Enzyme-Responsive Mesoporous Ruthenium for Combined Chemo

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Enzyme-Responsive Mesoporous Ruthenium for Combined ChemoPhotothermal Therapy of Drug-Resistant Bacteria Yanan Liu,†,‡,§ Ange Lin,‡,§ Jiawei Liu,‡ Xu Chen,‡ Xufeng Zhu,‡ Youcong Gong,‡ Guanglong Yuan,‡ Lanmei Chen,*,† and Jie Liu*,‡ †

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Guangdong Key Laboratory for Research and Development of Natural Drugs, School of Pharmacy, Guangdong Medical University, Zhanjiang 524023, China ‡ Department of Chemistry, Jinan University, Guangzhou 510632, China S Supporting Information *

ABSTRACT: The rapid mutation of drug-resistant bacteria and the serious lag of development of new antibiotics necessitate research on novel antibacterial agents. Nanomaterials with unique size effect and antibacterial mechanism could serve as an alternative for antibiotics, since they showed low possibility to develop drug-resistant bacteria. Here, an enzyme-responsive nanosystem, AA@Ru@HA-MoS2, with a synergistic chemo-photothermal therapy function is proposed to treat bacterial infections. Mesoporous ruthenium nanoparticles (Ru NPs) were used as nanocarriers, loading prodrug ascorbic acid (AA) and encapsulated by hyaluronic acid (HA). Then, molybdenum disulfide (MoS2) precoated with ciprofloxacin was used as a catalyst with targeting effect binding to the outer surface. When the nanosystem gathered at the infection site, Hyal secreted by bacteria could degrade the HA capping and trigger the release of AA and then generated hydroxyl radicals (•OH) in situ by the catalysis of MoS2. In addition, taking advantage of the good photothermal property of Ru NPs, combined chemo-photothermal antibacterial therapy could be achieved. The nanosystem exhibited potent bactericidal activity against drug-resistant Gram-positive and Gram-negative bacteria. Furthermore, it could break down the biofilm, inhibit the contained bacteria, and prevent the formation of a new biofilm. The in vivo bacterium-infected model also proved accelerated wound healing. The study showed a high potential of AA@Ru@ HA-MoS2 as a novel enzyme-responsive nanosystem for combating drug-resistant bacterial infection. KEYWORDS: enzyme-responsive, mesoporous ruthenium, combined chemo-photothermal therapy, drug-resistant bacteria, biofilm

1. INTRODUCTION Multidrug-resistant (MDR) bacterial infection is a stressful problem in the medical profession today.1 Bacteria exhibit rapid variation and short reproductive cycles to survive from antibiotics. However, the development of new antibiotics takes a long period of time, so it is unable to keep up with the mutation and proliferation rate of drug-resistant bacteria.2 MDR bacteria with severe infectivity and mortality have emerged worldwide,3 which pose a great threat to human health and may even lead to the end of the antibiotic era, thus entering the “post-antibiotic” era.4,5 The combination of antibiotics and drug-resistance inhibitors has been considered as a promising treatment for drug-resistant infections; however, no inhibitor has been approved for clinical use.6 Therefore, the development of an economical and effective antibacterial agent with low side effects is highly desirable. Compared with traditional antibiotics, nanomaterials have the advantages of broad spectrum and durability.7 Their antibacterial mechanisms are different from those of antibiotics, so nanomaterials are ideal substitutes for antibiotics to inhibit drug-resistant bacteria.8,9 Nevertheless, most of the persistent bacterial © XXXX American Chemical Society

infections and drug resistance are correlated to the biofilm formed in living tissues.10 A biofilm is a three-dimensional bacterial community in which microbes are present in the extracellular polymeric substance (EPS). Bacteria are well protected in EPS, which can prevent the influence from foreign substances, leading to inactivation of traditional antibiotics and extreme resistance.11,12 Nanoparticles (NPs) can enter into the bacterial cell wall and pass through EPS, so they are promising for killing bacteria and dispersing the biofilm.13−16 Also, by generating reactive oxygen species (ROS), or directly interacting with biomacromolecules (phospholipids, proteins, DNA, etc.), they are capable of destroying the biological functions of MDR bacteria as well as biofilm.17−19 Once gathered at the infection area, efficient antibacterial activity can be achieved using only small doses of nanoparticles. Furthermore, through controllable triggers responsive to the bacterium-infected microenvironment, a nano drug delivery Received: May 6, 2019 Accepted: July 2, 2019 Published: July 2, 2019 A

DOI: 10.1021/acsami.9b07866 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. (A) Preparation of the Enzyme-Responsive Antibacterial Nanosystem AA@Ru@HA-MoS2; (B) Schematic of the Combined Chemo-Photothermal Therapy of the Enzyme-Responsive Drug Delivery Nanosystem for Killing Bacteria and Dispersing the Biofilm

prodrug on the surface of bacteria to produce •OH in situ can be an ideal solution. Ascorbic acid (AA) is one of the essential nutrients in the human body, as well as a pro-oxidant that can be used as a prodrug of H2O2 for treating drug-resistant bacterial infections and cancer.36,37 Moreover, peroxidase is required to increase the conversion efficiency for the formation of •OH, for H2O2 is a low-activity antibacterial agent.38 It has been reported that MoS2 nanomaterials having peroxidase-like activity can efficiently transform low-concentration H2O2 to • OH. Therefore, MoS2 could serve as a catalyst for turning the prodrug AA into •OH.39−41 In this paper, we first prepared mesoporous Ru NPs as a nanocarrier carrying the prodrug AA, encapsulated it with HA, and then modified the surface with MoS2 to construct an enzyme-responsive delivery antibacterial system (AA@Ru@ HA-MoS2) for combined chemo-photothermal therapy for bacterial infections (Scheme 1). Besides, MoS2 nanoparticles having peroxidase-like activity were coated with ciprofloxacin (CIP), which has the ability to target Gram-positive as well as Gram-negative bacteria and accumulate the nanosystem at the infection area efficiently. After the targeted delivery system reached the infection position, the capping agent HA was decomposed by the Hyal secreted from bacteria, followed by the release of the encapsulated AA, which was directly catalyzed by the MoS2 attached to the bacterial cell to generate •OH. Meanwhile, mesoporous Ru NPs provided an excellent NIR photothermal effect for synergistic chemophotothermal antibacterial therapy. In short, taking advantage of the photothermal effect and the responsively produced • OH, the AA@Ru@HA-MoS2 nanosystem could inhibit drug-resistant Gram-positive and Gram-negative bacteria and

system can achieve enhanced local bactericidal concentration and eliminate premature release of the loaded drug.20,21 Our previous studies have shown that Ru NPs with different morphologies can deliver anticancer drugs to tumor cells and protect drugs from protein adsorption in the body during transport.22,23 In addition, we have previously reported that Ru NPs had a good photothermal effect and can be used for antibacterial photothermal therapy.24 As is known, photothermal therapy is an effective antibacterial strategy without involving drug resistance.25−27 Additionally, among the various kinds of nanoparticle-based delivery systems, mesoporous nanomaterials are one of the most promising nanocarriers.28,29 Thus, Ru NPs with a mesoporous structure are designed and synthesized, combining their photothermal properties with a large pore volume. Hyaluronic acid (HA), as a harmless and degradable extracellular matrix component, can interact with overexpressed CD44 of cancer cells and be decomposed by hyaluronidase (Hyal), so it is commonly used for targeting tumor cells.30−32 Therefore, HA can also be used as a capping agent to combat bacterial infectious because it is secreted in most bacterium-infected microenvironments. Reactive oxygen species (ROS) is an effective alternative to fight bacterial resistance. Hydrogen peroxide (H2O2), as a major and available ROS, can react with essential biomolecules effectively (such as nucleic acids, proteins, and polysaccharides).33 However, its utility is often limited by inefficiency, slow processing, and high concentration.34,35 •OH has a high antibacterial and biofilm-damaging activity, so the conversion of H2O2 to •OH can avoid the limitations mentioned above.18,19 However, delivering •OH directly to infected sites is demanding in vivo due to the specificity of bacterial cells, limiting its application. In that case, substituting a harmless B


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ACS Applied Materials & Interfaces disperse the stubborn biofilm synergistically. The novel nanosystem showed great application prospects in the biomedicine field. 1.1. Materials. Ruthenium trichloride (RuCl3) and molybdenum disulfide (MoS2) were purchased from J&K scientific. Diaminopolyoxyethylene (PEG), hyaluronic acid (HA), ascorbic acid (AA), N-methylpyrrolidone (NMP), acridine orange (AO), ethidium bromide (EB), and cetyltrimethylammonium bromide (CTAB) were purchased from Sigma & Aldrich Chemical Co. The Ru complex was prepared in our laboratory. The reactive oxygen species assay kit was bought from Beyotime Institute of Biotechnology, China. MDR Staphylococcus aureus and MDR Pseudomonas aeruginosa were obtained from Guangdong Microbiology Culture Center. Mice were purchased from Guangdong Medical Experimental Animal Center. 1.2. Synthesis and Characterization. 1.2.1. Synthesis of Ru NPs. Cetyltrimethylammonium bromide (CTAB; 0.1 g) was dissolved in 46 mL of distilled water and stirred for 1 h, and then 7 mM sodium hydroxide was added. The solution was warmed to 80 °C after being dissolved by stirring. Next, 2 mL of a RuCl3 (0.5 mM) solution was added to the resulting mixed solution while stirring, and the reaction mixture was further stirred at 80 °C for 2 h. Finally, 1.0 mL of sodium borohydride (NaBH4, 0.01 M) was added dropwise, and the reaction mixture was further stirred for 2 h. After the reaction was completed, the solution was centrifuged, washed with ethanol and water three times, and then resuspended in ethanol. Hydrochloric acid was added as an ion exchanger to remove CTAB, and the product was collected and washed successively with ethanol and water. Mesoporous Ru NPs were obtained after vacuum-drying. Mesoporous Ru NPs (20 mg) and AA (50 mg) were stirred at room temperature for 24 h in 10 mM phosphate-buffered saline (PBS, pH 7.4), centrifuged, and washed several times with PBS to obtain AA@Ru. 1.2.2. Synthesis of Ru@HA. First, 50 mg of PEG was dissolved in 10 mL of water. Then, the mesoporous Ru NPs obtained above were added into a 0.5 mM solution, and 2 mL of this solution was added to the above PEG aqueous solution, followed by stirring at room temperature overnight. After that, the resulting solution was activated with 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (25 mg) and N-hydroxysuccinimide (15 mg) for 3 to 4 h at room temperature and then added with 50 mg of HA, followed by stirring at room temperature for 12 h and centrifugation at 16 000 rpm for 10 min. Then, the precipitate was collected and washed with PBS three times. Furthermore, the precipitate was resuspended in water to obtain a Ru@HA solution. 1.2.3. Synthesis of MoS2. For this, 50 mg of MoS2 powder was dissolved in 1 mL of 50 mg/mL NMP in a mortar, ground for 30 min, then transferred to 3 mL of NMP solution and ultrasound-treated in an ice bath of 200 W for 2 h, and centrifuged at 8000 rpm for 10 min. The supernatant was poured with continuous grinding for 30 min and then transferred for ultrasound treatment followed by centrifugation. After repeating this process three times, the resulting supernatant was ground for 30 min, resuspended in 1.5 mL of NMP solution, sonicated in an ice bath for 2 h, and then centrifuged at 8000 rpm for 15 min. The supernatant was taken out, filtered through a 0.2 μm filter, and collected to obtain MoS2 nanosheets (MoS2 NSs). The target molecule ciprofloxacin (CIP) was coated on the surface of MoS2 NSs. Simply put, 20 mg of CIP was dissolved in the above-mentioned MoS2

nanosheet solution and then stirred at room temperature for 24 h. The solution was resuspended after centrifugation to obtain a MoS2−CIP aqueous solution, which is abbreviated as MoS2 hereinafter. 1.2.4. Synthesis of Ru@HA-MoS2. The MoS2 solution obtained above was added dropwise to the Ru@HA aqueous solution under constant stirring, and stirring was continued for 12−24 h to obtain Ru@HA-MoS2, which was left at room temperature for 24 h. Byproducts and unreacted chemicals were removed by centrifugation, repeated washings, and vacuum-drying. The size and morphology of the obtained nanoparticles were observed by transmission electron microscopy (TEM, Hitachi H-7650). ζ-Potential and size distribution were measured by dynamic light scattering (Malvern Instruments, U.K.), and elemental composition was analyzed by energy-dispersive X-ray spectrometry (Horiba, Japan). Fourier transform infrared spectroscopy (FTIR, Nicolet 6700) and ultraviolet−visible spectroscopy (UV−vis, UV-3600 SHIMADZU) were also used for characterization. Mesoporous closure was detected by nitrogen adsorption−desorption isotherm. 1.3. Peroxidase-like Activity of Ru@HA-MoS2. The kinetic measurements19 were performed in a time-course mode using a solution of 12 μg/mL Ru@HA-MoS2, 10 mM H2O2, and 1 mM tetramethylbenzidine (TMB) in 10 mM PBS (pH 4.0). The apparent kinetic parameters were calculated using the Lineweaver−Burk curve: 1/v = (Km/Vmax)/[S] + 1/Vmax, where v is the initial velocity, Vmax is the maximum reaction velocity, Km is the Michaelis constant, and [S] is the substrate concentration. The •OH generated by AA with the catalysis of nanocarrier was studied using fluorescent probe terephthalic acid (TA). The nonfluorescent compound TA can capture •OH to form the hydroxylated product 2-hydroxyterephthalic acid to indirectly detect the amount of •OH in the solution. After co-incubation of 4 mM TA with AA, Ru@HA-MoS2, and a mixed solution of AA and Ru@HA-MoS2 for 30 min in the dark, the above solution was centrifuged. Then, the supernatant was analyzed at 435 nm using a microplate reader. TA and AA components of the same concentration detected separately were set as controls. 1.4. Responsive Release of AA from AA@Ru@HAMoS2. The cumulative release of AA in the AA@Ru@HAMoS2 system in response to Hyal and MDR bacteria was detected by recording cumulative release over time. AA@Ru@ HA-MoS2 was co-incubated with Hyal (PBS, pH 7.4) and MDR S. aureus or MDR P. aeruginosa culture (OD 600 nm 0.5) respectively at 37 °C with gentle shaking for 12 h. At predetermined intervals, a small amount of mixture was collected to analyze the AA released from AA@Ru@HA-MoS2 by high-performance liquid chromatography. (An equal volume of the fresh medium was added each time after removal.) 1.5. NIR-Mediated Photothermal Analysis. To study the photothermal effect of the nanocarrier, the nanoparticles were prepared in different concentrations (0−20 μg/mL) and placed in a 1 mL Eppendorf. Irradiation was performed using an 808 nm near-infrared laser of different powers (0.5−1.0 W/ cm2) followed by imaging using a digital thermometer FLIR E40 near-infrared imaging system to record temperatures at different time intervals. 1.6. Bacterial Uptake of Ru@HA-MoS2. To investigate the uptake of Ru@HA-MoS2 by MDR S. aureus and P. aeruginosa, the Ru(II) complex was loaded into the C


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ACS Applied Materials & Interfaces mesoporous Ru NPs as a nanoparticle imaging tracer.42 The Ru(II) complex used herein was prepared in our laboratory and has been shown to have stable fluorescent properties in previous work. Specifically, the log-phase bacteria were incubated with Ru@HA-MoS2 (200 μL) loaded with the Ru(II) complex in Luria-Bertani (LB) medium at 37 °C for 2 h. After centrifuging, washing, and resuspending, the fluorescent images of the bacteria were taken by confocal laser scanning microscopy (CLSM, TCS SP5, Germany). In addition, flow cytometry was used to compare fluorescence intensities at different times.43 1.7. In Vitro Antibacterial Activity. MDR S. aureus and MDR P. aeruginosa were selected and cultured in LB medium for 6 h at 37 °C. The diluted bacteria (10 μL, 106 CFU/mL) were added to a 96-well plate. Then, 100 μL of PBS (as a blank control) and different components were separately added to a 96-well plate and cultured overnight; then, the absorbance at 600 nm was measured. In the plate experiment, nanoparticles of various groups were mixed with a diluted bacterial suspension (106 CFU/mL) and incubated for 30 min. Subsequently, 100 μL of the mixed solution was divided into no light and light groups, which were plated onto solid agar plates, and the light groups were irradiated with an 808 nm near-infrared laser. 1.8. Bacterial Live/Dead Assay. The log-phase bacteria in the LB medium were collected, washed, resuspended, and then divided into five groups with each treated with different samples. Each group was added with 100 μL of dye (AO and EB), stained for 15 min in the dark, washed with PBS, resuspended, and then observed for bacterial cell fluorescence using CLSM.44 1.9. Cell Membrane Integrity Assay. MDR S. aureus and P. aeruginosa diluted 10 times in log phase were treated with different nanosystem groups for 0.5−2 h and stained with propidium iodide (PI, 3 μM). In addition, the log-phase bacteria were diluted, added with 4 μM 3,3′-dipropylthiadicarbocynine iodide (DiSC3-5), incubated with them for 1 h, and then treated with different groups for 0.5−2 h. 3,3′Dipropylthiadicarbocynine iodide (DiSC3-5) can be selfquenched after entering the intact membrane. When the membrane permeability altered, the dye can be released and recover its fluorescence. Scanning electron microscopy (SEM) and TEM were used to directly detect the morphology of the bacteria.45−47 Logphase bacteria were co-incubated with different groups; then, the bacteria were collected by centrifugation, washed, and fixed with 2.5% glutaraldehyde solution for 2 h at 4 °C. Bacterial cells were continuously treated with ethanol at different concentration gradients for 30 min for gradient dehydration. After gold spraying, imaging was performed using SEM. The TEM samples were also treated in the same manner. The bacteria after PBS washing were collected, fixed in 2.5% glutaraldehyde solution for 2 h at 4 °C, washed with PBS three times to wash away excess glutaraldehyde, and then added with 0.1% hydrazine. The sample was co-incubated with acid, and after 2 h, it was continuously dehydrated with different concentration gradients of ethanol, stained by 2% uranyl acetate and 0.2% lead citrate, and observed by TEM. In the above experiment, the untreated experimental group was used as a control, and the different components were further divided into no light and light groups. Then, the light group was irradiated with an 808 nm near-infrared laser for 7 min.

1.10. Dispersion and Inhibition of Nanoparticles on Biofilms in Vitro. MDR S. aureus was chosen to form the biofilm for studying the biofilm dispersion and inhibition effect of the nanosystem.48,49 First, in the in vitro experiment, the LB medium containing S. aureus in the logarithmic growth phase was added into a 24-well plate and incubated at 37 °C for 48 h in air. Unbound bacteria and medium were removed by washing with PBS buffer. The biofilm dispersion assay was performed by co-incubating the obtained MDR S. aureus biofilm with different groups for 12 h and then rinsing with PBS (1.0 mL), and the residual biofilm was evaluated by 1.0% crystal violet. The stained biofilm was rinsed three times, then ethanol (1.0 mL) was added, and the residual biofilm was detected by determining the absorbance at 590 nm. To inhibit biofilm formation, S. aureus was treated with different components, and they were incubated for 48 h. The resulting biofilm was dyed with crystal violet and quantified by the above method. The components in which the physiological saline was added were used as a blank control, and the different components were further divided into no light and light groups, followed by irradiation with an 808 nm near-infrared laser for 7 min. 1.11. Animal Studies. Female Kunming mice (6−8 week old, ∼32.0 g) were selected for animal experiments and used for biofilm dispersion inhibition in vivo. All animal experiments were in accordance with the guidelines of the Care and Use of Laboratory Animals of Jinan University. 1.11.1. Biofilm Dispersion and Inhibition in Vivo. The MDR S. aureus biofilm model was established in mice using implant-related prosthetic infection,48 and the commercial vacutainer tubes “21 G × 0.75 in. × 7 in.” were cut into 10 mm sections and soaked for 24 h in 75% ethanol. Then, these were incubated in LB medium containing log-phase MDR S. aureus for 48 h to ensure successful biofilm formation on the catheter surface. Last, the culture medium was taken out, rinsed with PBS solution, and implanted into the outer thigh of the anesthetized mouse immediately. On the second day after catheter implantation, different groups (200 μL, 12 μg/mL) were injected into the mice and the control was treated with saline. The different groups were subdivided into no light and light groups, and the light group was irradiated with an 808 nm near-infrared laser for 7 min. After 7 days, the mice were euthanized, the implanted catheter was removed, and the wound changes at the implant site were observed. The biofilm dispersion and inhibition of nanoparticles in mice were evaluated via observing the catheter surface biofilm by SEM as well as histopathological analysis of implant site wounds by hematoxylin and eosin (H&E) staining. 1.11.2. In Vivo Antibacterial and Wound Healing Studies. To evaluate the anti-infective properties of drug delivery systems in vivo, a mouse wound model that has been reported has been used to evaluate efficacy.50 Five groups of mice were subjected to wound modeling with a wound diameter of 3 cm followed by treatment with PBS containing 108 CFU of MDR S. aureus and P. aeruginosa, respectively. Suspensions (100 μL) were placed in the left and right wound sites of the mice to induce bacterial infection. The wound area was treated with different groups at a fixed time every day after surgery. Briefly, the mice were separated into five groups: control (PBS), NIR (0.5 W/cm2, 7 min), Ru@HA-MoS2 (200 μL, 1 mg/mL) + NIR (0.5 W/cm2, 7 min), AA@Ru@HA-MoS2 (200 μL, 1 mg/ mL), and AA@Ru@HA-MoS2 (200 μL, 1 mg/mL) + NIR (0.5 D


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Figure 1. (A) TEM micrograph of Ru NPs, MoS2NSs, and Ru@HA-MoS2. (B) Variation of ζ-potentials of Ru NPs, Ru@HA, and Ru@HA-MoS2 during the coating process. (C) Nitrogen adsorption−desorption isotherm of Ru NPs and Ru@HA-MoS2. Inset: pore diameter distribution curve. (D) FTIR spectra of Ru@HA, MoS2, and Ru@HA-MoS2. (E) UV−vis absorption spectra of Ru NPs, PEG, HA, MoS2NSs, CIP, and Ru@HAMoS2. (F) Energy-dispersive X-ray spectroscopy (EDS) analysis of Ru@HA-MoS2. (G) Hydrodynamic diameter of Ru@HA-MoS2 in aqueous and PBS solutions during 28 days of storage.

W/cm2, 7 min) groups. The mice were intravenously administered only during the first treatment. Photographs of wounds from five different groups of mice were taken with a mobile phone to observe the infection. During the days of treatment, the infected tissue of mice was collected and evaluated by standard plate-counting to detect the antibacterial activity. All mice were euthanized after 14 days, and wound tissue was collected and assessed by hematoxylin and eosin (H&E) staining. 1.12. Biocompatibility Studies. 1.12.1. In Vivo Biocompatibility. To study the biocompatibility of the nanosystem in vivo, mice used to assess wound infection in vivo were weighed daily during the course of treatment and their body weight changes were observed. On the 1st, 7th, and 14th day, the blood of the mice treated with AA@Ru@HA-MoS2 + NIR was taken to evaluate the liver, kidney, and blood functions according to different indicators.51 After euthanasia, the mice were subjected to inductively coupled plasma atomic emission spectroscopy (ICP-AES) to measure the residual amount of nanoparticles in the main organs and the main organs were taken out for staining for histomorphometric analysis. 1.12.2. In Vitro Biocompatibility. 293T cells with different concentrations of nanoparticles were used for cytotoxicity

analysis. The 293T cells were cultured in 96-well plates, and then several NPs of different concentrations (0−320 μg/mL) were added into the supernatant and incubated for 24 h. This was followed by treatment with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) solution for 4 h. After that, MTT was removed and dimethyl sulfoxide was added to dissolve the formazan crystals. OD at 545 nm was measured. The cell survival rate experimental formula is as follows: cell viability (%) = ODt/ODn × 100% (ODt: absorbance of the experimental group, ODn: absorbance of the blank control).52 Fresh human blood (5 mL) was collected for hemolysis tests.53 Erythrocytes were separated by centrifugation and washed with PBS. Then, these were diluted and mixed with different concentrations of samples and co-incubated for 1 h at 37 °C. Hemolytic activity was evaluated by measuring OD405nm. Erythrocytes in 0.1 M PBS and acetic acid (HAc) were used as negative and positive controls, respectively. Hemolysis (%) = [(AS − A−)/(A+ − A−)] × 100%, where AS is the absorbance of NPs, A+ is the absorbance of the positive control, and A− is the absorbance of the negative control. The different components were subdivided into no light and light groups, and the light group was treated with an 808 nm nearinfrared laser for 7 min. E


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Figure 2. (A) Temperature- and (B) pH-dependent peroxidase-like activities with TMB (1 mM), H2O2 (10 mM), and Ru@HA-MoS2 (12 μg/ mL). (C) H2O2 concentration-dependent peroxidase-like activity with TMB (1 mM) and Ru@HA-MoS2 (12 μg/mL). (D) Ru@HA-MoS2 concentration-dependent peroxidase-like activity with TMB (1 mM) and H2O2 (10 mM). (E) Fluorescence spectra of TA; AA; TA and AA; TA and Ru@HA-MoS2; and TA, AA, and Ru@HA-MoS2 in PBS solution. (F) Cumulative release of AA from AA@Ru@HA-MoS2 in PBS in the presence of Hyal, MDR S. aureus, and MDR P. aeruginosa within 12 h.

1.13. Statistical Analysis. Data were gained from three independent experiments, and all statistical analyses were performed using Student’s t-test. *p < 0.05 suggests significant differences, and **p < 0.01 represents very significant differences.

grafting of PEG and HA on Ru NPs, respectively. The absorption peak at 3438 cm−1 in the MoS2 spectrum was attributed to the −COOH group on the CIP, which proved that CIP was loaded on the MoS2 NSs successfully. Ultraviolet spectroscopy results illustrated that Ru NPs had broad absorption in the 190−900 nm wavelength range. The characteristic absorption peak of HA is at 196 nm, and 219 and 258 nm are the characteristic absorption peaks of MoS2 and PEG, respectively. During the assembly process, the characteristic absorption peaks of the individual components were shown, with individual peaks cross-fused, implying the successful synthesis of the nanosystem. In addition, highresolution TEM−EDS showed the elemental composition of Ru@HA-MoS2 (Figure 1F). Ru, Mo, and Cu elements were detected, and the contents of Ru and Mo were 28.65 and 9.17%, respectively. The Cu atomic signal was mainly derived from the carrier copper mesh. All of the above results indicated the successful synthesis of the nanosystem and its drug-loading potential. In addition, Ru@HA-MoS2 exhibited high stability and excellent solubility in an aqueous environment since there was no significant increase in hydrodynamic size when stored in aqueous solution and PBS buffer for 28 days (Figure 1G). 2.2. Excellent Catalysis and Photothermal Effect of the Nanosystem. Figure 2A−D shows the peroxidase-like activity of Ru@HA-MoS2. Similar to other reported peroxidase-like nanomaterials, its catalytic activity was related to temperature, pH, H2O2, and concentration. The optimum temperature, pH, and H2O2 concentrations were 25 °C, 4.0, and 15 mM, respectively, and were concentration-dependent. Since the high concentration of H2O2 leads to high toxicity, the catalytic activity of Ru@HA-MoS2 was studied at low concentration of H2O2 (10 mM) with a similar effect together with the optimum temperature and pH. A typical Michaelis− Menten curve was obtained at a range of H2O2 concentrations. The Michaelis constant (Km) and the maximum initial velocity (Vmax) were 0.2671 × 10−3 M and 7.948 × 10−8 M/s,

2. RESULTS AND DISCUSSION 2.1. Morphology and Characterization of Nanosystem. In this paper, HA-blocked mesoporous Ru NPs were used as nanocarriers, AA was used as a prodrug, and MoS2 was used as a catalyst to construct an antibacterial drug delivery nanosystem AA@Ru@HA-MoS2 with a controllable enzyme-responsive drug release function. The morphology and size of mesoporous Ru NPs, MoS2NSs, and Ru@HA-MoS2 were characterized by TEM and hydrodynamic diameter distribution (Figures 1A and S1). The results showed that the mesoporous Ru NPs were spherical with a particle size of about 100 nm and a visible mesoporous structure, MoS2 NSs were sheetlike structure with a particle size of about 53 nm, and Ru@HA-MoS2 were pompon-like particles with a particle size of around 206 nm. The results of TEM, ζ-potential (Figure 1B), and nitrogen adsorption−desorption isotherm (Figure 1C) demonstrated that the pore size of mesoporous Ru NPs was about 3.7 nm, which has drug-loading potential. After coating with HA, the mesoporous structure could not be detected in the TEM image of Ru@HA-MoS2 and the Brunauer−Emmett−Teller surface area was also reduced from 898.63 to 85.43 m2/g. Meanwhile, the alternating potential changes in the ζ-potential indicated that HA had blocked the mesopores present on the mesoporous Ru NPs surface successfully. The infrared and ultraviolet spectra were further used to study the structural composition of the nanoparticles (Figure 1D,E). In the infrared spectrum of Ru@HA, 1653 and 1536 cm−1 were the CO stretching vibration peak and N−H bending vibration peak of the −CO−NH− bond formed by F


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ACS Applied Materials & Interfaces respectively, according to the Lineweaver−Burk equation. Accordingly, the catalytic reaction process followed the regulation of conventional enzyme kinetics, and the nanosystem had favorable catalytic activity. Subsequently, it was tested by the TA whether the nanosystem can catalyze the production of •OH. The nonfluorescent compound TA can capture •OH to form the hydroxylated product 2-hydroxyterephthalic acid to detect the amount of •OH in the solution indirectly. Figure 2E shows the fluorescence spectra of TA, AA, Ru@HA-MoS2, and their mixed solutions. An increase in fluorescence intensity was observed when AA solution was incubated with Ru@HA-MoS2 (Figure S2), indicating that a large amount of •OH was formed at that time. In contrast, the AA-treated group only showed weak intensity, suggesting that a small amount of •OH was produced. The release of AA from the HA-terminated nanosystem can be triggered by bacteria to treat bacterial infection. Also, premature leakage of AA can be avoided to achieve controllable release. We examined the total accumulation of AA in AA@Ru@HA-MoS2 in MDR S. aureus, MDR P. aeruginosa, hyaluronidase Hyal, and PBS (pH7.4) solutions. As a result, the release situation can be judged. It can be seen from Figure 2F that within 12 h, the AA release by the nanosystem in PBS buffer was negligible. When hyaluronidase (Hyal) or bacteria were present, a significant release of AA could be detected with a cumulative release of more than 80%. These results revealed that HA could effectively block the drug in the mesopores and release the loaded drug in the presence of Hyal. Thus, Ru@HA-MoS2 can be used to load AA and achieve controllable bacterium-triggered release. Ru NPs have excellent near-infrared absorption properties and have been used in photothermal therapy for thermal ablation of tumor cells. To explore the potential of Ru@HAMoS2 for photothermal treatment of bacterial infection, nanoparticles were irradiated with an 808 nm laser to test the photothermal effect. The results revealed that the photothermal effect of the nanoparticles was dependent on the laser power intensity and concentration. When the power intensity was 0.5 W/cm2, the concentration was 4 μg/mL and the temperature reached 43 °C after 7 min of irradiation, while when the concentration was 12 μg/mL, the solution temperature reached 52 °C. In contrast, the temperature of distilled water increased by 4 °C under the same conditions (Figure 3A,B). The photothermal effect of Ru@HA-MoS2 changed with time similar to the above results (Figure 3E). To investigate the photothermal stability of the nanosystem, the temperature profile of Ru@HA-MoS2 solution (12 μg/mL) at 808 nm (0.5 W/cm2) laser on/off was measured (Figure 3C). It was observed that the temperature of the solution rose rapidly within 7 min after the laser was turned on and that it returned to normal temperature quickly after the laser was turned off; the same result was obtained in the case of six laser on/off cycles (Figure 3D), indicating that it had favorable photothermal effect and near-infrared photothermal stability and that it could be used as a photothermal agent for synergistic photothermal and chemotherapy therapy of bacterial infection. 2.3. Specificity of Bacterial Uptake. The uptake of Ru@ HA-MoS2 by MDR S. aureus and P. aeruginosa was investigated using a Ru(II) complex with excellent and stable fluorescence as a nanoparticle imaging tracer. The results of CLSM observation showed that the nanoparticles could detect strong fluorescent signals after incubation with bacteria, implying that

Figure 3. (A) Temperature plots of different concentrations of Ru@ HA-MoS2 under 808 nm laser irradiation for 7 min. (B) Temperature plots of Ru@HA-MoS2 (12 μg/mL) under 808 nm NIR laser irradiation for 7 min. (C) Temperature−time plots of Ru@HA-MoS2 (12 μg/mL) during laser turned-on and laser turned-off. (D) Temperature−time plots of Ru@HA-MoS2 (12 μg/mL) under 808 nm laser irradiation during the continuous six cycles of heating− cooling. (E) Thermal image of various concentrations of Ru@HAMoS2 under 808 nm laser irradiation for 7 min.

they had good targeted ability and could be taken up by bacteria. This may be due to the fact that MoS2 nanoparticles were precoated with ciprofloxacin, which targeted Grampositive and Gram-negative bacteria (Figure 4A). The similar results were observed in flow cytometry, where Ru@HA-MoS2 (12 μg/mL) loaded with the Ru(II) complex was incubated with both bacteria for 0−9 h. The intracellular fluorescence intensity gradually increased with time compared to the control group, showing that the nanoparticles were well absorbed by the bacterial cells (Figure 4B). 2.4. Synergistic Chemo-Photothermal Antibacterial Effect of AA@Ru@HA-MoS2. Given the excellent peroxidaselike activity of Ru@HA-MoS2, it can catalyze the conversion of prodrug AA to •OH with excellent antibacterial activity. Thus, a drug-loaded nanosystem, AA@Ru@HA-MoS2, with controllable drug release and high antibacterial effect was designed and tested for its in vitro antibacterial ability against MDR S. aureus and P. aeruginosa. To rule out the antibacterial effect of the target agent ciprofloxacin, we tested its accumulation, release, and antibacterial effect (Figures S3 and S4). The results showed that ciprofloxacin used in our experiment could not inhibit the bacteria as an antibiotic when used alone. Next, we compared the sterilization efficiency of the photothermal therapy group (Ru@HA-MoS2 + NIR), the chemotherapy group (AA@Ru@HA-MoS2), and the combined chemoG


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Figure 4. (A) CLSM images of MDR S. aureus and P. aeruginosa after incubation with Ru@HA-MoS2 (Ru complexes loaded) for 2 h. (B) Flow cytometry curves by measuring Ru complex fluorescence of MDR S. aureus and P. aeruginosa incubated with Ru@HA-MoS2 (Ru complexes loaded).

number of colonies produced on LB agar plates. As shown in Figure 5C, there was no obvious difference between the NIRirradiated group and the control group, in which viable colonies were observed, showing negligible antibacterial effect; the number of colonies in the plate treated with AA@Ru@HAMoS2 alone was lower than in that treated with Ru@HA-MoS2 + NIR, indicating that the antibacterial effect of chemotherapy was higher than that of photothermal therapy. However, an obvious antibacterial effect was observed on the plate containing AA@Ru@HA-MoS2 and irradiated with NIR, which almost prevented the formation of colonies completely, and the number of colonies on the plate was significantly less than that in plates treated with other groups. The results above showed that AA@Ru@HA-MoS2 + NIR had the strongest synergistic antibacterial effect. To further confirm the high photothermal synergistic antibacterial property of the nanosystem, the bacteria was dyed by AO and EB and observed by CLSM. Fluorescence images of bacterial samples treated with different groups were displayed in the CLSM images respectively with the untreated group served as a control. As shown in Figure 6A,B, there was no difference in the control group and the NIR treatment group, with most of the bacteria survived because there was almost no red fluorescence. However, the AA@Ru@HA-MoS2 and NIR-irradiated groups showed an increase in red fluorescence compared with the separate Ru@HA-MoS2 +

photothermal therapy group (AA@Ru@HA-MoS2 + NIR). The results demonstrated that (Figure 5A,B) the bactericidal efficiency of the three groups was concentration-dependent and that the antibacterial effect of AA@Ru@HA-MoS2 + NIR was the most significant. Compared with Ru@HA-MoS2 + NIR, the AA-loaded Ru@HA-MoS2 had improved the antibacterial activity significantly under NIR irradiation, and the antibacterial activity could be observed at even lower concentrations. When the concentration was 12 μg/mL, its bactericidal rates against MDR S. aureus and P. aeruginosa were 89.2 and 81.9%, respectively. In contrast, the prodrug AA could only display minor antibacterial activity in a concentrationdependent manner (Figure S5); it inhibited the viability of bacteria only at high concentration (4500 μg/mL). This may be because of the fact that the AA-loaded delivery nanosystem can target the surface of bacteria and then release AA in situ, which was then catalyzed by the nanosystem to generate •OH with a higher antibacterial ability. It increased local concentration and had a synergistic antibacterial effect with photothermal therapy produced by external laser irradiation. Therefore, Ru@HA-MoS2 was used as a targeted catalyst and a photothermal agent. An effective synergistic antibacterial effect can be achieved by the photothermal effect and by improving the antibacterial properties of the prodrug AA in the system. In addition, the antibacterial performance of the different treatment groups was visually assessed by measuring the H


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Figure 5. (A) Antibacterial activity detected by MDR S. aureus and P. aeruginosa treated with the three groups at various concentrations (from 0 to 12 μg/mL) at 60 min. (B) Antibacterial activity detected by MDR S. aureus and P. aeruginosa treated with the three groups at different times (from 10 to 60 min) and at the same concentrations (12 μg/mL) (*p < 0.05, **p < 0.01; AA@Ru@HA-MoS2 + NIR-treated group vs untreated group). (C) Remaining colonies of MDR S. aureus and P. aeruginosa treated with four groups: NIR, Ru@HA-MoS2 + NIR, AA@Ru@HA-MoS2, and AA@ Ru@HA-MoS2 + NIR at the same concentrations (12 μg/mL) at 60 min. The untreated group is used as the control.

between the nanosystem and the bacterial cell membrane system. The bacterial cells of the control group showed clear edges and smooth morphology. However, the aggregated nanoparticles could be found around the bacteria and some bacteria exhibited morphological lysis and bacterial debris after being treated with the other test groups (Figure 8A). The morphological changes of the bacteria treated with AA@Ru@ HA-MoS2 + NIR were most pronounced. Not only the nanoparticles entered the bacteria, but also the cell walls of the bacteria were destroyed, and the bacterial deformation caused the content leakage. Next, the morphological changes of bacteria before and after different treatments were further observed by SEM (Figure 8B). For untreated bacterial cells, the cell walls were smooth and integral with extremely sharp edges. After the treatment with NIR, the surface of only some of the bacteria deformed and wrinkled. Although most of the bacteria were destroyed, the bacteria cannot be completely inhibited when treating bacterial cells with Ru@HA-MoS2 + NIR and AA@Ru@HA-MoS2 alone. In stark contrast, almost all bacterial cells treated with AA@Ru@HA-MoS2 + NIR lost cell integrity and showed the strongest antibacterial activity. These results indicate that the AA@Ru@HA-MoS2 + NIR nanosystem could target and adhere to the cell membrane of bacteria and oxidatively damage bacterial cell membranes by converting AA to •OH at the site of infection because of the presence of the targeting molecule ciprofloxacin. It then

NIR and AA@Ru@HA-MoS2, indicating that almost all of the bacteria died, and the antibacterial activity was the strongest (similar results were obtained in both groups of bacterial experiments), showing excellent synergistic chemo-photothermal antibacterial effect. 2.5. Effect of Nanosystem on Bacterial Cell Membrane. To study the mechanism of the interaction between the prepared nanosystem and biofilm, bacteria treated with NIR, Ru@HA-MoS2 + NIR, AA@Ru@HA-MoS2, and AA@ Ru@HA-MoS2 + NIR were stained by two kinds of fluorescent dyes, PI and DiSC3-5. Fluorescence intensity was used to detect the damage of the bacterial membrane, where PI can detect the integrity of the bacterial cell membrane, while DiSC3-5 can detect the integrity of the plasma membrane. The results of Figure 7A,B showed that the fluorescence intensity of bacteria treated with AA@Ru@HA-MoS2 + NIR increased significantly with time, indicating that the integrity of the cell membrane and plasma membrane of the bacteria was severely damaged. Although other treatment groups also damaged the bacterial cell membrane system with varying degrees, the fluorescence intensity was significantly lower than that for the synergistic treatment group. Therefore, the combined chemophotothermal therapy group could destroy the integrity of the bacterial membrane system extremely. The changes of bacterial cell morphology were visually observed by TEM and SEM to further explore the interaction I


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Figure 6. CLSM images of MDR S. aureus (A) and P. aeruginosa (B) after being treated with four groups: NIR, Ru@HA-MoS2 + NIR, AA@Ru@ HA-MoS2, and AA@Ru@HA-MoS2 + NIR at the same concentrations (12 μg/mL) at 2 h, respectively. The untreated group is used as the control. The green AO labels both live and dead cells, while EB produces red fluorescence and only penetrates cells with compromised and damaged membranes.

Figure 7. Detection of MDR bacterial membrane integrity using PI (A) and DiSC3-5 (B) (*p < 0.05, **p < 0.01; treated group vs control group).

J


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Figure 8. (A) TEM and (B) SEM images of MDR bacteria treated with NIR, Ru@HA-MoS2 + NIR, AA@Ru@HA-MoS2, and AA@Ru@HA-MoS2 + NIR for 2 h, respectively.

Figure 9. Efficacy of the nanosystem for elimination (A) and inhibition of MDR S. aureus biofilm formation (D). The remaining biofilms were quantified by crystal violet staining. Biofilms were visualized by photographs (B, E) and stained with crystal violet (C, F). The untreated group was used as the control (components in the four figures are processed from left to right with control, NIR, Ru@HA-MoS2 + NIR, AA@ Ru@HA-MoS2, and AA@Ru@HA-MoS2 + NIR treatment, respectively).

destroyed the integrity of the bacterial cell membrane system

cause leakage of bacterial contents, which in turn caused bacterial death. 2.6. High Dispersion and Inhibition of Biofilm of the Nanosystem. Persistent infections are always related to the formation of biofilm, which is also a major cause of antibiotic

in a more rapid and efficient manner under the action of nearinfrared laser irradiation and •OH by a specific controlled release effect and finally entered the interior of the bacteria to K


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Figure 10. (A) Photograph of the wound when the catheter was implanted on the first day. (B (a)) Photograph of the wound when the catheter was removed from the wound after 7 days. (B (b)) Histological analysis of the tissue around the wound at the site of implantation. (B (c)) SEM images of the removed implanted catheters after 7 days. The untreated group is used as the control.

resistance. Since MDR S. aureus forms biofilm on the surface of biomaterial implants easily, we used it as a bacterial model to evaluate the effect of the drug delivery nanosystem on biofilm dispersion and inhibition. The biofilm quality was quantitatively evaluated using a standard crystal violet colorimetric assay. As shown in Figure 9A, use of NIR irradiation alone had almost no dispersing effect on the biofilm, the biofilm was still fixed on the surface of the pore integrally, and a clear biofilm band could be observed (Figure 9B,C). Ru@HA-MoS2 + NIR and AA@Ru@HA-MoS2 could only remove the biofilm quantity of about 26 and 51%, only causing a moderate effect on biofilm damage, and the remaining biofilm bands could be observed clearly. However, AA@Ru@HA-MoS2 + NIR removed about 91% of the biofilm, showing the greatest biofilm damage, and the biofilm was almost completely eradicated. Similarly, the same conclusion has been reached for the inhibition of biofilm by the nanosystem (Figure 9D− F). Separate NIR irradiation had the least effect on biofilm inhibition, and it still exhibited a relatively intact morphology. The coexistence of AA@Ru@HA-MoS2 and NIR inhibited the formation of MDR S. aureus biofilm strongly, which may be due to the toxic •OH generated by AA. It could effectively oxidize the nucleic acid, protein, and polysaccharide of the biofilm, thereby producing high damage and inhibition effects.

Therefore, the nanosystem constructed in this paper could efficaciously disperse the formed biofilm and inhibit the formation of a new biofilm. 2.7. Model of Nanosystem Inhibiting Biofilm in Mice. The experiment was based on a previous report using the implant-associated prosthetic infection model to assess the effects of combined chemo-photothermal therapy on biofilm in mice. A medical catheter was incubated with MDR S. aureus to form a biofilm on its surface, which was then subcutaneously implanted into the mice. Seven days later, the mice were euthanized and the implanted catheter was removed to compare the efficacy of the different groups. As shown in Figure 10A,B(a), the control group was protected against bacteria by the biofilm substrate, so significant swelling and purulence were observed at the wound both just prior to implantation and after 7 days post-implantation. The effect of NIR irradiation alone was not obvious, and obvious rotting and edema were observed. The groups treated with Ru@HA-MoS2 + NIR and AA@Ru@HA-MoS2 also showed different degrees of ulceration. However, wounds treated with AA@Ru@HAMoS2 + NIR showed no signs of abscesses, but a tendency to heal gradually. At the same time, the healing effect of the infected wound was evaluated by histological analysis (Figure 10B(b)), and a severe inflammatory reaction in the untreated L


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Figure 11. (A) Photographs of the MDR bacterium-infected mice within 10 days after being treated with NIR, Ru@HA-MoS2 + NIR, AA@Ru@ HA-MoS2, and AA@Ru@HA-MoS2 + NIR, respectively. (B) Related areas of infectious wounds shown in (A). Error bars represent the standard deviation of three repeated measurements (*p < 0.05, **p < 0.01 for AA@Ru@HA-MoS2 + NIR-treated group vs control group; &p < 0.05, &&p < 0.01 for AA@Ru@HA-MoS2-treated group vs control group; #p < 0.05, ##p < 0.01 for Ru@HA-MoS2 + NIR-treated group vs control group). (C) Photographs of bacterial cultures from the skin tissue of MDR bacterium-infected mice in control and AA@Ru@HA-MoS2 + NIR-treated wounds. (D) Related histological analysis of the infected skin tissues (on day 10).

biofilm and the inactivation of the bacteria embedded in it. All of these results illustrated that the synergistic therapeutic nanosystem AA@Ru@HA-MoS2 + NIR could target the infected site and control the release of the drug, which not only eliminated the biofilm but also achieved efficient sterilization. 2.8. Model of Nanosystem Promoting Wound Healing in Mice. The skin is essential to protect the body from the threat of bacterial infection. Once the skin is damaged, the microorganisms are easily invaded, leading to serious wound infections and even fatal illness. Therefore, a mouse back wound infection model was created to evaluate the potential of the nanosystem for antitraumatic infection in vivo. Mice wounds were treated with different groups, and the wound healing conditions and wound area of the infected ones were quantitatively measured from 1 to 10 days. Figure 11A,B (the left and right wounds were infected with MDR S. aureus

group was observed, while the wound treated with the AA@ Ru@HA-MoS2 + NIR group had no inflammatory response, but the same muscle texture as that of the healthy tissue. In addition, Figure S6 shows the morphology of the catheter removed after 7 days. It was found that the surface of the untreated group was yellowed and distorted due to the decay caused by bacterial infection. The surface of the catheter treated with the AA@Ru@HA-MoS2 + NIR group did not show serious contamination, and no deformation was observed. At the same time, SEM was used to study the remaining biofilm on the surface of the catheter. In Figure 10B(c), a large number of intact biofilms were observed on the surface of the control catheter, but the biofilm of the catheters treated with Ru@HA-MoS2 + NIR and AA@Ru@HA-MoS2 separately showed some fragmentation and reduction. In particular, in the AA@Ru@HA-MoS2 + NIR group, almost all of the biofilm was broken, indicating the dispersion of the M


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Figure 12. Biocompatibility evaluation of the nanosystem. (A) Changes with time in body weight achieved from mice in different groups during the treatment. (B) Time-dependent biodistribution of nanoparticles after treatment. (C) Hemolysis and (D) cell viabilities of 293T cells treated with different concentrations of nanoparticles. (E) Alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST) levels in the blood at different time points after AA@Ru@HA-MoS2 + NIR treatment. (F, G) Time-course changes of BUN and CRE. (H−L) Time-course changes of platelets (H), red blood cells (I), white blood cells (J), hemoglobin (K), and hematocrit (L) from control mice and AA@ Ru@HA-MoS2 + NIR-treated mice.

and P. aeruginosa) shows that all wounds had severe inflammatory reactions on the second day after surgery and the skin around the wound showed redness and ulceration, implying that the infection model had been established successfully. On the fourth day, erythema and edema were still observed in the control group and a biofilm was formed. The wounds in the group treated with NIR alone were similar, and different degrees of decay were observed in the wounds treated with Ru@HA-MoS2 + NIR and AA@Ru@HA-MoS2 alone, indicating that inhibition of bacterial infection was relatively lower. In the group treated with AA@Ru@HA-MoS2 + NIR, no obvious ulceration but the symptoms of scarring were observed, and the size of the left and right wounds was

reduced by about 37% (Figure 11B), showing that it can prevent wound infection effectively and promote the wound healing process significantly compared with other groups. On the 10th day after surgery, the wounds treated with AA@Ru@ HA-MoS2 + NIR completely healed and newly grown hair could be seen around the wound. In contrast, wounds treated with other groups were not completely cured and some showed a little redness. To further assess the actual efficacy of the synergistic therapeutic nanosystem for bacterial infection, bacteria at the wound tissue from untreated and AA@Ru@HA-MoS2 + NIRtreated mice collected at each treatment were counted by standard bacterial culture methods (Figure 11C). The CFU N


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3. CONCLUSIONS This study demonstrates an example of an enzyme-responsive delivery antibacterial system, AA@Ru@HA-MoS2, using photothermal mesoporous Ru NPs as nanocarriers to load the prodrug AA. The nanosystem combat resistant bacterial infections by combining bacterial microenvironment-responsive chemotherapy with photothermal therapy. A series of in vitro antibacterial experiments showed that the AA@Ru@HAMoS2 nanosystem not only had high efficiency and broadspectrum bactericidal properties against drug-resistant Grampositive and Gram-negative bacteria but also dispersed the biofilm and inhibited the formation of new biofilms effectively. The same conclusion was also obtained in the in vivo antibacterial experiment. Additionally, an accelerated wound healing activity was proved by the mice infection model. The results of the biocompatibility evaluation experiments elucidated that the nanosystem had neither obvious cytotoxicity to normal cells nor long-term toxicity in the organism. We believe that the AA@Ru@HA-MoS2 nanosystem has the potential to treat drug-resistant bacteria more effectively with its combined therapy.

count showed that the group treated with AA@Ru@HA-MoS2 + NIR decreased the number of bacteria significantly on the sixth day, and almost no bacterial colonies were observed on the tenth day, showing a complete recovery, which was consistent with the in vivo experiments results. To further illustrate the superior wound healing efficacy of the nanosystem, histological analysis of bacterially infected wound sections was performed by H&E staining (Figure 11D). As can be seen in the figure, after treatment with AA@Ru@HA-MoS2 + NIR, no symptoms of infection were observed and a fully thickened epidermis was observed, showing normal vascular and hair follicle morphological characteristics, which were similar to those of healthy muscle tissue. However, the other groups showed significant infection and morphological features of the rupture. These results showed that our nanosystem not only killed planktonic bacteria and eradicated the antibioticresistant biofilm but also resisted wound infection effectively. 2.9. Biocompatibility Determination. From the change of mouse body weight in the treatment time range, Figure 12A, it could be seen that the weight of mice treated with the synergistic treatment nanosystem was similar to that of the control group, revealing low toxicity of the nanosystem. Subsequently, the biodistribution in vivo was assessed by ICPMS. In Figure 12B, the nanoparticles in the heart, spleen, and lungs were cleared quickly over time, while the liver and kidneys had some residual, meaning that they are metabolized by liver and kidney. Moreover, the results of hemolysis experiments (Figure 12C) showed no obvious hemolysis with the increase of the concentration of nanoparticles, suggesting that the nanosystems had low hemolysis effects and good biocompatibility. In addition, cytotoxicity experiments were performed on 293T cells with different concentrations of the nanosystem (Figure 12D). The results showed that the cell viability was still high even at high concentrations. In addition, the in vivo toxicology of the synergistic therapeutic nanosystem AA@Ru@HA-MoS2 + NIR was investigated. The liver and kidney function indicators and blood parameters from the body of the control and treated mice were collected. Among them, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) are the major indicators of liver function, and their concentrations were within the reference range and are not much different from the control group, indicating that the nanosystem had no obvious liver toxicity. As an indicator of renal function, the levels of urea nitrogen (BUN) and creatinine (CRE) in the blood of mice were also within the normal range (Figure 12E−G). For hematology assessment (Figure 12H−L), white blood cells, red blood cells, platelet count, hemoglobin, and hematocrit were selected. The results in the figure demonstrated that the above indicators were normal in the treatment group compared with the control group, representing that the nanosystem had no significant toxicity in vivo. Finally, we investigated the histological changes in the main organs (heart, liver, spleen, lungs, and kidneys) to assess the long-term toxicity of our nanosystem (Figure S7). The results showed that under our experimental conditions, no significant histological damage or adverse reactions were observed in the main organs of the mice, supporting that the nanosystem had no obvious toxic side effects on the mice.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b07866.

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Size distributions of Ru NPs, MoS2NSs, and Ru@HAMoS2, cumulative release of ciprofloxacin, antibacterial activity of ciprofloxacin, antibacterial activity of AA, photographs of the removed implanted catheters after 7 days, and H&E-stained slice images of major organs (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.C.). *E-mail: [email protected]. Tel/Fax: +86-20-85220223 (J.L.). ORCID

Jie Liu: 0000-0003-2237-9309 Author Contributions §

Y.L. and A.L. contributed equally to the work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21877051, 81803027, 21701034), the Natural Science Foundation of Guangdong Province (2018A030310628), the Planned Item of Science and Technology of Guangdong Province (2016A020217011), Projects of Special Innovative of Department of Education of Guangdong Province (2017KTSCX078), and Project of Young Innovative Talents in Universities and Colleges of Department of Education of Guangdong Province (2018KQNCX100).

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