New Strategy for Specific Eradication of Implant-Related Infections

Jul 2, 2019 - Especially when they are utilized in antibiofilm, the expression levels of biofilm-related genes (icaA, fnbA, atlE, and sarA for Staphyl...
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New Strategy for Specific Eradication of Implant-Related Infections Based on Special and Selective Degradability of Rhenium Trioxide Nanocubes Wenlong Zhang,†,⊥ Chuang Yang,§,⊥ Ziyu Lei,† Guoqiang Guan,† Shu-ang He,† Zhenbo Zhang,∥ Rujia Zou,*,† Hao Shen,*,§ and Junqing Hu*,†,‡

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†

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, International Joint Laboratory for Advanced Fiber and Low-Dimension Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China ‡ College of Health Science and Environmental Engineering, Shenzhen Technology University, Shenzhen 518118, China § Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, China ∥ Reproductive Medicine Center, Department of Obstetrics and Gynecology, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, China S Supporting Information *

ABSTRACT: The greatest bottleneck for photothermal antibacterial therapy could be the difficulty in heating the infection site directly and specifically to evade the unwanted damage for surrounding healthy tissues. In recent years, infectious microenvironments (IMEs) have been increasingly recognized as a crucial contributor to bacterial infections. Here, based on the unique IMEs and rhenium trioxide (ReO3) nanocubes (NCs), a new specific photothermal antibacterial strategy is reported. These NCs synthesized by a rapid and straightforward spaceconfined on-substrate approach have good biocompatibility and exhibit efficient photothermal antibacterial ability. Especially when they are utilized in antibiofilm, the expression levels of biofilm-related genes (icaA, fnbA, atlE, and sarA for Staphylococcus aureus) can be effectively inhibited to block bacterial adhesion and formation of biofilm. Importantly, the ReO3 NCs can transform into hydrogen rhenium bronze (HxReO3) in an aqueous environment, making them relatively stable within the low pH of IMEs for photothermal therapy, while rapidly degradable within the surrounding healthy tissues to decrease photothermal damage. Note that under phosphatebuffered saline (PBS) at pH 7.4 without assistant conditions, these ReO3 NCs have the highest degradation rate among all known degradable inorganic photothermal nanoagents. This special and IME-sensitive selective degradability of the ReO3 NCs not only facilitates safe, efficient, and specific elimination of implant-related infections, but also enables effective body clearance after therapy. Solely containing the element (Re) whose atomic number is higher than clinic-applied iodine in all reported degradable inorganic photothermal nanoagents under the PBS (pH 7.4) without any assistant condition, the ReO3 NCs with high X-ray attenuation ability could be further applied to X-ray computed tomography imaging-guided therapy against implantrelated infections. The present work described here is the first to adopt degradable inorganic photothermal nanoagents to achieve specific antibacterial therapy and inspires other therapies on this concept. KEYWORDS: infectious microenvironments, specific photothermal eradication, implant-related infections, selective degradability, rhenium trioxide nanocubes

1. INTRODUCTION

resistant biofilms has motivated a worldwide effort to search for alternative therapeutic means able to eliminate implantrelated infections.7−10 The past decade has witnessed an everincreasing passion in application of nanomaterials with intrinsic antibacterial ability to fight bacteria and their biofilms.11−14 Nanomaterials have been found to overcome bacterial resistance.15 Additionally, many nanomaterials could achieve killing bacteria and

Implant-related infections, once they occur after implant replacement surgeries, usually require a second operation to take out the infected implant or even amputation for serious infections.1 It brings patients great pains and economic losses. Conventional antibiotics are known to be capable of selectively inhibiting or killing bacteria.2,3 However, in implant-related infections, bacterial biofilms often form and adhere to the surface of the implant as well as permeate into the surrounding tissues;4 they impede antibiotic penetration, stimulate the development of antibiotic resistance, and help bacteria evade host immune responses.5,6 The emerging threat of antibiotic© XXXX American Chemical Society

Received: April 27, 2019 Accepted: July 2, 2019 Published: July 2, 2019 A

DOI: 10.1021/acsami.9b07359 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces promoting wound healing at the same time.16,17 Nonetheless, the utilization of nanomaterials themselves as antibacterial agents is considerably restricted to their nonspecific biological toxicity. Another feasible avenue as the alternative to current antibiotic therapy is resorting to photothermal therapy (PTT).18−24 PTT, via physical heat generated by low-toxic photothermal agents to induce bacterial membrane disruption and protein denaturation, has broad-spectrum antibacterial capacity and will not cause bacterial resistance.1,25−28 Hence, a growing number of photothermal agents have recently sprung up for antibacterial therapy.18−24,29 Among them, inorganic nanoagents, due to their innate optical, electrical, acoustic, magnetic, and thermal properties, benefit multifunctionalization to detect and combat bacteria.19−22,26,29 More importantly, similar to enhanced permeation and retention effect of tumor, the blood vessels are also penetrable in the inflammation region derived from mediation of various inflammatory factors, contributing to passive targeting of inorganic nanoagents to bacteria-infected site for precise antibacterial therapy.22,30 Although various inorganic photothermal nanoagents have been established for preventing bacterial infections, however, their actual clinical practice is still very limited thus far. The major obstacle is that photothermal nanoagents are hard to be totally localized in the infection site during antibacterial therapy, thereby producing nonlocalized heating damage to the surrounding healthy tissues. Moreover, in general, most of inorganic nanoagents are nondegradable and could not be cleared from body after fulfilling their PTT task, which inevitably gives rise to long-term toxicity. To address these issues, a commonly pursued tactic is conjugating inorganic photothermal nanoagents with bacteriaspecific antibodies26,31 or bacterial pretreated membrane.22 Such conjugated nanoagents could acquire actively targeted photothermal ablation against bacteria and their biofilms in the localized infection site with decreased nonspecific damage to normal tissues. But still, the further application of bacteriatargeted inorganic photothermal nanoagents is limited to their targeting efficiency. Besides, these inorganic nanoagents could not be cleared from body after the PTT. An innovative alternative route is constructing magnetic photothermal antibacterial nanoagents.20,32 With assistance of an external magnet, the magnetic properties of the photothermal nanoagents benefit enriching the nanoagent−bacteria aggregation as a localized heating source so as to decrease toxic adverse effect to healthy tissues. Furthermore, after the PTT, these magnetic photothermal nanoagents could be easily recycled using a magnet. Unfortunately, in view of clinical translation, this route involving manipulation of nanoagents by an external magnetic field is too complicated and time-consuming. It is getting necessary and fascinating to explore safe and efficient photothermal antibacterial approaches with recognition capacity. As has been unequivocally demonstrated, a bacterial infection lesion brings about unique acid infectious microenvironments (IMEs).33,34 Allowing for the acid feature of IMEs different from that of normal tissues around them, most recently, IMEs-sensitive photothermal antibacterial nanoagents have been designed and synthesized.23,24,35,36 They can specially encapsulate bacteria together in acidic IMEs via surface charge inversion,23,24,35 or be confined in acid IMEs through colloidal−gel transformation,36 favoring specific photothermal therapy. On the other hand, our group has recently discovered rhenium trioxide (ReO3) as an admirable

photothermal nanoagent.37 However, this new study did not involve antibacterial applications. Inspired by these previous reports, herein, in a rapid and straightforward space-confined on-substrate way, ReO3 nanocubes (NCs) have been obtained for PTT of implant-related infections (Scheme 1). The asScheme 1. Schematic Illustration for Synthetic Procedure of ReO3 NCs and PTT of Implant-Related Infections

obtained ReO3 NCs, owing to their high near-infrared (NIR) localized surface plasmon resonance (LSPR) absorbance, prove to be a high-efficiency photothermal antibacterial nanoagent against planktonic bacteria and biofilms in vitro. More fascinatingly, the ReO3 NCs turn to hydrogen rhenium bronze (HxReO3) when dissolved in aqueous solution, rendering them relatively stable in acidic IMEs, while rapidly degradable in surrounding healthy tissues. Despite their high degradation rate, the ReO3 NCs still display favorable biocompatibility and biosafety. Without damaging the normal tissues, these NCs show potent antibacterial efficacy to eradicate implant-related infections. Simultaneously, the degradability of the ReO3 NCs enables their effective body clearance after therapy, further easing their adverse reactions and gaining safety. In addition, the ReO3 NCs, from their highZ element Re, aroused X-ray computed tomography (CT) imaging capabilities and could function as a CT imagingguided antibacterial agent, which further improves the precision of therapy for implant-related infections. To the best of our knowledge, this study represents the first paradigm of IME-sensitive selectively degradable inorganic photothermal nanoagents to eradicate implant-related infections and thus offers a specific, simple, safe, and efficient pathway toward precise antibacterial therapy.

2. MATERIALS AND METHODS 2.1. Fabrication and Characterization of ReO3 NCs. The fabrication and characterization of the ReO3 NCs were according to our previous report (for details, see the Supporting Information and Figure S1).37 2.2. Photothermal Performance of ReO3 NC Aqueous Dispersions. The assessment of photothermal performance for the aqueous dispersions of ReO3 NCs was performed according to our previous report (see the Supporting Information for details).37 2.3. In Vitro Cytotoxicity Evaluation. The evaluation for in vitro cytotoxicity of the ReO3 NCs was conducted according to our previous report (see the Supporting Information for details).37,38 2.4. Hemolysis Assay. The hemolysis assay for the ReO3 NCs was performed according to our previous report (see the Supporting Information for details).37 2.5. In Vitro Photothermal Antiplanktonic Bacteria. Gramnegative Escherichia coli (ATCC 35218) and Gram-positive methicillin-resistant Staphylococcus aureus (ATCC 43300) were selected to appraise the photothermal antibacterial effect of our ReO3 NCs. These two kinds of bacteria were incubated in fresh trypticase soy broth (BD Biosciences, Franklin Lakes, NJ) medium at 37 °C B

DOI: 10.1021/acsami.9b07359 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

intercalated. Subsequently, the incisions were sutured. Finally, 100 μL of prepared 107 CFU/mL S. aureus suspension was injected subcutaneously upon the washers (Figure S2). 2.8. In Vivo Photoacoustic (PA) Imaging. In the course of the experiments, mice 3 days after implant-related infections as well as healthy mice were first anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection). In virtue of Vevo LAZR PA imaging system (VisualSonics Company, Canada), PA imaging at both the subcutaneous tissues and infectious abscess was achieved. PA images were captured at both positions right after subcutaneous (healthy mice, on their anterior upper backs) and intra-abscess (infected mice) injection for 200 μL of 200 ppm ReO3 NC PBS solutions and at 30 min post injection. To measure PA signal intensities, three repeated experiments were carried out. 2.9. In Vivo CT Imaging. For CT imaging implant-related infections, mice with implant-related infections, before and at 0.5 h post injection with 400 μL of ReO3 NC PBS solutions (6000 ppm) at the infection site, were scanned in a CT imaging system. By means of high-resolution X-ray microCT scanning (Quantum FX, PerkinElmer, Hopkinton, MA), three-dimensional images of mouse inner structure in detail were acquired. 2.10. In Vivo Photothermal Antibacterial Therapy. Typically, mice 24 h after implant-related infections were divided into four groups (n = 8): PBS only (100 μL), PBS + NIR laser (100 μL), ReO3 NCs only (1000 ppm, 100 μL), and ReO3 NCs + NIR laser (1000 ppm, 100 μL). Laser irradiation (808 nm, 0.7 W/cm2) was performed on the PBS + NIR laser group and ReO3 NCs + NIR laser group in 0.5 h post injection at the infection site for 7 min. The temperature and thermal images of the mice from the above four groups were recorded on the IR thermal camera. Every 2 days after treatment, diameters of infected areas were monitored by a digital caliper. To quantify in vivo bacteria, subcutaneous polyethylene washers from every group were sterilely extracted and adherent bacteria were separated and collected. Then, the whole living numbers of adherent bacteria were identified by SPM. Additionally, the peri-implant soft tissues from all four groups were dissected, immersed in PBS, and homogenized with a high-speed homogenizer, and the viable bacteria within the peri-implant soft tissues were also assessed using SPM. For histological observation, 14 days after treatments, the soft tissues around the implants from every group were collected to make paraffin sections and stained with hematoxylin and eosin (H&E) and Giemsa; the major organs (heart, liver, spleen, lung, and kidney) of mice from the therapy group were dissected to make paraffin sections and stained with H&E. A Leica DMi8 fluorescence microscope was employed to examine these sections. 2.11. In Vivo Toxicity, Biodistribution, and Metabolism. In vivo toxicity, biodistribution, and metabolism experiments were carried out according to our previous report (see the Supporting Information for details).37 2.12. Statistical Analysis. The mean value ± standard deviation was adopted to express experimental data. To appraise significance among acquired data, P values were estimated employing one-way analysis of variance statistical analysis approach. P value of 0.05 is regarded significant. The outcomes were labeled in figures as * P < 0.05, ** P < 0.01, and *** P < 0.001.

overnight. Then, the concentration of both bacterial suspensions was adjusted to ∼107 colony-forming units (CFU)/mL. Next, 100 μL of 107 CFU/mL bacterial suspension (E. coli or S. aureus) and 100 μL of 200 ppm ReO3 in phosphate-buffered saline (PBS, pH was adjusted to 6.0 before) or PBS without ReO3 were added into 96-well plates. The above mixtures were illuminated under the 808 nm laser (Shanghai Xilong Optoelectronic Technology Co., Ltd., China) with a power density of 0.7 W/cm2 and a spot area of ∼12.57 cm2 for 0, 1, 5, and 10 min. After PTT, the viable counts of planktonic bacteria were determined by the spread-plate method (SPM). 2.6. In Vitro Photothermal Antibiofilm. Each medical titanium metal plate (10 × 10 × 1 mm3) with 1 mL of 107 CFU/mL prepared bacteria suspension (E. coli or S. aureus) was statically incubated in 24-well plates at 37 °C overnight. Each incubated titanium plate was gently washed with sterile PBS three times to get rid of nonadherent bacteria on the surface and was put into new 24-well plates containing 500 μL of 200 ppm ReO3 in PBS (pH was adjusted to 6.0 before) or PBS without ReO3. After that, the incubated titanium plates immerging in the ReO3 dispersions were illuminated by the 0.7 W/ cm2 808 nm laser with a spot area of ∼12.57 cm2 for 0, 1, 5, and 10 min. The above photothermally treated titanium plates were lightly washed using sterile PBS three times, and thus any nonadherent bacteria on the plates were gently rinsed away. For quantitative evaluation of an efficiency of the photothermal antibiofilm, each plate was placed in a sterile vial with 1 mL of PBS. Thereafter, the vial was sonicated for 10 min (150 W, 50 kHz) and rapidly mixed for 1 min to separate the residual adhesion bacteria. The dispersion was harvested and live bacteria were quantified by SPM. Data were collected from three separated tests. For qualitative evaluation of the photothermal antibiofilm by scanning electron microscopy (SEM), the plates were fixed at 4 °C for 4 h with 2.5% glutaraldehyde, then consecutively dehydrated by a graded ethanol series (i.e., 50, 70, 80, 90, 95, and 100% v/v) in new 24-well plates for 10 min, and finally freeze-dried and sprayed with gold, for SEM observation. For qualitative evaluation of the photothermal antibiofilm by fluorescence microscope, each plate was put into new 24-well plates and then stained for 20 min by 1 mL of mixed dye (LIVE/DEAD BacLight bacteria viability kits, Invitrogen), lightly washed using sterile PBS once, and eventually imaged through a Leica DMi8 fluorescence microscope. Live bacteria were stained green, while dead bacteria were stained red. For evaluation of the photothermal antibiofilm for the whole biofilms on the titanium plates by crystal violet staining, each plate was placed in new 24-well plates and then stained with 500 μL of 0.4% crystal violet solution in water for 40 min. After staining, the plates were rinsed four times using sterile PBS to detach unbound dye. At this point, typical experimental results were photographically recorded to qualitatively evaluate biofilms. Next, for quantitative evaluation of biofilms, 200 μL of 96% ethanol was added to wash out the biofilms and the absorption of the resulting violet solution was monitored by the microplate reader at 595 nm. Data were collected from four separated experiments. For real-time polymerase chain reaction (RT-PCR) analysis of related gene transcription for S. aureus biofilms, the genes involving the formation of S. aureus biofilms were quantitatively tested via RTPCR according to our previous report [see Table S1 (Supporting Information) for details].9 2.7. Mouse Implant-Related Soft Tissue Infection Model. All animal experiments were supervised by the Laboratory Animal Center of Shanghai Sixth People’s Hospital, and procedures were conducted according to protocols approved by the Animal Care and Use Committee at Shanghai Sixth People’s Hospital. Male BALB/c mice (8 weeks, 18−23 g) were purchased from Shanghai Sixth People’s Hospital. During our experiments, the mice were anesthetized intraperitoneally with pentobarbital sodium (40 mg/kg) in the beginning, and the skin on anterior upper backs (the subcutaneous surgical site) was shaved and disinfected. Afterward, the skin was sheared layer by layer under sterile conditions, and ready polyethylene washers (6 mm in diameter, 1 mm in thick) were subcutaneously

3. RESULTS AND DISCUSSION 3.1. Characterization, Absorption, and Degradation of ReO3 NCs. The ReO3 NCs have been fabricated through a rapid and straightforward approach (Scheme 1, see the Supporting Information for details). ReO3, under ambient conditions, is known to crystallize as a cubic perovskite-type structure,37,39,40 and its crystal lattice is shown in Figure 1a. The electronic structure of rhenium(VI) in ReO3, with an unpaired electron in the band associated with d orbital, could be conveyed by [Xe] 6s04f145d1.37 For this reason, metallic ReO3 owns a strong LSPR absorption comparable to that of gold.37,41 Figure 1b exhibits a scanning electron microscopy C

DOI: 10.1021/acsami.9b07359 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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A high NIR absorption is the prerequisite for photothermal nanoagents. The aqueous dispersion of our ReO3 NCs presents strong absorption from 700 to 1100 nm (Figure 1g), which can be ascribed to the LSPR originated from unique metallic feature of ReO3, as mentioned above. It has been shown that ReO3 reacts with water to form a kind of typical hydrogen insertion compounds of transition-metal oxides, hydrogen rhenium bronze (HxReO3), and such HxReO3 could further be neutralized to [ReO3]− followed by oxidization into [ReO4]−, finally leading to the degradation of ReO3.37,39−43 The proposed mechanism could be described using the following equations (eqs 1−3) ReO3 + H 2O → HxReO3

(1)

HxReO3 + OH− → [ReO3]− + H 2O

(2)

[ReO3]− + O2 → [ReO4 ]−

(3)

Thus, it is attractively found that the ReO3 NCs progressively degrade in physiological solutions, including PBS (pH 7.4), DMEM, and FBS, with their colors becoming shallow until transparent (Figure 1h). The degradation of the ReO3 NCs in PBS (pH 7.4) can be further confirmed by the corresponding time-dependent absorption spectra (Figure 1i). As can be seen clearly, the absorbance intensity of ReO3 NCs reduces significantly over dispersion time, indicating they degrade rapidly and efficiently under normal PBS. On the other hand, HxReO3, an intermediate product from the reaction of ReO3 with water (eq 1), is acidic; under acidic conditions, it should be more stable and the degradation may be slower (see eq 2). As a proof of concept, we also investigated the time-dependent absorption spectra of the ReO3 NCs in PBS at pH 5.0 and pH 6.0 (Figure S3). As expected, the absorbance of the ReO3 NCs in acidic environment is more stable, especially in PBS with lower pH. Figure 1j further exhibits the variation of the ReO3 NCs under different pH values in the absorption ratios (A/A0) at 808 nm. Notably, the absorbance of these ReO3 NCs in PBS with pH 7.4 at 808 nm (A) decreases by 68.5% compared to the original value (A0) after initial only 0.5 h, and up to 94.8% after 3 h. In stark contrast, after 3 h, the absorbance intensities under pH 6.0 and 5.0 drop by 77.5 and 53.5%, respectively. Such pH-dependent selective degradability of the ReO3 NCs could be directly and clearly observed in their degradation photographs as well (Figure 1k). Moreover, no large particles were found from the TEM image for the ReO3 NCs after 3 h of incubation in PBS (pH 7.4), except for a few ultrasmall nanoparticles (2.8 ± 1.1 nm) that can be excreted out of the body through kidney filtration (Figure 1l). Also, it is noted that in PBS with pH 7.4 without any assistant condition, our ReO3 NCs are very special for having the highest degradation rate among all known degradable inorganic photothermal nanoagents (Table S2). 3.2. Photothermal Performance of ReO 3 NCs. Furthermore, the ReO3 NC aqueous dispersions with various concentrations (0−200 ppm) under an NIR laser (808 nm, 0.7 W/cm2) were employed to assess their photothermal performance. As a result, the ReO3 NCs could serve as an efficient photothermal agent, displaying drastic temperature rise in a concentration-dependent way (Figure 2a,b). Considering faster degradation results in greater optical absorption loss as exhibited, the influence of special and pH-dependent degradability for the ReO3 NCs on their photothermal performance was further examined. As can be seen from

Figure 1. (a) Crystal structure of ReO3. Re and O atoms are represented by gray and red balls, respectively. (b) SEM and (c) TEM images (inset: a size distribution histogram) of ReO3 NCs. (d) TEM and (e) HRTEM images (inset is the corresponding FFT pattern) of a single NC. (f) XRD spectrum of ReO3 NCs with scanning transmission electron microscopy-EDS elemental mapping as the inset. (g) UV−vis−NIR absorption spectrum of the ReO3 NC aqueous dispersion (inset shows a photograph of this dispersion). (h) Photographs exhibiting degradation course of ReO3 NCs under PBS, Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS). (i) UV−vis−NIR absorption spectra of ReO3 NCs over time in PBS (166 ppm) with pH 7.4. (j) Variation of the absorption ratios (A/A0) at 808 nm for the ReO3 NCs dispersed in PBS (166 ppm) of varying pH with time tested via UV−vis−NIR absorption spectra and (k) the corresponding photographs. (l) TEM image of the degradation product for ReO3 NCs.

(SEM) image of such prepared ReO3 sample and indicates that the sample consists of large-scale uniform NCs. Further identified via transmission electron microscopy (TEM) images (Figure 1c,d), these ReO3 NCs have a mean edge length of 31.7 ± 9.1 nm (Figure 1c, inset). The high-resolution TEM (HRTEM) image of an individual NC is depicted in Figure 1e, where cubic ReO3 crystal is confirmed by the labeled interplanar d-spacings of ∼0.38 nm. Moreover, fast Fourier transform (FFT) diffraction pattern, shown in the inset of Figure 1e, could be assigned to the [001] zone axis of the ReO3 crystal. Furthermore, we utilized X-ray diffraction (XRD) to verify the crystallographic architecture. Seen from Figure 1f, the XRD spectrum of these NCs can be accurately indexed to the cubic ReO3 phase (JCPDS no. 33-1096). The energydispersive spectrometer (EDS) elemental mapping (Figure 1f, inset) manifests that in the NCs, Re and O elements are homogeneously distributed. All of these definitely evidence that the ReO3 NCs were synthesized successfully. It should be noted that in this study, by only heating for a very short time (5 min), high-quality and low-loss ReO 3 NCs were synthesized. Considering such simplicity compared to other reported approaches, our ReO3 NCs exhibit promising feasibility for large-scale production of the ReO3 NCs. D

DOI: 10.1021/acsami.9b07359 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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temporal and spatial resolution to visualize and supervise the degradation of the ReO3 NCs in both subcutaneous tissues and infectious abscess in real time.45 Healthy and infected mice received subcutaneous and intra-abscess injection with the ReO3 NCs in PBS solutions (200 ppm, 200 μL), respectively. PA imaging was conducted at both places of mice at immediately post injection of the ReO3 NCs’ solution and at 30 min post injection (Figure 3a). Interestingly, ∼35.8% of PA

Figure 2. (a) Temperature and (b) IR thermal images of the dispersions containing ReO3 NCs under the NIR laser (808 nm, 0.7 W/cm2) irradiation for 3 min. (c) Temperature variations of ReO3 NCs in PBS (50 ppm) with varying pH values under irradiation of the 808 nm laser (0.7 W/cm2) for 10 cycles (3 min of irradiation for each cycle).

Figure 2c, initially, the temperature of these ReO3 NCs in PBS under pH 7.4 increases by 16.1 °C after 3 min of 808 nm laser irradiation (0.7 W/cm2) in the first cycle, but just after 9 min in the second cycle, the temperature rise is sharply down to 11.1 °C due to the rapid degradation of the ReO3 NCs. Afterward, the temperature is accompanied by increasingly severe deterioration of the photothermal performance with time, and after 57 min in the 10th cycle, it only increases by 3.7 °C. Conversely, the photothermal performance for the ReO3 NCs dispersed in PBS with pH 5.0 is much better. Somewhat surprisingly, cycles are repeated 10 times almost stably, implying that the degradability of the NCs is not affected by the photothermal treatment significantly. After 57 min, the temperature increases by 17.4 °C with 3 min irradiation and it is close to the initial value of 19.2 °C. Beyond that, the ReO3 NCs in PBS at pH 6.0 also exhibit gradually decreased temperature increase. The difference in the photothermal performance among the ReO3 NCs under varying pH values is basically consistent with optical absorbance, while the extraordinary stability for the ReO3 NCs at pH 5.0 might be due to that their degradation reaches equilibrium more easily under lower concentration and stronger acidic condition. 3.3. Biodistribution and Clearance of ReO3 NCs. Ascribed to a combination of low oxygen tension triggering anaerobic fermentation and the production of organic acids, the IMEs typically show acidic characteristics (pH 6.0−6.6) different from normal tissues (pH 7.4) around them.33,36,44 Because of the special and pH-dependent selective degradability for our ReO3 NCs, they are expected to degrade slowly in acidic IMEs while rapidly in the surrounding healthy tissues. Furthermore, it has been confirmed that faster degradation distinctly leads to greater photothermal loss. Therefore, in in vivo photothermal antibacterial applications, it is reasonable to predict that our ReO3 NCs with such unique degradability could restrict the generated heat to the site of infection and reduce thermal damage to the surrounding healthy tissues for highly selective therapy. The ReO3 NCs with high NIR absorbance could provide strong contrast under PA imaging.37 Thus, to assess their degradability in vivo, we used PA imaging possessing high

Figure 3. (a) Ultrasound and PA imaging of the subcutaneous tissues and infectious abscess 0 and 30 min after injection of the ReO3 NCs’ solution. (b) Relative PA signal intensities 0 and 30 min after injection in the subcutaneous tissues and infectious abscess. Timedependent (c) biodistribution and (d) excretion patterns at different time points post intravenously (i.v.) injection with the ReO3 NCs.

signals at subcutaneous tissues are left just after 30 min (Figure 3b) in stark contrast to PA signals at the abscess, where ∼79.4% of their initial signals remain 30 min later, evidencing that the ReO3 NCs have high degradation rate in healthy subcutaneous tissues and apparently degrade slower in the infectious abscess. Such a significantly tissue-selective degradation feature of these NCs will enable specific photothermal antibacterial therapy. As a rule of thumb, inorganic photothermal agents are mostly nondegradable and would accumulate in the body for a long period of time, which may induce undesired long-term toxicity, inflammatory response, or even fibrosis and cancer.46 Since our ReO3 NCs are degradable, we further investigated their bioelimination behaviors in vivo. ReO3 NCs (20 mg/kg) were intravenously (i.v.) injected into mice, which were sacrificed on the 1st, 3rd, 7th, 15th, and 30th days, with their main organs harvested for biodistribution study (Figure 3c). It is found that after 1 day, a majority of the Re retains in liver and spleen, where the Re content gradually decreases in the following time points. The Re levels in all examined organs are very low after 30 days, suggesting that such degradable ReO3 NCs can be efficiently cleared from the mouse body. Note that after injection of the ReO3 NCs’ solution, high levels of Re could be tested in feces of mice (Figure 3d). These results are consistent with our previous studies.37 From these results, it is suggested that after being phagocytosed by reticuloendothelial systems (RESs), the ReO3 NCs might be progressively degraded into ions or small nanoparticles to accelerate their excretion out of RES (Figure S4).37,47−49 3.4. Toxicity and Biocompatibility Evaluation. Next, to explore if such degradable ReO3 NCs would bring about any side effect, we performed a series of careful experiments. First, the Cell Counting Kit-8 assay with Rat bone marrow E

DOI: 10.1021/acsami.9b07359 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. (a) Cell viability of rBMSCs with various concentrations of ReO3 NCs before and after degradation. (b) Hemolysis percent of RBCs at various concentrations of ReO3 NCs. (c) Time-dependent body weight curve within a period of 30 days. (d−g) Blood biochemistry and (h−o) complete blood counts of mice treated with ReO3 NCs. The data were harvested at various time points post i.v. injection.

complete blood panel assays were conducted on the 1st, 3rd, 7th, 15th, and 30th days. The parameters concerning the serum biochemistry (Figure 4d−g) and complete blood panel test (Figure 4h−o) have no meaningful changes. Moreover, from the histological examination by H&E staining, no notable sign of inflammation or tissue damage is observed in major organs, including heart, liver, spleen, lung, and kidney (Figure 4p). Given the efficient body clearance after 30 days, it is possible to conclude that long-term in vivo toxicity will not be caused by our ReO3 NCs, highlighting their large potential for clinical applications. 3.5. Photothermal Antibacterial Activity of ReO3 NCs in Vitro. After confirming the beneficial biocompatibility of the ReO3 NCs, their photothermal antibacterial activities against Gram-negative E. coli and Gram-positive S. aureus planktonic bacteria was evaluated using SPM (Figures 5a,b and S5). Clearly visible in Figure 5a, after treatment with the ReO3

mesenchymal stem cells (rBMSCs) was utilized to verify the cytotoxicity of the ReO3 NCs. As shown in Figure 4a, the samples with a Re concentration below 200 ppm, whether degradation happens or not, exhibit a higher cell viability (>75%). Then, in the hemolysis assay, the ReO3 NCs incubated with red blood cells (RBCs) were used to examine their blood biocompatibility (Figure 4b). It is hard to observe and examine apparent hemolyzed RBCs, even at the higher concentration of 250 ppm, among the ReO3 NC incubation. Such lower cytotoxicity and hemolytic performance formulate a favorable biocompatibility of these NCs in vitro. In the final set of experiments, the toxicity of the ReO3 NCs in vivo was appraised. Previously, we have proven the biosafety of our ReO3 NCs at a dosage of 10 mg/kg.37 Here, healthy mice were i.v. injected with the ReO3 at 20 mg/kg. In the course of 30 days, no death, abnormalities in body weight (Figure 4c), or abnormal behaviors were noted. Serum biochemistry and F

DOI: 10.1021/acsami.9b07359 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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surface. Crystal violet staining was carried out as well to further verify that from the whole biofilms on the titanium plates, the numbers of both E. coli and S. aureus decreased evidently with the irradiation time (Figures S6 and S7). Also, the antibiofilm activity was quantified by SPM (Figure 5d). In agreement with the trend of live/dead staining, the survival of live bacteria dramatically decreases with increasing irradiation time after treatment of the ReO3 NCs and 808 nm laser. The antibacterial efficiencies of the ReO3 NCs against E. coli and S. aureus biofilms after 10 min of irradiation reach 99.2 and 99.0%, respectively, whereas the ReO3 NCs or laser exposure alone does not cause notable impact on biofilm viability. 3.6. Antibiofilm Mechanism Study of ReO3 NCs. To further investigate and interpret the antibiofilm behaviors described above, SEM was conducted to directly observe the destruction and morphology changes of biofilms. From the low-magnification SEM image in Figure 6a, evidently, after

Figure 5. (a) Photos and (b) quantitative analysis of bacterial colonies formed by E. coli and S. aureus planktonic bacteria incubated with the ReO3 NCs after 808 nm laser irradiation (0.7 W/cm2) for different times. (c) Live/dead staining images and (d) quantitative analysis of E. coli and S. aureus biofilms on the titanium metal plate surfaces treated with the ReO3 NCs after exposure to 808 nm laser irradiation (0.7 W/cm2) for different times. (e) Bacterial count based on (c).

NCs and 808 nm laser irradiation, the numbers of colonies for both E. coli and S. aureus decrease significantly with irradiation time. Within only 1 min of exposure to 808 nm laser, the bacteria survival rates quickly decrease to 13.9 and 12.1% for E. coli and S. aureus, respectively, and the bacteria inactivation percentages up to 99.1 and 98.4% are achieved for E. coli and S. aureus, respectively, after 10 min of 808 nm laser irradiation (Figure 5b). As a control, the ReO3 NCs or 808 nm laser alone exhibits negligible antibacterial efficacy. This finding demonstrates that our ReO3 NCs possess superior photothermal antibacterial performance against planktonic bacteria. As stated above, biofilm formation plays a critical role in the pathogenesis of implant-related infections. Further, the photothermal therapeutic effect of the ReO3 NCs on biofilms in vitro toward E. coli and S. aureus was investigated. The antibiofilm performance was first assessed using a live/dead (green/red) staining assay. Figure 5c depicts fluorescence images of E. coli and S. aureus on the surfaces of titanium metal plates under 808 nm laser for different times. With ReO3 NCs alone, both E. coli and S. aureus biofilms are densely colonized and stained bright green, revealing that the ReO3 NCs do not display antibiofilm ability without 808 nm laser and the surface of the titanium plate is fit for bacterial growth. However, increasingly more red fluorescence appears on the surfaces of the titanium plates after being treated with the ReO3 NCs and 808 nm laser irradiation. After irradiation for 10 min, almost all bacteria show red fluorescence and negligible green fluorescence in the pretreated biofilms, implying that these bacteria are destroyed. In the meantime, we observe sharply reduced bacterial cells in the biofilms with irradiation time (Figure 5c,e), demonstrating that the PTT by the ReO3 NCs effectively block bacterial colonization on the titanium plate

Figure 6. (a) Low- and (b) high-magnification SEM images of E. coli and S. aureus biofilms on the titanium metal plate surfaces treated with the ReO3 NCs after 808 nm laser irradiation (0.7 W/cm2) for different times. (c) RT-PCR of mRNA of icaA, fnbA, atlE, and sarA in S. aureus biofilms treated with the ReO3 NCs after 808 nm laser irradiation (0.7 W/cm2) for different times. (d) Schematic illustration for photothermal antibiofilm process of the ReO3 NCs.

treatment with the ReO3 NCs and 808 nm laser irradiation, both the numbers of E. coli and S. aureus in biofilms decrease with irradiation time, which is in accordance with the exhibited tendency of bacteria live/dead staining and crystal violet staining assays. On the other hand, the high-magnification SEM image (Figure 6b) shows that both the bacteria can maintain the natural morphology after treatment with the ReO3 NCs without irradiation, revealing that the ReO3 NCs alone have no influence on the morphology of bacteria. By contrast, after exposure to 808 nm laser irradiation, the cell walls become wrinkled and rough, and disruption appears. G

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ACS Applied Materials & Interfaces Notably, after treatment with the ReO3 NCs followed by 10 min of 808 laser irradiation, the bacterial surfaces show much more violent damage with many bacteria completely broken, which is in agreement with previous reports.1,25−28 As is known, S. aureus is a major cause of implant-related infections.50,51 To further clarify that the PTT by the ReO3 NCs is responsible for biofilm eradication, S. aureus biofilmassociated gene expressions of icaA, fnbA, atlE, and sarA were investigated by RT-PCR. The result in Figure 6c shows that their expressions all decrease significantly with irradiation time compared to group without irradiation, verifying that the PTT suppresses the expressions of icaA, fnbA, atlE, and sarA. These genes are involved in encoding enzymes required for polysaccharide intercellular adhesin synthesis,50 encoding cell wall-anchored proteins,52 inducing cell lysis to produce extracellular DNA for adhesive and strengthened biofilms,53 and activating biofilm development by enhancing the ica operon transcription,54 respectively. Such an RT-PCR result agrees well with SEM imaging, live/dead staining assay, as well as crystal violet staining assay. Consequently, all of these demonstrate that the PTT by our ReO3 NCs could prevent bacterial adhesion and biofilm formation, as well as kill bacteria for rapid and highly effective biofilm eradication (Figure 6d). 3.7. Photothermal Antibacterial Therapy in Vivo. After understanding the excellent antibacterial effect of the ReO3 NCs in vitro, we further explored their ability against bacteria in vivo. In our above studies, we have demonstrated that these NCs possess special and significantly tissue-selective degradability. Under the NIR irradiation, we hope that they could realize specific photothermal eradication of implantrelated infections by restraining the generated heat to the infection site and decreasing thermal damage to the surrounding normal tissues (Figure 7a). On the other hand, in view of their rapid degradability in normal PBS (pH = 7.4) that possibly weakens the targeting efficiency of infection site after i.v. injection, herein, in vivo photothermal antibacterial therapy by the ReO3 NCs was conducted via in situ injection. Mice 24 h after S. aureus-induced implant-related infections were divided into four groups (eight mice each) with different treatments: PBS only (100 μL), PBS + NIR laser (100 μL), ReO3 NCs only (1000 ppm, 100 μL), and ReO3 NCs + NIR laser (1000 ppm, 100 μL). During the therapy process, by means of the thermal camera to record the temperature and thermal images of the mice, we continuously obtained IR thermal imaging. According to Figure 7b,c, only the infection site receiving treatment of both ReO3 NCs and laser irradiation displays rapid increase at temperature to 57.2 °C in 7 min. Moreover, possibly benefiting from the tissue-selective degradation of the ReO3 NCs, we note that the temperature in the surrounding normal tissues has no obvious increase. Over the whole therapy course, the body weights of the mice in the four groups vary slightly (Figure 7d), implying that the therapy strategy is safe. Furthermore, we also recorded the changes of infection area after different treatments (Figure 7e). The infection area from the ReO3 NC injection and laser irradiation group shows only slight increase in the first 2 days, but a continuous decrease up to 14 days. This trend suggests the infection eradication 14 days after the PTT. In discrepancy with the therapy group, the other three groups grow sharply after 2 days and show slower decrease in the following days, which manifests that the immune system alone is difficult to eradicate implant-related infections. The photos of mice with implant-related infections before and 7 and 14 days after

Figure 7. (a) Schematic illustration for specific photothermal eradication of implant-related infections. (b) IR thermal imaging of mice with S. aureus-induced implant-related infections treated with PBS, PBS + NIR laser, ReO3 NCs, and ReO3 NCs + NIR laser group and (c) the corresponding temperature change curves of the infected site in mice with irradiation time. (d) Changes in body weights and (e) quantitative measurement of infection area during the treatment from different groups. The numbers of bacteria (f) on the implants and (g) in the peri-implant soft tissues 14 days after treatments. The results are expressed as the actual numbers of CFU retrieved, and the horizontal line shows the median value. Each group includes eight mice. (h) Photos of mice with implant-related infections before and 7 and 14 days after treatments.

different treatments are shown in Figure 7h. It can be observed that the infected site treated by the ReO3 NC injection and laser irradiation is markedly smaller than others on day 7 and even disappears at the initial infection site on day 14; however, the other three control groups show different levels of infection over the whole process. To fully appraise the antibacterial activity of our ReO3 NCs, the subcutaneous implants (polyethylene washers) and the soft tissues around the implants from all of the groups were harvested after 14 days of treatments, and the numbers of bacteria extracted from implants and peri-implant soft tissues were counted using SPM (Figure 7f,g). Similar to the in vitro antibacterial result, these NCs display a superior antibacterial effect with nearly 100% killing ratio toward S. aureus infection on implants and periimplant soft tissues under NIR light irradiation. The above results indicate that the ReO3 NCs could eliminate implantrelated infections with high performance. 3.8. Histological Evaluation of Antibacterial Effect. To further evaluate the antibacterial effect of this strategy, histological examinations using H&E and Giemsa staining of the peri-implant soft tissues 14 days after different treatments were carried out (Figure 8a). The H&E staining images show the typical signs of soft tissue infections in three control groups, including acute inflammation, exudation, synovial tissue necrosis, and neutrophil infiltration into tissues. In marked contrast, few inflammatory cells emerge in the ReO3 NC injection and laser irradiation group, and fresh granulation tissues with mature fibroblasts and newly formed vessels can be H

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Figure 9. CT imaging of implant-related infections before (pre) and after (post) injection with the ReO3 NCs.

activity. In particular, the high antibiofilm performance is demonstrated via significant inhibition for the expression levels of biofilm-related genes (icaA, fnbA, atlE, and sarA for S. aureus) to prevent bacterial adhesion and the formation of the biofilm, as well as bacterial killing. Interestingly, the ReO3 NCs could change into HxReO3 in an aqueous environment so that they can keep relatively stable in acidic IMEs for PTT, but degrade rapidly in surrounding healthy tissues to decrease photothermal damage. Distinctively, in phosphate-buffered saline with pH 7.4, our ReO3 NCs have the higher degradation rate than any other reported degradable inorganic photothermal nanoagents. Such a special and IME-sensitive selective degradability of the ReO3 NCs not only facilitates safe, efficient, and specific elimination of implant-related infections but also enables clearance from the body in a reasonable period of time after fulfilling their therapy function. Additionally, these ReO3 NCs with high X-ray attenuation ability, different from other reported degradable inorganic photothermal nanoagents, are revealed to be able to act as a CT imaging-guided antibacterial agent for simultaneous diagnosis and therapy of implant-related infections. Overall, this study brings forward a minimally invasive antibacterial method and may have an impact on clinical therapy of bacterial infections.

Figure 8. (a) H&E and Giemsa staining images of peri-implant soft tissues 14 days after treatments. The red arrows represent the infectious bacteria. (b) H&E stained tissues of major organs (heart, liver, spleen, lung, and kidney) 14 days after the PTT.

observed. Moreover, in the Giemsa staining images, many bacteria (indicated by red arrows) in control groups can be found, whereas no bacteria can be observed from the group treated with the ReO3 NCs and NIR light. In addition to the in vivo robust antibacterial effect, it should be noted that the tissues around the implant were not damaged by the PTT, according to the results of the H&E staining, most likely due to the tissue-selective degradability of the ReO3 NCs as well as the regenerable tissues after the short-term light irradiation. Besides, H&E staining images of major organs from the ReO3 NC injection and laser irradiation group on day 14 indicate that such photothermal antibacterial therapy in vivo has high biocompatibility and negligible toxicity (Figure 8b). All of these suggest that this new antibacterial strategy using selectively degradable ReO3 NCs can eradicate the implantrelated infections specifically, efficiently, and safely. 3.9. CT Imaging of Implant-Related Infections with ReO3 NCs. Apart from their outstanding antibacterial efficacy, our ReO3 NCs should also be promising for CT imaging of implant-related infections, since Re with a higher atomic number (Z = 75) than clinic-applied iodine has strong X-ray attenuation ability. It should be noted that, compared to other known degradable inorganic photothermal nanoagents under PBS (pH 7.4) without any assistant condition, only ReO3 NCs have elements whose atomic number is higher than that of iodine. As expected, before injection of the ReO3 NCs, the infection site could not be detectable, while a clear contrast arises in the infection site after ReO3 injection (Figure 9).

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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.9b07359. Primers used in the present study for real-time polymerase chain reaction; summary of degradability for current degradable inorganic photothermal nanoagents; a photo of solid ReO3 NC thin film on glass substrate; photos of polyethylene washer and mouse implant-related soft tissue infection model; UV−vis− NIR absorption spectra of ReO3 NCs over time in PBS (166 ppm) with pH 5.0 and 6.0; illustration to describe the in vivo degradation and clearance process of the ReO3 NCs; IR thermal images of 96-well plates when the wells are incubated with ReO3 NCs and planktonic bacteria before and after exposure to an 808 nm laser; photos for crystal violet staining of E. coli and S. aureus biofilms on the titanium metal plate surfaces treated with the ReO3 NCs after exposure to 808 nm laser irradiation (0.7 W/cm2) for different times; and optical density at 595 nm for E. coli and S. aureus biofilms washed out after crystal violet staining (PDF)

4. CONCLUSIONS In summary, the ReO3 NCs were prepared through a rapid and straightforward space-confined on-substrate route. These prepared NCs with strong NIR LSPR absorbance and satisfied biocompatibility show excellent photothermal antibacterial I

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (R.Z.). *E-mail: [email protected] (H.S.). *E-mail: [email protected] (J.H.). ORCID

Rujia Zou: 0000-0001-5566-5091 Junqing Hu: 0000-0001-8422-7250 Author Contributions ⊥

W.Z. and C.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Fundamental Research Funds for the Central Universities, the National Natural Science Foundation of China (Grant Nos. 51472044, 51672049, 81772364, and 81472108), the Program Innovative Research Team in University (IRT_16R13), the Science and Technology Commission of Shanghai Municipality (18ZR1402000), Shanghai Municipal Science and Technology Committee of Shanghai outstanding academic leaders plan (19XD1423100), Shanghai Municipal Education CommissionGaofeng Clinical Medicine Grant Support (20181714), the DHU Distinguished Young Professor Program, the International Joint Laboratory for Advanced fiber and Low-dimension Materials (18520750400), the Shenzhen Science and Technology Research Project (Grant No. JCYJ20180508152903208), and the Shenzhen Pengcheng Scholar Program. In addition, Wenlong Zhang particularly thanks his parents who have been giving him powerful support for over 30 years.

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DOI: 10.1021/acsami.9b07359 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX