanoparticles (b). The areas without a bacterial infection are circled with white lines in the pictures. (c) The diameters were measured with a Vernier caliper. (d) MIC test results of ZnO@SN and ZnO nanoparticles after incubation with MRSA for 24 h. 1–6: the concentration of nanoparitcles is 250, 125, 50, 25, 12.5, 6.25 μg/mL, respectively; 7: the negative control; 8: the positive control. (e) K+ detection of MRSA treated with ZnO@SN nanoparticles and ZnO nanoparticles for 6 h. SEM images of normal MRSA (f) and the result of treatment with ZnO@SN nanoparticles showing that the cytoplasm leaked out after 6 h of incubation induced by the ZnO@SN nanoparticles (g, h). (i) The scheme of the antibacterial activity mechanism of the ZnO@SN nanoparticles.

The potassium ions released from bacterial cells were detected after receiving treatment with ZnO nanoparticles and ZnO@SN nanoparticles (Fig. 3e). Compared to the ZnO nanoparticle treatment, higher concentrations of potassium ions were found in the ZnO@SN nanoparticle group, and a concentration-dependent trend existed in both groups. Fig. 3f and g showed SEM images of MRSA cells from the negative control and ZnO@SN nanoparticle-treated groups, respectively. SEM images of the MRSA cells also indicated that the ZnO@SN nanoparticles strongly induced damage to the cytomembranes of the bacterial cells, which exhibited extensive cytoplasm leakage (Fig. 3g). Fig. 3h shows that many silica nanorattles adhered to the cells.

3.4 Anti-infection in skin inflammation model

9

Images of wounded animals after receiving MRSA and ZnO@SN nanoparticle treatment are shown in Fig. 4a. Compared to the control group, no inflammatory exudate was observed at the wound sites of the mice after treatment with ZnO@SN nanoparticles. Fig. 4a1 shows a photo of mice infected by MRSA with no treatment on the first day. The white thin arrow points at the infectious skin wound. Fig. 4a2 shows a serious inflammatory exudate (as indicated by the white bold arrow) on the wounds of the mice that did not receive any treatment after 10 d A photo of mice receiving ZnO@SN nanoparticles in a PBS gel (as indicated by the black thin arrow) is shown in Fig. 4a3. Fig. 4a4 shows a photo of mice receiving ZnO@SN nanoparticle treatment after 10 d, and there was no inflammatory exudate observed at the wound sites (as indicated by the black bold arrow). Detailed images of the mice after receiving different treatments are shown in Fig. S7 (online). After skin resection, there were white inflammatory exudates at the wound sites (as indicated by the white arrow). The blood routine examination results indicated that the amount of white blood cells (WBC) in the mice of the ZnO@SN nanoparticle groups remained in the normal range. However, the amount of WBC in the positive control group and ZnO nanoparticle group increased over 10,000 per mL at 3 d and 6 d after infection (Fig. 4b), respectively. These results suggest that MRSA not only caused a localized skin infection but also a systemic inflammatory response in mouse models.

Fig. 4 (Color online) (a) Photographs of mice after receiving different treatments: (1) positive control at 24 h. The white thin arrow is the infection wound; (2) positive control at 10 d. The white bold arrow is the inflammatory exudate; (3) mice after receiving ZnO@SN nanoparticles at 8 h. The black thin arrow shows the ZnO@SN nanoparticles; and (4) mice of the ZnO@SN nanoparticles group at 10 d. The black bold arrow shows no inflammatory exudate. (b) The results of the WBC amounts of different groups during the experiment.

The results of the bacterial counting at the wound sites indicated that the CFU levels of mice who received ZnO@SN nanoparticle treatment remained at a low level during the experimental period, and this result suggested that the elimination of bacteria was achieved with ZnO@SN nanoparticle treatment (Fig. S8 online). Colonies were found on the nutrient agar plate of the positive control groups at 3 days after infection (Fig. S9 online). However, no colonies existed on the plate of the ZnO@SN nanoparticle treatment group at the same time. The weight changes of the mice who received different treatments are shown in Fig. S10 (online). Compared to the blank control group, the

10

mice in the positive control group lost body weight until 6 d after infection. Mice in the ZnO@SN nanoparticle and ZnO nanoparticle groups kept increasing weight throughout the experimental period. As shown in Fig. 5a, compared to the healthy control animals, the morphological changes of the infected animals (Fig. 5b) included submucosal edema (white bold arrow), hyperemia (white thin arrow), inflammatory cell infiltration (white bold arrow) and fatty degeneration (black arrow). These lesions did not occur in the mice receiving ZnO@SN nanoparticles (Fig. 5c).

Fig. 5 (Color online) Histological analysis of tissues stained with H&E for the control (a row), positive control (b row) and ZnO@SN nanoparticles treated group (c row). The pathological change of the wound skin of the infected animals included submucosa edema (white bold arrow), hyperemia (white thin arrow), inflammatory cell infiltration (white bold arrow) and fatty degeneration (black arrow). No changes were observed in the group in which mice received ZnO@SN nanoparticles treatment.

4. Discussion and conclusions Hoon and coworkers previously produced mesoporous zinc silicate particles with a core-shell structure in which the core contained a mixed oxide of ZnO and SiO2 [26]. In the present study, we used different methods to produce ZnO@SN nanocomposites. The smaller apertures of the SNs formed from this recipe made them more suitable for loading ZnO nanoparticles because of the tiny size of the ZnO nanoparticles compared to common hollow silica materials, such as MCM-41 and SBA-15. Using the present method, the small size and high reactivity of the ZnO nanoparticles were reserved and tended to be more stable instead of agglomerating in the system. There were no obvious morphological differences between several cycles of washing, and this result indicates the stable properties of the synthesized ZnO@SN nanoparticles.

11

There are a few mechanism assumptions ranging from the influence of the reactive oxygen species (ROS), metal ion release, mechanical damage of the membrane cell wall through adhesion on the cell membrane to the influence of the pH conditions of the reaction system being discussed herein [27]. ROS, such as hydrogen peroxide [28], hydroxyl radicals [29], superoxide radicals [30], or singlet oxygen [31] are considered especially important factors in the mechanism of nanoparticle antibacterial activity. These reactive states of oxygen can induce damage to various biological substrates, such as DNA, RNA and proteins. The consequence is that cell membranes and cellular walls are broken, resulting in the death of cells [32,33]. The hydroxyl radical is one of the most powerful oxidizing agents and is able to react unselectively and instantaneously with surrounding chemicals, biological molecules and microorganisms [34]. Studies on the ZnO nanoparticle antibacterial mechanism have shown that ROS play an important role in the biocidal effect against a broad spectrum of microorganisms [35,36]. The holes on the ZnO nanoparticle surface can oxidize water molecules to form hydroxyl free radicals directly, as shown in formula (1). h+ZnO + OH−→ OH•

(1)

According to published data [37-39], the surface of nanosized quartz silica particles in an aqueous medium can generate hydrogen peroxide, singlet oxygen, hydroxyl radicals, and other ROS. However, our results indicate that SNs without ZnO nanoparticles were unable to produce hydroxyl radicals. The variation in the peak intensities demonstrates that at the same concentration of zinc oxide, more hydroxyl radicals were produced from the ZnO@SN nanoparticles than from the ZnO nanoparticles. Skorochod and coworkers suggested that nano-SiO2 can mediate the accumulation of hydroxyl radicals on the surface of silica nanoparticles [40]. We hypothesize that the special structure of the silica nanorattles could accumulate more hydroxyl radicals and improve the average efficiency of the hydroxyl radicals produced by ZnO nanoparticles under UV irradiation for a higher detection rate. This hypothesis was verified by the catalytic oxidation of a peroxidase substrate, TMB, as a chromogenic substrate in the presence of Fe3O4 nanoparticles and H2O2 at room temperature. Fe3O4 nanoparticles catalyzed the decomposition of H2O2, and hydroxyl radicals were formed from this reaction. Then, these hydroxyl radicals oxidized the TMB, as indicated by a change from no color to visual blue, which is the typical color of OXTMB. In other words, OXTMB is derived from the oxide form of TMB with the help of hydroxyl radicals after a eries of procedures. The results in Fig. 2 indicate that the hollow structure of the SNs affected the catalytic reaction by concentrating the hydroxyl radicals on the SNs. The schematic of TMB oxidation is shown in Fig. 2d. Grau-Atienza and coworkers reported that hybrid mesoporous Fe3O4/silica could achieve high catalytic activity and enhance the stability of the oxidation reaction of TMB [41]. They explained that the Fe3O4 nanoparticles maintained their catalytic activity when incorporated onto the silica and that they were freely accessible to the reactants. Unlike their study, we added silica nanoparticles into the reaction solutions temporarily for 5 min and could not form a hybrid incorporation structure. The Fe3O4 nanoparticles participating in this experiment were similar to SNs in size, so it was impossible to

12

incorporate Fe3O4 into the SN nanoparticle structure. A possible reason for this phenomenon is that the silica nanorattles afforded “safe spaces” for the hydroxyl radicals and enriched them at the silica surface, which promoted the reaction. The differences between the SNs and solid silica nanoparticles suggest that the hollow cavity of the SNs played a crucial role in the process. During past few decades, MRSA has attracted increasing public attention across the world for its ability to cause a wide range of infections, ranging from localized skin diseases to life-threatening pneumonia and sepsis [2-4,42]. Approximately 18,000 deaths have been recorded due to MRSA infections each year, and the cost associated with MRSA skin and soft tissue infections alone ranges from 108 to 343 million dollars annually in the United States [43,44]. Even though new types of antibiotics have been explored and applied in recent years, the emergence of resistant S. aureus strains has dramatically increased. Unique and efficient therapeutic strategies are urgently needed for current MRSA treatment. To evaluate the antibacterial activity of ZnO@SN nanoparticles and ZnO nanoparticles, MRSA collected from sputum specimens of patients at the PLA General Hospital was used in this study. The identifying codes for the strains are S. aureus 50. The strains are stored at the Department of Microbiology, PLA General Hospital. The results demonstrate that the ZnO@SN nanoparticles exhibited better antibacterial effects against MRSA in vitro compared to the ZnO nanoparticles, which agrees with our assumption that the SNs could enhance the antibacterial effects of ZnO nanoparticles by enriching the hydroxyl radicals due to the functionalized nanostructure. It is well known that the physical and chemical characteristics of nanomaterials, including size, surface charges, surface chemistry and topography, affect their biocidal activity [45,46]. Hong et al. [47] found that silver nanocubes and nanospheres could achieve close contact with bacterial cells because of their granulated shape and large specific surface area. It has been recognized that some natural antibacterial materials possess greater antibacterial properties as their particle size is reduced due to the increased surface area to volume ratio of a given mass of particles [48,49]. However, the way in which they interact with and penetrate into bacteria has been attracting more and more attention. The previous reports revealed that ZnO nanoparticles with a smaller size have a more powerful effect in antibacterial applications [50]. In our present study, silica nanorattles not only maintained the high activity of small-size ZnO nanoparticles but also collected them together, resulted in a collective efficiency for killing bacterial cells. The unique shell of the SNs and the radicals within the cavities formed a new assembly similar to an aircraft carrier loaded with many weapons. Radicals acted as a whole unit instead of performing alone. The outstanding antibacterial properties of the smaller ZnO nanoparticles were maintained, and at the same time, the volume of these complexes increased to a certain extent compared to the ZnO nanoparticles alone. This organization could lead to a larger damage area. Potassium ions are typical ions that exist in many cells, and detection of their release indicates damage to cell membranes. As shown in Fig. 3, enhancement of the antibacterial effect of ZnO@SN nanoparticles can be explained by the following two reasons: (1) The collective efficiency of the silica nanorattles on the ZnO nanoparticles brought the scattered active agents (ZnO

13

nanoparticles and hydroxyl radicals) together (the schematic on the left in Fig. 3i). (2) The ZnO@SN nanoparticles could induce worse damage to cytomembranes than ZnO nanoparticles due to the roughness of the nanoscale surface, providing an enlarged contact area and injured area size (the schematic on the right in Fig. 3i). In general, the ZnO@SN nanoparticles combined two mechanisms, enriched radicals and a rough nanoscale surface, which achieved remarkable antibacterial activity. Skin and soft tissue infections are the most common manifestation of MRSA infections in clinics [51]. Recent studies found that MRSA infections now account for approximately 60% of the SSTIs presented to emergency departments in the US [52]. Localized skin infections were established with MRSA according to the previously described method [53]. Images of animal infectious models and routine blood examinations also revealed that ZnO@SN nanoparticles had a better effect than free ZnO nanoparticles on the inhibition of systemic inflammation caused by MRSA in vivo. The following results suggest the low toxicity of ZnO@SN nanoparticles and ZnO nanoparticles to mice by skin exposure. The animals were sacrificed on the last day, and the skins at the wound sites were collected for histopathological examinations. These results indicate the excellent antibacterial properties and wound healing activity of ZnO@SN nanoparticles against MRSA during in vivo experiments. In summary, hybrid silica nanorattles with ZnO nanoparticles were synthesized to treat MRSA for the first time in this article. The ZnO@SN nanoparticles had remarkable antibacterial effects compared to the ZnO nanoparticles against MRSA in vitro and in vivo. To explore the mechanism of the ZnO@SN nanoparticles, the damage conditions of the cell membranes and the released radicals were tested. An assumption was proposed that introduction of the SNs enhanced the production of the radicals from the ZnO nanoparticles compared to the free ZnO nanoparticles and that the ZnO@SN nanoparticles enhanced the percentage of radicals in contact with the cell membranes. Furthermore, the area of damage was larger with the help of the SNs, and they made it easier for the cytoplasm to leak out. This effect might be a potential opportunity for utilizing such agents in the antibacterial field. Using this strategy, other nanoparticles or agents can be loaded into hollow nanocarriers to extend their antimicrobial effects in future clinical applications.

Acknowledgements This work was supported by the National Natural Science Foundation of China (61671435, 81630053), Beijing Natural Science Foundation (4161003), and CAS-DOE program.

Conflict of interests The authors declare that they have no conflict of interest.

References

14

[1] Joaquin N, Diekema D, Perencevich E et al. (2013) Long-term risk for readmission, methicillinresistant Staphylococcus aureus (MRSA) infection, and death among MRSA-colonized veterans. Antimicrob Agents Chemother 57: 1169-1172 [2] Kriegeskorte A, Peters G (2012) Horizontal gene transfer boosts MRSA spreading. Nat Med 18: 662-663 [3] McKenna M (2012) Man vs MRSA. Nature 482: 23-25 [4] Elhassan H, Dixon T (2012) MRSA contaminated venepuncture tourniquets in clinical practice. Postgrad Med J 88: 194-197 [5] Kuo D, Yu G, Hoch W et al. (2015) Novel quorum-quenching agents promote methicillinResistant Staphylococcus aureus (MRSA) wound healing and sensitize MRSA to β-lactam antibiotics. Antimicrob Agents Chemother 59: 1512-1518 [6] Lee B, Singh A, David M et al. (2013) The Economic burden of community-associated methicillinresistant Staphylococcus aureus (CA-RSA). Clin Microbiol Infect 19: 528-536 [7] Vojtech L, Neruda M (2013) Design of radiofrequency protective clothing containing silver nanoparticles. Fibres Text East Eur 21: 141-147 [8] Torad N, Hu M, Ishihara S (2014) Direct synthesis of MOF-derived nanoporous carbon with magnetic Co nanoparticles toward efficient water treatment. Small 10: 2096-2107 [9] Shi L, Li Z, Zheng W et al. (2014) Synthesis, antibacterial activity, antibacterial aechanism and food applications of ZnO nanoparticles: a review. Food Addit Contam Part A 31: 173-186 [10] Ghule K, Ghule A, Chen B et al. (2006) Preparation and characterization of ZnO nanoparticles coated paper and its antibacterial activity study. Green Chem. 8: 1034-1041 [11] Ocsoy I, Paret M, Ocsoy M et al. (2012) Nanotechnology in plant disease management: DNAdirected silver nanoparticles on graphene oxide as an antibacterial against xanthomonas perforans. ACS Nano 6: 8972-8980 [12] Meghana S, Kabra P, Chakraborty S et al. (2015) Understanding the pathway of antibacterial activity of copper oxide nanoparticles. RSC Adv. 5: 12293-12299 [13] Agnihotri S, Bajaj G, Mukherji S et al. (2015) Arginine-assisted immobilization of silver nanoparticles on ZnO nanorods: an enhanced and reusable antibacterial substrate without human cell cytotoxicity. Nanoscale 7:7415-7429 [14] Selvam S, Sundrarajan M (2012) Functionalization of cotton fabric with PVP/ZnO nanoparticles for improved reactive dyeability and antibacterial activity. Carbohydr Polym 87: 1419-1424 [15] Bai X, Li L, Liu H et al. (2015) Solvothermal synthesis of ZnO nanoparticles and anti-infection application in vivo. ACS Appl Mater Interfaces 7: 1308-1317 [16] Xiong H (2013) ZnO nanoparticles applied to bioimaging and drug delivery. Adv Mater 25: 5329-5335 [17] Hembury M, Chiappini C, Bertazzo S et al. (2015) Gold-silica quantum rattles for multimodal imaging and therapy. Proc Natl Acad Sci USA 112: 1959-1964

15

[18] Liu T, Liu H, Fu C et al. (2013) Silica nanorattle with enhanced protein loading: a potential vaccine adjuvant. J Colloid Interface Sci. 400: 168-174 [19] Jiao Y, Sun Y, Tang X et al. (2015) Tumor-targeting multifunctional rattle-type theranostic nanoparticles for MRI/NIRF bimodal imaging and delivery of hydrophobic drugs. Small 11:19621974 [20] Liu B, Zhang W, Feng H et al. (2011) Rattle-type microspheres as a support of tiny gold nanoparticles for highly efficient catalysis. Chem Commun. 47: 11727-11729 [21] Choi E, Kwak M, Jang B et al. (2013) Highly monodisperse rattle-structured nanomaterials with gold nanorod core-mesoporous silica shell as drug delivery vehicles and nanoreactors. Nanoscale 5: 151-154 [22] Zhai X, Yu M, Cheng Z et al. (2011) Rattle-type hollow CaWO4:Tb3+@SiO2 nanocapsules as carriers for drug delivery. Dalton Trans 40: 12818-12815 [23] Tang F, Li L, Chen D (2012) Mesoporous silica nanoparticles: synthesis, biocompatibility and drug delivery. Adv Mater 24: 1504-1534 [24] Chen D, Li L, Tang F et al. (2009) Facile and scalable synthesis of tailored silica “nanorattle” structures. Adv Mater. 21: 3804-3807 [25] Choi H, Um K, Im M et al. (2015) A simple method for nanostructure engineering of mesoporous zinc silicate particles. Chem Mater 27: 2343-2349 [26] Seil J, Webster T (2012) Antimicrobial applications of nanotechnology: methods and literature. Int J Nanomed. 7: 2767-2781 [27] Bai X, Li L, Liu H et al. (2015) Solvothermal synthesis of ZnO nanoparticles and anti-infection application in vivo. ACS Appl Mater Interfaces 7: 1308-1317 [28] Arakawa H, Takasaki M,Tajima N et al. (2014) Antibacterial activities of persimmon extracts relate with their hydrogen peroxide concentration. Biol Pharm Bull. 37: 1119-1123 [29] Xu Y, Zhou T, Huang S et al. (2015) Preparation of magnetic Ag/AgCl/CoFe2O4 composites with high photocatalytic and antibacterial ability. RSC Adv 5: 41475-41483 [30] Pratt A, DiDonato M, Shin D et al. (2015) Structural, functional, and immunogenic insights on Cu,Zn superoxide dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus. J Bacteriol 197: 3834-3847 [31] Sudhamani C, Naik H, Gowda K et al. (2015) Synthesis, DNA interactions and antibacterial PDT of Cu(II) complexes of phenanthroline based photosensitizers via singlet oxygen generation. Spectrochim Acta A 138: 780-788 [32] Jacquemin G, Margiotta D, Kasahara A et al. (2015) Granzyme B-induced mitochondrial ROS are required for apoptosis. Cell Death Differ 22: 862-874 [33] Zhang D, Wei Y, Chen K et al. (2015) Biocompatible reactive oxygen species (ROS)-responsive nanoparticles as superior drug delivery vehicles. Adv Healthc Mater 4: 69-76

16

[34] Gligorovski S, Strekowski R, Barbati S et al. (2015) Environmental implications of hydroxyl radicals (.OH). Chem Rev 115: 13051-13092 [35] Jones N, Ray B, Ranjit K et al. (2008) Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS Microbiol Lett. 279: 71-76 [36] Reddy K, Feris K, Bell, J et al. (2007) Selective toxicity of zinc oxide nanoparticles to prokaryotic and eukaryotic systems. Appl Phys Lett. 90: 213902-213904 [37] Lenz A, Krombach F, Maier K (1992) Oxidative stress in vivo and in vitro: modulation by quartz dust and hyperbaric atmosphere. Free Radical Biol Med. 12: 1-10 [38] Dalal N, Shi X, Vallyathan V (1990) Role of free-radicals in the mechanisms of hemolysis and lipid-peroxidation by silica-comparative ESR and cytotoxicity studies. J Toxicol Environ Health. 29: 307-316 [39] Konecny R, Leonard S, Shi X et al. (2001) Reactivity of free radicals on hydroxylated quartz surface and its implications for pathogenicity experimental and quantum mechanical study. J Environ Pathol Toxicol Oncol. 20: 119-132 [40] Skorochod I, Roy A, Kurdish I (2016) Influence of silica nanoparticles on antioxidant potential of Bacillus subtilis IMV B-7023. Nanoscale Res Lett 11: 139-144 [41] Grau-Atienza A, Serrano E, Linares N et al. (2016) Magnetically separable mesoporous Fe3O4/silica catalysts with very low Fe3O4 content. J Solid State Chem 237: 138-143 [42] Quezada Joaquin N, Diekema D, Perencevich E et al. (2013) Long-term risk for readmission, methicillin-resistant Staphylococcus aureus (MRSA) infection, and death among MRSA-colonized veterans. Antimicrob Agents Chemother 57: 1169-1172 [43] Kuo D, Yu G, Hoch W et al. (2015) Novel quorum-quenching agents promote methicillinResistant Staphylococcus aureus (MRSA) wound healing and sensitize MRSA to β-lactam antibiotics. Antimicrob Agents Chemother 59: 1517-1523 [44] Lee B, Singh A, David M et al. (2013) The economic burden of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Clin Microbiol Infect. 19: 528-536 [45] Uskokovic V, Batarni S, Schweicher J et al. (2013) Effect of calcium phosphate particle shape and size on their antibacterial and osteogenic activity in the delivery of antibiotics in vitro. ACS Appl Mater Interfaces 5: 2422-2431 [46] Guida F, Benincasa M, Zahariev S et al. (2015) Effect of size and N-terminal residue characteristics on bacterial cell penetration and antibacterial activity of the proline-rich peptide Bac7. J Med Chem. 58: 1195-1204 [47] Hong X, Wen J, Xiong X et al. (2016) Shape effect on the antibacterial activity of silver nanoparticles synthesized via a microwave-assisted method. Environ Sci Pollut Res 23: 4489-4497 [48] Lu Z, Rong K, Li J et al. (2013) Size-dependent antibacterial activities of silver nanoparticles against oral anaerobic pathogenic bacteria. J Mater Sci Mater Mater Med 24: 1465-1471

17

[49] Helmlinger J, Sengstock C, Gross-Heitfeld C et al. (2016) Silver nanoparticles with different size and shape: equal cytotoxicity, but different antibacterial effects. RSC Adv. 6: 18490-18501 [50] Jiang W, Mashayekhi H, Xing B (2009) Bacterial toxicity comparison between nano- and microscaled oxide particles. Environ Pollut 157: 1619-1625 [51] Stryjewsk M, Chambers H (2008) Skin and soft-tissue infections caused by community-acquired methicillin-resistant Staphylococcus aureus. Clin Infect Dis 46: S368-S377 [52] Talan D, Krishnadasan A, Gorwitz R et al. (2008) Comparison of Staphylococcus aureus from skin and soft-tissue infections in US emergency department patients, 2004 and 2008. Clin Infect Dis 53: 144-149 [53] Mohammad H, Cushman M, Seleem M (2015) Antibacterial evaluation of synthetic thiazole compounds in vitro and in vivo in a methicillin-resistant Staphylococcus aureus (MRSA) skin infection mouse model. PLoS One 10: e0142321

18