Surface-Adaptive and Initiator-Loaded Graphene as a Light-Induced

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 1766−1781

www.acsami.org

Surface-Adaptive and Initiator-Loaded Graphene as a Light-Induced Generator with Free Radicals for Drug-Resistant Bacteria Eradication Xunzhou Yu,† Danfeng He,† Ximu Zhang,*,§ Hongmei Zhang,§ Jinlin Song,§ Dezhi Shi,∥ Yahan Fan,‡ Gaoxing Luo,*,† and Jun Deng*,†

ACS Appl. Mater. Interfaces 2019.11:1766-1781. Downloaded from pubs.acs.org by UNIV OF KANSAS on 01/21/19. For personal use only.

†

Institute of Burn Research, Southwest Hospital, State Key Lab of Trauma, Burn and Combined Injury and ‡Department of Blood Transfusion, Southwest Hospital, Army Medical University, Chongqing 400038, China § Chongqing Key Laboratory of Oral Disease and Biomedical Sciences, Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education & Stomatological Hospital of Chongqing Medical University, Chongqing 401174, China ∥ Key Laboratory of Three Gorges Reservoir Region’s Eco-Environment, Ministry of Education, Faculty of Urban Construction and Environmental Engineering, Chongqing University, Chongqing 40005, China S Supporting Information *

ABSTRACT: Since generating toxic reactive oxygen species is largely dependent on oxygen, bacteria-infected wounds’ hypoxia significantly inhibits photodynamic therapy’s antibacterial efficiency. Therefore, a novel therapeutic method for eradicating multidrug-resistant bacteria is developed based on the light-activated alkyl free-radical generation (that is oxygen independent). According to the polydopamine-coated carboxyl graphene (PDA@CG), an initiator-loaded and pH-sensitive heat-producible hybrid of bactericides was synthesized. According to fluorescence/thermal imaging, under the low pH of the bacterial infection sites, this platform turned positively charged, which allows their accumulation in local infection site. The plasmonic heating effects of PDA@CG can make the initiator decomposed to generate alkyl radical (R•) under the followed near-infrared light irradiation. As a result, oxidative stress can be elevated, DNA damages in bacteria can be caused, and finally even multidrug-resistance death can be caused under different oxygen tensions. Moreover, our bactericidal could promote wound healing in vivo and negligible toxicity in vivo and in vitro and eliminate abscess. Accordingly, this study proves that combination of oxygen-independent free-radical-based therapy along with a stimulus-responsiveness moiety not only can be used as an effective treatment of multidrug-resistant bacteria infection, but also creates a use of a variety of free radicals for treatment of multidrug-resistant bacteria infection wounds. KEYWORDS: hypoxia, free radicals, pH responsive, drug-resistant bacteria, graphene cancer therapy9,10 and bacteria eradication.11−13 Photodynamic therapy (PDT) is recognized as the best strategy for the current free-radical treatment model due to its tumor that is invasive14,15 or bacteria localized features.11−13 In general, light energy in PDT can undergo transfer by photosensitizers (PSs) to produce reactive oxygen species (ROS) (e.g., 1O2, •OH) for ablating tumor or bacteria.16 Nevertheless, ROS generation in PDT largely depends on oxygen, significantly limiting its therapeutic effects in hypoxic bacteria-infected wounds.17−19 Thus, many efforts have been made to overcome these problems, such as self-generation of O2 and combining PSs with other therapeutic agents.20−22 However, the use of these assistive strategies on PDT does not completely solve the problem of oxygen dependence and may also be accompanied by other side effects.20−22 Carbon-based free radicals are also highly reactive and can be formed without

1. INTRODUCTION Bacterial infectious disease, in particular multidrug resistance, has turned into a big global health problem and attracted people’s wide concern.1,2 Recently, one study suggested that failure to control drug-resistant bacterial infections can cause the death of more than 10 million patients a year by 2050 and losses of up to 100 trillion dollars.3 However, at a time of slow antimicrobial development, the world could one day face a pandemic of incurable bacterial infections, which could be catastrophic in severity.4−6 Such worrying factors have hastened the improvement of alternative efficient therapeutic approaches to treat drug-resistant bacterial infections. Free radicals are mostly highly reactive molecular or molecule fragments with unpaired valence electrons.7 Generally, free radicals, especially oxygen-related radicals, have wide existence and maintain homeostasis in living organisms.8 Free radicals have a positive effect on cell metabolism in the normal state; however, excessive free radicals can have a direct interaction/reaction with lipids, proteins, and DNA to induce cell dysfunction.8 Thus, free radicals have been applied in © 2018 American Chemical Society

Received: July 29, 2018 Accepted: December 7, 2018 Published: December 7, 2018 1766

DOI: 10.1021/acsami.8b12873 ACS Appl. Mater. Interfaces 2019, 11, 1766−1781

Research Article

ACS Applied Materials & Interfaces

Figure 1. (A) Schematic illustration of AIBI-GCS-PDA@CG fabrication and (B) photothermal ablation of bacteria in vivo. (C) Schematic description of the antibacterial mechanism of AIBI-GCS-PDA@CG.

oxygen-independent strategy is developed for multidrugresistant ablation, and the potential of many free radicals for biomedical use is revealed.

oxygen. However, controlling these radicals’ generation at the bacteria infection sites still remains a great challenge. Thermal degradation azo initiator is a popular free-radical generator, which can be used for free-radical polymerization and for inducing oxidative stress in biological systems (OS).23 Nevertheless, these initiators’ decomposition of that induces cell death at physiological temperatures is restricted, and the in situ heater is ideal for accelerating the production of free radicals. Graphene-based nanomaterials are agents of photothermal conversion (PTC) that have been broadly explored, showing excellent biocompatibility.24−26 Additionally, as a class of two-dimensional nanomaterials, graphene has aroused wide attention in drug delivery fields due to its large specific surface area.24−26 Thus, graphene nanosheets are well suited as the heat source and initiator carrier. Therefore, a new therapeutic strategy for the eradication of multidrug-resistant bacteria is proposed in this study according to alkane free radicals’ light-induced generation. As shown in Figure 1, the polydopamine-coated carboxyl graphene (PDA@ CG) nanosheets were improved by using glycol chitosan (GCS) (GCS-PDA@CG), a water-soluble chitosan derivative with stealth properties and a pH-sensitive switchable charge (pKa ∼ 6.5).27−29 Finally, an initiator of 2,2-azobis(2-(2imidazolin-2-yl)propane)dihydrochloride (AIBI) was selected as the source of radicals, and it was loaded on GCS-PDA@CG for the formation of a benign and efficient antimicrobial depot (AIBI-GCS-PDA@CG). The plasmonic heating of PDA@CG will cause AIBI decomposition under NIR irradiation for the generation of alkyl radicals (R•), as well as break the coordination bond or hydrophobic interaction between the AIBI and the PDA for releasing the R•. Under various oxygen tensions, the antibacterial efficacy of AIBI-GCS-PDA@CG in vivo and in vitro and the mechanism therapy of the generated free radicals were assessed. On the basis of this system, a novel

2. EXPERIMENTAL SECTION 2.1. Materials. Carboxyl graphene (CG) was obtained from Nanjing XFNANO Materials Tech Co. Ltd. (Nanjing, China). Aladdin Reagent Co. Ltd. (China) provided dopamine hydrochloride (C8 H11O2N·HCl, DA, >98%), glycol chitosan (GCS, ≥60% deacetylation), and α-(4-pyridyl N-oxide)-N-tert-butylnitrone (POBN, 99%). Sigma-Aldrich provided 2-methyl-2-nitrosopropane dimmer (MNP, 98%) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO, 97%). J&K Scientific Ltd provided N-hydroxysuccinimide (C4H5NO3, NHS, 98%), 2,2′-azobis(2-(2-imidazolin-2-yl)propane)dihydrochloride (C12H22N6·2HCl, AIBI, 98%), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (C8H17N3·HCl, EDC, 99%). Beyotime Institute of Biotechnology (China) provided 2′,7′-dichlorofluorescin diacetate (DCFH-DA). Dongren Chemical Technology Co. Ltd. (Shanghai, China) provided Cell Counting Kit-8 (CCK-8). LIVE/DEAD BacLight Bacterial Viability Kit was bought from Semmerfeld Technology Co. Ltd. (China). Unless specially mentioned, all the rest of the chemicals were of analytical grade and were used without further purification. Milli-Q water was used during the entire experiment. Cy5 NHS Ester (Cy5-SE) was provided by MedChem Express Co., Ltd. (Shanghai, China). Multidrug-resistant Staphylococcus aureus (MRSA, ATCC 43300) and S. aureus (ATCC 25923) isolated from human clinical specimen were provided by Clinical Microbiology Laboratory, Institute of Burn Research, Southwest Hospital, Army Medical University (Chongqing, China). 3T3 cells were provided by the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences. Nutrient agars used in the experiment were bought from Pang Tong Medical Instrument Co. Ltd. (Chongqing, China). Experimental Animal Department of the Army Medical University provided BALB/c mice (20−25 g, 6−8 weeks). All animals were handled following the ethical principles of the Institutional Animal Care and Use Committee of Army Medical University. 1767


Research Article

ACS Applied Materials & Interfaces 2.2. Synthesis of AIBI-GCS-PDA@CG. 2.2.1. Synthesis of PDAModified CG (PDA@CG). Briefly, we introduced 2 mg of CG into 20 mL of dopamine solution (0.2 mg/mL, pH 8.5, 10 mM Tris−HCl). After 30 min of sonication, we concentrated and collected PDA@CG by centrifugation (15 000 rpm, 60 min). 2.2.2. Preparation of GCS-Coated PDA@CG (GCS-PDA@CG). Using the chemical reaction between the quinone group in PDA and the amine group in GCS, the GCS was coated on the PDA@CG. Typically, we dissolved 50 mg of GCS first in 10 mL of 0.1 M HCl. Subsequently, 0.4 mL of GCS (5 mg/mL) and 20 mg of NHS were introduced into the solution of PDA@CG and stirred for 2 h. Next, we introduced 50 mg of EDC into the mixture and stirred overnight at 30 °C. Then, the GCS-PDA@CG was obtained by centrifugation. 2.2.3. Loading GCS-PDA@CG with AIBI (AIBI-GCS-PDA@CG). Briefly, 10 mg of AIBI (5 mg/mL) was mixed with the GCS-PDA@ CG solutions concentrated under stirring for 3 h at 30 °C. After that, the free AIBI molecules were removed three times for 60 min by centrifugation at 15 000 rpm, achieved through dialysis for 2−3 days. 2.3. Characterizations. The surface size and morphology of the prepared samples were characterized under an atomic force microscope (AFM, JPK NanoWizard 4, German) and a transmission electron microscope (TEM-1400 PLUS, Japan). The hydrodynamic diameters of the samples were measured by dynamic light scattering (DLS, ZEN5600, Malvern Instruments, U.K.). The contents of PDA, GCS, and AIBI on AIBI-GCS-PDA@CG were acquired using thermogravimetric analysis (TGA, TG-Q500) in a N2 environment. Using an IR Prestige-21 infrared spectrophotometer (Shimadzu, Japan), Fourier transform infrared (FTIR) spectra were recorded. To analyze the pH-dependent changes of GCS-PDA@CG’s and AIBIGCS-PDA@CG’s surface charges, the test samples were dispersed in phosphate buffer saline (PBS, pH 5.0−7.4, 100 μg/mL) and their ζpotentials were measured with Zetasizer (ZEN5600, Malvern Instruments, U.K.). With the use of a UV−vis−NIR spectrophotometer (UV-3600 SHIMADZU), the UV−vis−NIR optical properties of CG, PDA@CG, GCS-PDA@CG, and AIBI-GCS-PDA@CG in PBS were recorded at pH 6.3. To elucidate GCS-PDA@CG’s and AIBI-GCS-PDA@CG’s photothermal abilities, test samples were dispersed in PBS at different concentrations (0−1 mg/mL) at pH 6.3 in a 96-well plate and subsequently under irradiation with 808 nm NIR laser (VLSM-808-B, Connet). The temperature of the above suspensions was monitored by using a thermocouple thermometer (FLIR, Sweden). The near-infrared fluorescence (NIRF) imaging and biodistribution in vivo were visualized using IVIS Lumina II (Xenogen IVIS Spectrum, Perkin Elmer). Finally, we calculated the photothermal conversion efficiency (η) with the method reported in previous studies.30,31 The Raman spectra of graphene composites were recorded using a LabRAMHR800 spectrometer (Horiba JobinYvon, France). To synthesize the fluorescence-labeled AIBIGCS-PDA@CG (f-AIBI-GCS-PDA@CG), 2 mg of Cy5-SE was dissolved in dimethyl sulfoxide (4 mL). Subsequently, the prepared Cy5-SE solution was added to 4 mL of AIBI-GCS-PDA@CG suspension. For the removal of the free Cy5-SE, after about 4 h of stirring, the mixture was centrifuged three times for 8 min at 10 000 rpm. All of these steps need to be operated in the dark. The fluorescence spectra of graphene composites were recorded using a spectrophotometer (RF-5310PC, Japan). 2.4. Electron Spin Resonance (ESR) Measurements. The ESR signal was detected using an ESR spectrometer (Bruker X-band A200). In brief, the solution of AIBI-GCS-PDA@CG (0.2 mg/mL) and the spin traps (POBN, 100 mM) were mixed in sealed tubes. After bubbling with air at ambient temperature for 30 min, the mixed solution was irradiated by an 808 nm NIR laser with 0.75 W/cm2 power for 2 min. Immediately, the irradiated solution (∼200 μL) was added to a quartz capillary tube, and the ESR signal was detected with an ESR spectrometer (Bruker X-band A200) at ambient temperature. Then, 200 μL of AIBI-GCS-PDA@CG (1 mg/mL) and POBN (100 mM) was mixed in a tube under the irradiation of an NIR laser light (0.75 W/cm2, 2−11 min) to monitor the releasing process of free radicals. After being irradiated for a certain time, 200 μL of the solution was transferred to a quartz capillary tube and was detected by

an ESR spectrometer (Bruker X-band A200). Control experiments were conducted in parallel with PBS (pH 6.3, 0.2 mg/mL), AIBI (0.2 mg/mL), and GCS-PDA@CG (0.2 mg/mL). 2.5. Hemolysis Assay of AIBI-GCS-PDA@CG. Resh human blood was used in the experiment, and the blood sample was from the Southwest Hospital, Third Military Medical University, with the consent of the patient. The erythrocytes were collected via centrifugation (1500 rpm, 15 min) and washed with saline three times. Next, centrifuged erythrocytes (3 mL) were added to saline (11 mL) to prepare the stock dispersion, and 200 μL of the stock dispersion was added to 1 mL of AIBI-GCS-PDA@CG dispersions. The final red blood cell hematocrit level was nearly about 4%. We incubated the combined solutions for 3 h at the temperature of 37 °C. Afterward, by UV−vis analysis of the supernatant (540 nm), the percentage of hemolysis was measured after centrifugation (12 000 rpm, 15 min). Pure water was the positive control, and saline served as the negative one. In the same way, hemolysis of AIBI-GCS-PDA@ CG was assayed. Hemolysis percentage was calculated by hemolysis (%) = (A S − AN)/(AP − AN) × 100% where AP denotes the absorbance as a positive control following the addition of deionized water, AN denotes the absorbance as a negative control following the addition of saline, and AS denotes the absorbance resulting from the addition of AIBI-GCS-PDA@CG to the erythrocyte suspension. 2.6. In Vitro Experiments. 2.6.1. Bacteria Culture. The cultures of both the standard S. aureus and MRSA bacterial strains were in 4 mL of liquid Luria-Bertani medium and shaken at 200 rpm for 12−16 h at 37 °C. Subsequently, during the exponential growth phase of the resultant bacteria, the resultant bacteria were collected and then rinsed by using PBS buffer (pH 6.3 or 7.4) by centrifuging at 3500 rpm. 2.6.2. In Vitro GCS-PDA@CG’s and AIBI-GCS-PDA@CG’s Specific Targeting to Bacteria. By incubating them together in an acidic environment (pH 6.3) and a physiological environment (pH 7.4), the interactions between synthetic nanomaterials (GCS-PDA@CG or AIBI-GCS-PDA@CG) and bacteria were assessed in vitro. Typically, 200 μL of GCS-PDA@CG (1 mg/mL) or AIBI-GCS-PDA@CG (1 mg/mL) and 800 μL of the test bacteria were mixed together. For the complete removal of free GCS-PDA@CG or AIBI-GCS-PDA@CG, the mixture solution, incubated at ambient temperature for 30 min, was centrifuged (3000 rpm, 5 min) and washed thrice by PBS. Subsequently, the sediment (bacteria together with nanomaterials) was suspended in 1 mL of the relative PBS buffer, and its ζ-potentials were recorded with Zetasizer Nano. Meanwhile, aggregated pellets’ morphology was detected under a scanning electron microscope (SEM, Inspect F, Philips, The Netherlands). The model host cell NIH 3T3 fibroblasts were coincubated with test nanomaterials (GCS-PDA@CG or AIBI-GCS-PDA@CG) at pH 7.4 for 24 h to verify whether the GCS-PDA@CG or AIBI-GCSPDA@CG will interact with the host cells in physiological cases. Then, the cells were washed two times with PBS and were observed under SEM (Inspect F, Philips, The Netherlands) according to our previous methods. 2.6.3. In Vitro Free-Radical Generation Analysis. In vitro freeradical generation analysis was performed following the previously reported methods with little modifications.32 Briefly, 200 μL of AIBIGCS-PDA@CG (1 mg/mL) and 800 μL of the test bacteria were mixed together for 30 min. Subsequently, the mixture solution was incubated in spin trap solution (0.5 M POBN in PBS) at room temperature. Thirty minutes later, at a light density of 0.5 W/cm2, we irradiated the combined solution under NIR laser light for 7 min in a tube. Afterward, the solution was transferred to a quartz capillary tube immediately, and then its ESR spectra were monitored at room temperature. 2.6.4. In Vitro ROS Generation Assay. The intracellular ROS level was detected using DCFH-DA under a confocal laser scanning microscope (CLSM, Zeiss, lsm780). After the test bacteria were incubated with DCFH-DA (1 × 10−5 M) for 30 min at 37 °C in the dark, they were cultured with 0.2 mg/mL AIBI-GCS-PDA@CG for 4 1768


Research Article

ACS Applied Materials & Interfaces h, and the mixture was washed three times with PBS. Afterward, the bacteria were irradiated with 808 nm NIR laser light for 7 min with a power of 0.5 W/cm2. In parallel with AIBI (0.2 mg/mL) and GCSPDA@CG (0.2 mg/mL), control experiments were conducted. To confirm whether the ROS level was detected under hypoxic condition (1% O2), the bacteria incubated with AIBI-GCS-PDA@CG for 4 h were also measured via CLSM. 2.6.5. In Vitro Antibacterial Efficiency of AIBI-GCS-PDA@CG. With the standard plate count, AIBI-GCS-PDA@CG’s in vitro antibacterial efficiency was assessed.33 In brief, 200 μL of AIBI-GCSPDA@CG (1 mg/mL) solution was mixed with 800 μL of bacteria solution (pH 6.3, OD600 0.4−0.5) and shaken sufficiently for 30 min. Afterward, we irradiated the solution by 808 nm NIR laser (0.5 W/ cm2, 7 min). The processed suspension of resultant bacteria was fully shaken and subsequently diluted and then uniformly spread onto nutrient agars. After the culture at 37 °C for 16−18 h, and the bacterial colonies were recorded and counted with an automatic colony counter (Supcre, Shineso, Hangzhou, China). In parallel with AIBI and GCS-PDA@CG, control experiments were performed. To assess the function of alkyl free radicals in depth, the antibacterial effect of AIBI-GCS-PDA@CG after adding alkyl free radicals scavenger (vitamin C) was also examined with the use of an automatic colony counter (Supcre, Shineso, Hangzhou, China). Briefly, vitamin C solution (0.4, 0.8, 1.2 mg/mL) and 200 μL of AIBIGCS-PDA@CG (1 mg/mL) solution were mixed with 800 μL of bacteria solution (pH 6.3, OD600 0.4−0.5). Then, the mixture was shaken sufficiently for 30 min. Afterward, the solution was irradiated for 7 min with an 808 nm NIR laser (0.5 W/cm2). The processed suspension of the resultant bacteria was shaken fully and subsequently diluted and evenly spread onto nutrient agars. Moreover, to further evaluate the thermal antibacterial efficiency of the nanomaterials, a 50 °C water bath was used. The mixture solution of AIBI-GCS-PDA@CG and bacteria as-prepared above was deposited in a thermostat water bath cauldron (50 °C) under shaking for different times (8, 10, and 12 min). Then, following the standard plate count method, the number of bacteria colonies was ascertained. Control experiments were also conducted in parallel with AIBI and GCS-PDA@CG. 2.6.6. In Vitro Live/Dead Bacteria Staining Assay. Bacteria suspensions cultured with AIBI-GCS-PDA@CG were irradiated under NIR laser (0.5 W/cm2, 7 min) in normal or hypoxic condition. Then, the suspensions were stained with live/dead dye solution (0.5 mL, 5 μM of propidium iodide, and 1 μM of SYTO 9) simultaneously for 30 min. The green SYTO 9 dye entered both bacteria with damaged cell structure (cell membrane and wall) and intact bacteria; yet, the red propidium iodide dye only entered bacteria with the damaged cell membrane or wall. 2.6.7. Glutathione (GSH) Assay. Bacteria cells were seeded in an Eppendorf tube and were collected at the exponential growth phase (OD600 = 0.4, ∼5 × 107 cells/mL). After incubating with AIBI (0.2 mg/mL), GCS-PDA@CG (0.2 mg/mL), and AIBI-GCS-PDA@CG (0.2 mg/mL) for 4 h, the bacteria were irradiated for 7 min with 808 nm NIR laser light (0.5 W/cm2). After the mixture was washed three times with PBS, 200 μL of Triton-X 100 lysis buffer (0.4%) was introduced to the mixture and incubated on ice for 20 min. Afterward, 50 μL of supernatant was mixed with 200 μL of Ellman’s reagent (0.5 mM 5,5′-dithiobis-2-nitrobenzoic), and the lysates were centrifuged for 5 min at 6000 rpm. In the end, with a microplate reader (Thermo Varioskan Flash), we measured the absorbance (405 nm) of the samples. 2.6.8. DNA Damage Assay. The DNA damage ability of the free radicals generated by AIBI-GCS-PDA@CG was studied using the method reported before.34 Briefly, 200 ng of pUC 18 DNA was introduced to 1 mL of AIBI-GCS-PDA@CG (0.2 mg/mL) in PBS. The solution was bubbled for 30 min with air or nitrogen and irradiated for 7 min with 808 nm NIR laser light (0.5 W/cm2). Then, the solution was lyophilized, dissolved in 20 μL of PBS, and mixed with the loading buffer (0.25% bromophenol blue). After that, we loaded the sample on 1% agarose gel with GelRed. The gel was run in 0.5× Tris-borate−ethylenediaminetetraacetic acid (TBE, Tris boric

acid) buffer at 85 V for 1.5 h and photographed through the Vilber Lourmat imaging system (BIO-RAD Segrate Italy). 2.7. In Vivo Experiments. 2.7.1. Animal Model. To study the antibacterial effect of AIBI-GCS-PDA@CG in vivo, subcutaneous abscess on BALB/c mice (6−8 weeks, 20−25 g) was established. Mice were anesthetized with 1% pentobarbital to form subcutaneous abscesses. Then, 100 μL of MRSA (5 × 107 CFU/mL) subcutaneous injection was administered on each test mouse’s disinfected and shaved back, and at the same time the left side as control was injected with PBS. Twenty-four hours later, a focal infection formed as a subcutaneous abscess. 2.7.2. In Vivo NIRF and Fluorescent Imaging of Bacterial Infection. The infected mice were injected with AIBI-GCS-PDA@CG nanomaterials (200 μL, 1 mg/mL) through the tail vein for in vivo NIFR imaging. Afterward, the infected mice were anesthetized with isoflurane (0.2−0.3 L/min) and NIRF images were recorded with an IVIS Lumina II imaging system (Xenogen IVIS Spectrum, Perkin Elmer) at scheduled time points. Under an IVIS Lumina imaging system, near-infrared fluorescence (NIRF) images were taken at ascertained time points (0, 2, 4, 6, and 24 h). Then, at 24 h post injection, the kidney, lung, spleen, liver, heart, abscess, and other organs of the mouse were taken and imaged by the IVIS cavity imaging system. Each side of the injection side in the back of the mice was irradiated with 808 nm near-infrared laser (0.5 W/cm2, 7 min) at preascertained time intervals (0, 2, 4, 6, and 24 h), and thermographic images were taken with an IR thermal camera (FLIR-E49001, Estonia) for demonstrating AIBI-GCS-PDA@CG’s in vivo targeting capacity. Additionally, to assess the capability of GCSPDA@CG and AIBI-GCS-PDA@CG for the production of in vivo hyperthermia at abscess, we intravenously injected 100 μL of PBS, GCS-PDA@CG (0.8 mg/kg), or AIBI-GCS-PDA@CG (0.8 mg/kg) to the infected mice (10 mM, pH 6.3). Six hours later, we irradiated abscess of each mice for 0−12 min under an 808 nm NIR laser (0.5 W/cm2) and monitored the temperature with an IR thermal camera (FLIR-E49001, Estonia). 2.7.3. In Vivo Antimicrobial Activity Evaluation. Our hybrid bactericides’ in vivo antimicrobial activity was studied in mice showing subcutaneous abscesses infected by MRSA. Briefly, male BALB/c mice with abscesses were split into six groups (five mice per group): control (PBS), NIR (0.5 W/cm2, 7 min), GCS-PDA@CG (200 μL, 1 mg/mL), AIBI-GCS-PDA@CG (200 μL, 1 mg/mL), GCS-PDA@CG (200 μL, 1 mg/mL) + NIR (0.5 W/cm2, 7 min), and AIBI-GCS-PDA@CG (200 μL, 1 mg/mL) + NIR (0.5 W/cm2, 7 min) groups. The mice were intravenously administered only at the first time. After 10 days of treatment, all the mice were scratched, and the infected tissue was collected and assessed via standard platecounting approach and hematoxylin and eosin (H&E) staining. 2.8. In Vitro and in Vivo Biocompatibility Evaluation of AIBI-GCS-PDA@CG. By a cell counting kit-8 (CCK-8) assay, AIBIGCS-PDA@CG’s in vitro cytotoxicity was ascertained following the manufacturer’s specification. Typically, the researcher seeded 3T3 fibroblasts cells on a 96-well plate at 3000 cells per well in density and allowed them to be attached for 24 h. Next, we replaced the culture medium with 10% fetal bovine serum/Dulbecco’s modified Eagle’s medium containing different AIBI-GCS-PDA@CG concentrations (0.1−1 mg/mL). After coculture for 24 h, the plates were washed with PBS three times. Next, we introduced the CCK-8 reagent into each well and cultured them for another 3 h at 37 °C. Finally, with a microplate reader (Thermo Varioskan Flash), we measured the optical density (OD) value of the medium at 450 nm. We acquired the cell viability as follows: cell viability = (OD(samples)/OD(control)) × 100%, where OD(samples) and OD(control) denote the absorbance value at 450 nm in the absence and presence of AIBI-GCS-PDA@CG, respectively. Control experiments were also conducted in parallel with Hacat cells. With the use of healthy BALB/c mice (20−25 g, 6−8 weeks, five mice each group), the in vivo biotoxicity of AIBI-GCS-PDA@CG was assessed. After injecting with 0.5 mL of AIBI-GCS-PDA@CG (0.2, 0.5 mg/mL) through the tail vein and without NIR irradiation, the mice were sacrificed for a certain time (1 or 30 days). The major 1769


Research Article


Figure 2. Characterization of AIBI-GCS-PDA@CG. (A) TEM and (B) AFM images of AIBI-GCS-PDA@CG nanosheets. (C) Hydrodynamic diameter of AIBI-GCS-PDA@CG after 10 h of ultrasound bath. (D) ζ-Potential of AIBI-GCS-PDA@CG under different pH values (5.0, 5.5, 6.0, 6.3, 6.5, 7.0, and 7.4). (E) UV−vis−NIR spectra of PBS, CG, PDA@CG, GCS-PDA@CG, and AIBI-GCS-PDA@CG. (F) Photothermal effect of AIBI-GCS-PDA@CG under NIR (808 nm) irradiation with different powers (0.5, 0.75, and 1 W/cm2). (G) Photothermal effect and (H) thermographic images of AIBI-GCS-PDA@CG with different concentrations (0.05−0.2 mg/mL) under NIR irradiation (808 nm, 0.5 W/cm2). (I) Thermal decomposition of AIBI (i1) and schematic illustrating the reaction of free radicals with spin traps (POBN) (i2). (J) ESR spectra of 100 mM POBN in 0.20 mg/mL of AIBI-GCS-PDA@CG under NIR irradiation (808 nm, 0.5 W/cm2) for different times (4, 6, 8, and 10 min). The arrows represent the changes of the spectra with increase of irradiation time. (K) Plot of ESR peak intensity versus irradiation time. Solutions of AIBI (0.20 mg/mL) and GCS-PDA@CG (0.20 mg/mL) under the same conditions were used as negative controls. organs, for example, lung, heart, spleen, kidney, and liver, were gathered, washed by PBS, and immobilized in 4% paraformaldehyde for 24 h. After being treated with paraffin embedding, serial section, and hematoxylin and eosin (H&E) staining, the samples, with the use of an optical microscope (CTR600, Leica, Germany), underwent histological examination. To assess the potential toxicity of AIBIGCS-PDA@CG in depth, the blood of each test mouse was drawn from the axillary artery and detect the blood routine examination, liver function, kidney function and myocardial enzyme spectrum. PBS was used to treat mice as the control group. 2.9. Statistical Analysis. All data are denoted mean ± standard deviation. With the use of one-way analysis of variance (ANOVA)

(for two groups) and two-way ANOVA (for more than two groups) in Origin software, we analyzed remarkable diversification between groups. The statistical significance was established as p < 0.05 and p < 0.01.

3. RESULTS 3.1. Characterizations of AIBI-GCS-PDA@CG. In this study, XFNANO Company (Nanjing, China) provided the CG nanosheets. Following sonication and purification, under FTIR spectroscopy, the obtained CG nanosheets were studied. Carboxyl OC−O (1351 cm−1), aromatic CC (1618 1770


Research Article

ACS Applied Materials & Interfaces cm−1), and surface carboxylic groups CO−O (1726 cm−1) are shown in the spectrum of CG. Then, the hydrodynamic size and morphology of CG were, respectively, ascertained with DLS and TEM. CG had a sheetlike structure (Figure S2) with a size of 122−220 nm (Figure S2A). The size of ∼164 nm represents the largest amount of sheets with this size. To assess the thicknesses of GCS and CG, AFM was employed. As shown in Figure S2A2, the thickness of CG was ∼1.0 nm. Through dopamine self-polymerization in the alkaline case (pH 8.5), PDA coating was deposited on CG nanosheets, enabling further modification on CG.35 PDA@CG nanosheets were analyzed by FTIR, AFM, and ζ-potential for a reliable demonstration of finished synthesis of PDA@CG. The spectrum of PDA revealed peaks at 1512 and 1600 cm−1, following the indoline or indole structures in PDA36 (Figure S1A). In the meantime, there was a peak of C−H stretching vibration in the aromatic ring at 2962 cm−1. A broad peak spanning 3200−3600 cm−1 followed the presence of hydroxyl groups in PDA. The spectrum of PDA@CG showed all the mentioned spectral features for the PDA sample, giving evidence of the successful deposition of PDA coating on CG (Figure S1A). In comparison with the TEM image of CG, there were basically no significant size and morphological changes after PDA coating, suggesting that the PDA deposits uniformly on CG, and its thickness is relatively thin (Figure S2B1). The AFM images also supported this (Figure S2B2). After PDA coating, the thickness was increased by only 0.2 nm. After PDA coating, CG’s ζ-potential was reduced from −38.1 ± 3.5 to −44.9 ± 1.1 mV (Figure S3A). ζ-Potential reduction was primarily attributed to the less negatively charged hydroxyl groups in PDA coating. GCS, a water-soluble chitosan derivative featuring pHsensitive charge reversal,37 has been reported capable of ameliorating the in vivo behavior of CG by our previous works.26 Here, by the reaction between the quinone groups in PDA and the amine groups of GCS based on the Schiff base reaction, GCS was transfected on [email protected],39 According to Figure S1B, the successful synthesis of GCS-PDA@CG was demonstrated by the appearance of C−O−C (1120 cm−1) for GCS in the spectrum of GCS-PDA@CG and the reduced intensity of CO (1730 cm−1) for PDA. The morphology was almost constant (Figure S2C1) in comparison with the TEM images of PDA@CG before and after the modification with GCS. However, the AFM images showed that the thickness was increased up to 1.7 nm after the GCS grafting (Figure SC2). These results certified that the GCS molecules were successfully grafted on the PDA@CG. Using a Zetasizer (Figure S3B), the ζ-potential of GCS-PDA@CG was examined to assess its pH-sensitive performance in depth. Because the free amino group in GCS is protonated by the combination of free amino group and proton under acidic conditions, GCSPDA@CG’s ζ-potential changed to a net-positive one with the decrease in pH from a negative charge (−3.77 ± 0.39 mV) at pH 7.4.37 After GCS modification, AIBI was loaded onto GCS-PDA@ CG as a free-radical-generating agent through coordination and hydrophobic and hydrogen-bond interaction. To select a reaction concentration of AIBI that is economic and efficient, the loading amount of AIBI with various concentrations was studied using TGA (Figure S4). With the increase in AIBI concentration, the loading amount of AIBI increased. When the concentration of AIBI increased to 0.3 mg/mL, 60 μg of AIBI was loaded on 1 mg of AIBI-GCS-PDA@CG. Never-

theless, by further improving the concentration of AIBI, there was very slight change in the loading amount of AIBI. Therein, 0.3 mg/mL was the optimal reaction concentration for AIBI loading. In the AIBI-GCS-PDA@CG spectrum (Figure S1C), we found a novel peak at 1576 cm−1 in comparison with GCSPDA@CG’s FTIR spectrum, as observed in the spectrum of the AIBI alone as well, showing a good presentation of successful AIBI loading on GCS-PDA@CG nanosheets. Compared with GCS-PDA@CG, the TEM and AFM images showed no obvious variation in the morphology and thickness of AIBI-GCS-PDA@CG nanosheets (Figure 2A,B). The thickness of AIBI-GCS-PDA@CG was ∼2.1 nm, indicating that the AIBI-GCS-PDA@CG nanosheets were still nearly single layer. The hydrodynamic size of AIBI-GCS-PDA@CG was assessed by in-depth DLS analysis. According to Figure 2C, AIBI-GCS-PDA@CG exhibited a size of 164−295 nm. Moreover, the ζ-potential results proved that the AIBI-GCSPDA@CG possesses the ability of being pH-triggered chargereversible (Figure 2D), indicating that the AIBI loading could not influence the pH-sensitive performance of our bactericidal depot. 3.2. Optical and Photothermal Properties of AIBIGCS-PDA@CG. Because of its low absorbance by water, blood, and biological tissue,40 an NIR laser of 808 nm in wavelength is considered to be an optimal light source for photothermal therapy (PTT), which can enter the skin up to a depth of 10 nm. An effective PTT reagent should react strongly to near-infrared light that focuses on the target position. With a UV−vis−NIR spectrometer, the optical absorbance of CG, PDA@CG, GCS-PDA@CG, and AIBIGCS-PDA@CG at pH 6.3 (complying with that at the sites of skin abscesses) was studied. As shown in Figure 2E, after the PDA coating, at the same concentration of nanosheet, the absorption value at 808 nm increased significantly, which may be caused by the introduction of lone pair electrons in auxochrome and of auxochrome in PDA (−OH) for increasing the conjugate system of molecules via resonance, thus increasing the electron activity range.41 Note that the GCS grafting could optimize the surface of our bactericidal depot and significantly improved the absorption of NIR light (808 nm) possibly because of the hydrogen-bond interactions between GCS and PDA, improving electrons’ motion.42 The AIBI loading had no obvious effects on the absorption at 808 nm. To further ascertain its photothermal ability, a variety of concentrations of AIBI-GCS-PDA@CG in PBS at pH 6.3 was exposed to 808 nm NIR laser; pure PBS (pH 6.3) served as the control. With an IR thermal camera, we ascertained the temperature profiles of the test samples and their heat maps. The low-power intensities of 0.5 and 0.75 W/cm2 were selected as high-power intensity of NIR laser exposure is likely to lead to skin damage. As expected, our nanomaterial suspensions exhibited both laser energy- and concentrationdependent temperature change (Figures 2F,G and S5). The temperatures of GCS-PDA@CG and AIBI-GCS-PDA@CG solutions increased rapidly in the case of the NIR light (0.5 or 0.75 W/cm2), achieving concentration-dependent plateaus in 7 min. However, the PBS slightly changed in temperature from 28.8 to 31.5 °C (0.5 W/cm2) and 29 to 32.6 °C (0.75 W/cm2) even after 10 min of irradiation. Notably, AIBI-GCS-PDA@ CG’s and GCS-PDA@CG’s photothermal effects showed no obvious difference upon light irradiation. According to these effects, both GCS-PDA@CG and AIBI-GCS-PDA@CG are 1771


Research Article


Figure 3. (A) ζ-Potential of bacteria before and after incubation with AIBI-GCS-PDA@CG at pH 6.3. (B) Representative SEM micrographs of bacteria (pH 6.3 and 7.4) and (C) 3T3 fibroblasts (pH 7.4) before and after incubation with AIBI-GCS-PDA@CG.

trone (POBN). We observed particular alkyl radical (R•) signals with POBN (αN = 15.1 G and αβ‑H = 2.8 G), as shown in Figure 2I. This study plotted the ESR peak intensity to reflect the number of R• versus NIR irradiation time (Figure 2J,K) for monitoring the R• release kinetics. In the initial 3 min, the ESR peak intensity showed a slow increase with irradiation, certifying the temperate rise at this time. Hereafter, the ESR peak increased sharply in the following 6 min, suggesting that AIBI decomposed rapidly under photothermal action. In the end, a phase can be observed in the figure, indicating that most AIBI is exhausted within 11 min. As expected, we did not observe the ESR signals in the group of GCS-PDA@CG or AIBI-GCS-PDA@CG without NIR irradiation (Figure S11). Comparatively, there were no ESR signals in AIBI and GCS-PDA@CG groups, suggesting that AIBIGCS-PDA@CG’s free-radical generation ability results from the plasmonic heating of initiators (Figures 2K and S11). 3.5. pH-Dependent Bacterium-Specific Interactions of AIBI-GCS-PDA@CG. Owing to the high content of anionic phospholipids on the cell walls of bacteria, the surface has a net negative charge;44 however, because of the protonation/ deprotonation of their free amino groups, GCS-PDA@CG and PDA-GCS-PDA@CG have special surface charge switches, which are dependent on their local pH value, and are conducive to their capability to be involved in the bacteriaspecific interaction in the acidic pus of focal infection (pH 6.3) while eliminating their direct contact with neighboring host cells (pH 7.4). To test this, the model bacteria MRSA and S. aureus were treated with synthetic nanomaterials at various pH values (7.4 or 6.3). Through incubating bacteria with GCSPDA@CG or AIBI-GCS-PDA@CG at pH 6.3, the mixture rapidly condenses and precipitates to the tube’s bottom in 30 min (Figure S12). Yet in the pH 7.4 treatment (Figure S12), we saw no such phenomena. The mentioned phenomena possibly resulted from the potent binding ability of GCSPDA@CG or AIBI-GCS-PDA@CG for bacteria through electrostatic force in acidic condition. The ζ-potential results also confirmed these. As presented in Figures 3A and S13A, the pH-responsive GCS-PDA@CG and AIBI-GCS-PDA@CG showed a net-positive surface charge under the aforementioned acidic environment (pH 6.3), causing close electrostatic interaction with the MRSA membrane charged negatively, which converts their surface charge from negative to positive. A larger number of supporting evidence on the acidity-triggered bacterium-targeting properties of GCS-PDA@CG or AIBIGCS-PDA@CG is provided in SEM analysis. GCS-PDA@CG or AIBI-GCS-PDA@CG nanosheets are attached to the bacterial surface at pH 6.3, through the surface of bacteria at pH 7.4 showed no GCS-PDA@CG or AIBI-GCS-PDA@CG nanosheets (Figures 3B and S13B).

able to efficiently convert optical energy into heat. Moreover, AIBI-GCS-PDA@CG’s photothermal conversion (PTC) was ascertained in accordance with the method reported previously.31 The AIBI-GCS-PDA@CG has a high PTC of 41.96%, better than GCS-CG (39.6%) in our previous work43 possibly due to the PDA coating, as shown in Figure S6. Considering that thermal ablation of most microbes happens over 50 °C as well as the skin’s tolerance to the laser power, a 0.2 mg/mL GCS-PDA@CG or AIBI-GCS-PDA@CG concentration with 0.5 W/cm2 laser intensity irradiation for 7 min is applicable to the in vitro experiments. 3.3. Stabilities of AIBI-GCS-PDA@CG. The absorbance and temperature change of AIBI-GCS-PDA@CG were determined before and after irradiation at 0.5 W/cm2 at the concentration of 0.2 mg/mL for 7 min to verify whether heating could damage the morphology of our antibacterial depot. According to Figure S7A, after five cycles of irradiation at 0.5 W/cm2, AIBI-GCS-PDA@CG suspensions’ absorbance (808 nm) loss was below 5%, illustrating AIBI-GCS-PDA@CG had a prominent photothermal stability. After five cycles of laser irradiation (808 nm, 0.5 W/cm2; Figure S7B), the AIBIGCS-PDA@CG suspensions maintained consistent temperature changes as expected, demonstrating the good photothermal stability of AIBI-GCS-PDA@CG. AIBI-GCS-PDA@ CG’s integrity under biological conditions of infection (pH 6.3) must be considered since its final product was created as a therapeutic agent for the treatment of infection. The TGA results indicated that no obvious change was observed after AIBI-GCS-PDA@CG deposited in the acidic medium (pH 6.3, Figure S8). Overall, these results demonstrated that AIBIGCS-PDA@CG presented high photothermal conversion efficiencies and good stability even under acidic condition, which render our bactericidal depot very promising as a photothermal therapeutic agent. Subsequently, the stability of AIBI-GCS-PDA@CG in serum-containing media was evaluated using DLS (Figure S9) and Raman spectroscopy (Figure S10). After being cultured in 10% serum-containing PBS, the hydrodynamic diameter of AIBI-GCS-PDA@CG showed negotiable change with the incubation time increasing, indicating that the nanosheets in serum-containing medium were very stable for at least 7 days. Moreover, the Raman spectra of AIBI-GCSPDA@CG also confirmed the presence of graphene in those 7 days. 3.4. Identification of the Generated Free Radicals. With the electron spin resonance (ESR) technique, free-radical generation ability of AIBI-GCS-PDA@CG was investigated. To capture the highly free radicals, we employed 5,5-dimethyl1-pyrroline N-oxide (DMPO), 2-methyl-2-nitrosopropane dimmer (MNP), and α-(4-pyridyl N-oxide)-N-tert-butylni1772


Research Article


Figure 4. Images of bacterial colony forming units (CFUs) of AIBI (0.10 mg/mL), GCS-PDA@CG (0.10 mg/mL), and AIBI-GCS-PDA@CG (0.10 mg/mL) under (A) normoxic and (B) hypoxic conditions with or without NIR irradiation in vitro. Quantitative analysis of MRSA treated with AIBI (0−0.50 mg/mL), GCS-PDA@CG (0−0.50 mg/mL), and AIBI-GCS-PDA@CG (0−0.50 mg/mL) under (C) normoxic and (D) hypoxic conditions, separately. The images of live/dead staining assay of AIBI (0.20 mg/mL), GCS-PDA@CG (0.20 mg/mL), and AIBI-GCSPDA@CG (0.10 mg/mL) under (E) normoxic and (F) hypoxic conditions, separately. The bacteria were irradiated with NIR laser light (808 nm, 0.5 W/cm2) for 7 min. (G) DCFH-DA determining the OS of MRSA treated with (G1, G4, G7) AIBI (0.20 mg/mL), (G2, G5, G8) GCS-PDA@CG 1773


Research Article

ACS Applied Materials & Interfaces Figure 4. continued

(0.20 mg/mL), and (G3,G6,G9) AIBI-GCS-PDA@CG (0.20 mg/mL) immediately and with light irradiation under (G1−G3) normoxic and (G4− G9) hypoxic conditions for 4 h. Scale bar: 50 μm. (H) GSH depletion measurement using Ellman’s reagents. MRSA were treated with PBS, AIBI (0.20 mg/mL), GCS-PDA@CG (0.20 mg/mL), and AIBI-GCS-PDA@CG (0.20 mg/mL) (808 nm, 0.5 W/cm2, 7 min), separately.

= 15.410 G and αβ‑H = 24.841 G), but under air environment, four signal peaks with intensity ratio of 1:2:2:1 (αN = 14.145 G and αβ‑H = 14.605 G) were observed, which can be assigned as alkoxyl radical (RO•) signals. This can be explained by the fact that DMPO compared with oxygen exhibits a slower reaction rate with R•. In addition, R• prefers to react with oxygen to form RO•; then, RO• is captured by DMPO under air environment. However, POBN and MNP react faster with R• than oxygen, and as a result, R• is captured by POBN and MNP before the formation of RO•. Overall, we can conclude that the generation of R• is oxygen independent and can easily be transferred to RO• by oxygen under air environment because of its high reactivity. As a fluorescent probe, dichlorofluorescein diacetate (DCFH-DA) was used for assessing the intracellular ROS level to further assess the OS effects of AIBI-GCS-PDA@CG. Only AIBI-GCS-PDA@CG showed dramatic fluorescence, as shown in Figure 4G3, suggesting that the generated free radicals can lead to OS in the case of normoxia. All three materials showed weak ability to induce OS immediately after light irradiation under hypoxia (Figure 4G4−G6). When bacteria were further cultured for 4 h, green fluorescence was seen in the AIBI-GCS-PDA@CG group (Figure 4G9), suggesting that the produced R• under hypoxia could induce cell OS as well but showing a distinct system. Antioxidant systems, for example, glutathione (GSH) in bacteria cells, are vital for reducing the damaging effect of other oxidizers like free radicals. Therefore, we assumed that R• could directly consume GSH in bacteria cells due to its high reactivity. GSH depletion will damage the redox balance of the bacteria and cause OS. In this regard, the GSH consumption ability of AIBI-GCS-PDA@CG was ascertained under various tensions of oxygen with Ellman’s reagent. According to Figure 4H, only AIBI-GCS-PDA@CG (0.2 mg/mL) under the laser irradiation group indicated remarkable ability of GSH depletion, which was not obviously (p > 0.05) influenced by various oxygen tensions, indicating that both R• and RO• free radicals could cause GSH consumption and OS. By a supercoiled (SC) plasmid pUC 18 DNA assay, this paper also assessed the intracellular DNA-damaging activity of our bactericidal depot. After the single strands were cleared by free radicals, the plasmid pUC 18 DNA can be covered from the supercoiled (SC) form to the open circular (OC) form, which can be distinguished using agarose gel electrophoresis. As shown in Figure S16A,B, the SC form was effectively covered to the OC form in AIBI-GCS-PDA@CG under the laser irradiation group, especially under hypoxic condition; however, in the conditions of AIBI and GCS-PDA@CG (808 nm, 0.5 W/cm2), the DNA still remained in the SC form, indicating that the AIBI or GCS-PDA@CG alone could not induce the single-strand breaking of DNA. These results indicated that the generated alkyl radicals (R•) could effectively cleave the DNA strands under both normoxic and hypoxic conditions, indicating the high DNA-damaging activity of AIBI-GCS-PDA@CG in bacteria. 3.7. In Vivo Bacterium-Targeting Ability and Antibacterial Activity. Inspired by the significant therapeutic

Additionally, the test AIBI-GCS-PDA@CG or GCS-PDA@ CG nanosheets had a little negative charge and were not attached to the cultured cells charged negatively when exposed to 3T3 fibroblasts at 7.4the pH of host cells under physiological conditions (Figure 3C). The above results reveal that our bactericidal depots can be used for the specific targeting of various bacteria in the acidic environment of local infection. 3.6. In Vitro Antimicrobial Property. The free-radical generation ability of AIBI-GCS-PDA@CG has been confirmed, and its therapeutic potential under normoxic (21% O2) and hypoxia (1% O2) conditions was examined using MRSA bacteria. As presented in Figure 4A,B, the materials showed no antibacterial effect. Under NIR irradiation, the AIBI group still exhibited no bactericidal effects. Taking GCS-PDA@CG and AIBI-GCS-PDA@CG as comparison, higher antibacterial effect was observed in the AIBI-GCS-PDA@CG group, in particular at a concentration of 0.2 mg/mL; the antibacterial efficiency was ∼25% for the GCS-PDA@CG group, whereas it was about 100% for the AIBI-GCS-PDA@CG group, revealing that the bactericidal effect of AIBI-GCS-PDA@CG ( 0.05). On the contrary, the GCS-PDA@CG + NIR group and the AIBIGCS-PDA@CG + NIR group suggested excellent antibacterial effects. More importantly, the AIBI-GCS-PDA@CG + NIR group showed excellent in vivo antibacterial activity (since their CFU count decreased to merely 1% of that of the untreated control group, p < 0.05), and this decrease was significantly larger than that of the GCS-PDA@CG + NIR group (p < 0.05), indicating the efficient antibacterial effect of PDT in the treatment of abscess. Moreover, histological analysis in skin tissue infected by the model bacteria MRSA was further conducted by H&E staining. The skin with MRSA infection still displayed a significant infected skin lesion with a huge amount of aggregated inflammatory cells compared with the healthy skin, as shown in Figure 6D. However, the group of AIBI-GCS-PDA@CG + NIR had a considerably decreased inflammatory cells’ infiltration. These results suggest that therapy of free radicals combined with photothermal therapy can serve as a good alternative therapeutic agent for treatment in vivo. 1777


Research Article


Figure 8. Hematology and blood biochemistry analyses of healthy BALB/c mice sacrificed at 30 days intravenously injected with AIBI-GCS-PDA@ CG at a concentration of 0.2 or 0.5 mg/mL (n = 5). The PBS-treated ones were considered as controls. (A) White blood cells, (B) red blood cells, (C) blood urea nitrogen, (D) platelets, (E) hemoglobin, (F) creatinine (Cr), (G) alanine aminotransferase, (H) aspartate aminotransferase, (I) albumin, (J) lactate dehydrogenase, (K) α-HBDH, and (L) CK-MB. NS denotes no significant difference (p > 0.05).

Considering these issues together, we developed NIR lightactivatable bactericidal depots as well as bacterial-specific targeting and assessed their efficacy to eradicate the subcutaneous MRSA infection in the mouse model in Figure 1. Our sterilization warehouse is given a shell comprising glycol units in GCS, significantly avoiding their interaction with proteins in the biological liquid and making the warehouse biologically invisible at physiological pH 7.4. If we reach the site of bacterial infection and create an environment that is more acidic, the bactericidal warehouse here exposes the free amino acid groups in the shell, forming a network charged positively with strong interaction, with bacterial cell surface charged negatively to prevent scour and keep it there. The PDA@CG acted as the photothermal agent; the 808 nm light activation is going to generate more heat and subsequently produce the alkane free radical (R•) to eliminate the bacteria under the hypothermia conditions mainly through damaging the intracellular DNA and depleting the intracellular GSH. After the comprehensive consideration, these unique characteristics so that our sterilization warehouse completely ablation table infection. The center here is that the bactericidal efficiency of the lightactivated AIBI-GCS-PDA@CG was significantly higher than that of the light-activated GCS-PDA@CG (Figure S20) or the heat-activated AIBI alone (Figure S21). This enhancement effect is partly due to the synergistic effect of photothermal and free-radical sterilization. As shown in Figures 4 and S16, the main bactericidal effect for MRSA is caused by generated free

caused by AIBI-GCS-PDA@CG in mice. Overall, the excellent biocompatibility of AIBI-GCS-PDA@CG makes it a promising antibacterial agent for biomedical applications.

4. DISCUSSIONS Bacterial infections, especially rapid emergence of resistance of drug, has turned into one of the world’s largest health problems and poses a huge medical and financial burden.46−48 For example, methicillin-resistant S. aureus (MRSA) is one of the most feared pathogenic bacteria (superbugs) clinically, which can lead to life-threatening diseases, for example, septicemia and acute endocarditis, with high morbidity and mortality clinically.49−52 Photodynamic therapy (PDT) is known for its characteristics of minimally invasive and high microflora localization, and it is a promising supplement to traditional bacteria and even multidrug-resistant bacteria therapy in recent years.53 In general, PDT includes the production of reactive oxygen species (ROS) (mostly 1O2, • OH) activated by PSs, which induces cell death and vascular injury.54 However, some of the serious bacterial infections, particularly the inner organ infections (i.e., lung, liver), are located a few millimeters below the human skin. Owing to the limited penetration depth of tissues, roughly all PSs cannot be effectively stimulated by ultraviolet or visible light, leading to the unsatisfactory clearance efficiency of bacteria. Moreover, mostly of the environment of bacterial infection diseases are hypoxia,55 the therapeutic efficacy of the traditional PSs may be limited by oxygen availability of surrounding molecules. 1778



■

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (51673171, 31500789), the Science and Technology Innovation Plan of Southwest Hospital (Nos. SWH2016ZDCX2014, SWH2016JCYB-04, and SWH2017ZDCX1001), the Third Military Medical University (Grant No. 2016XPY12), The Chongqing Yuzhong District Science and Technology Plan Project grants (20170124), Nature Science Foundation of Chongqing, China (Nos. cstc2018jcyjAX0807, cstc2017jcyjAX0020, and cstc2015shmszx00012), and Program for Innovation Team Building at Institutions of Higher Education (No. CXTDG201602006) funded by the Chongqing Municipal Education Commission of China in 2016.

radicals, whereas the antibacterial effect of the photothermal action (GCS-PDA@CG + NIR) alone was very weak (bactericidal efficiency 24.3%). Likely, compared to the heatactivated AIBI alone, our pH-adaptive bactericidal library is directly targeted at the bacterial cell surface, which is one of the reasons for improving the antibacterial effect. Previous work also observed that R• or RO• was very short lived,56 which possibly possesses not enough time to reach the bacteria, resulting in significantly reduced bactericidal efficiency. More importantly, the strong antibacterial effect caused by our bactericidal depots was similar under the normal oxygen and hypoxia conditions, indicating that our bactericidal depots could overcome the hypothermia of the subcutaneous. Compared to previous strategies of self-generation of oxygen through the catalytic water/H2O2 in the bacterial infection sites54,57 or delivering O2 to the diseases sites58 to overcome the hyperthermia, our novel strategy did not change the hypothermia environment of the subcutaneous layer, which is beneficial to wound healing.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b12873.

■

REFERENCES

(1) Levin, B. R.; Antia, R. Why We Don’t Get Sick: The WithinHost Population Dynamics of Bacterial Infections. Science 2001, 292, 1112−1125. (2) Andersson, D. I.; Hughes, D. Antibiotic Resistance and its Cost: Is It Possible To Reverse Resistance? Nat. Rev. Microbiol. 2010, 8, 260−271. (3) Shankar, P. R. Book review: Tackling Drug-Resistant Infections Globally. Arch. Pharma. Pract. 2016, 7, 110−111. (4) Stewart, P. S.; Costerton, J. W. Antibiotic Resistance of Bacteria In Biofilms. Lancet 2001, 358, 135−138. (5) Taubes, G. The Bacteria Fight Back. Science 2008, 321, 356− 361. (6) Mah, T.-F.C.; O’Toole, G. A. Mechanisms of Biofilm Resistance To Antimicrobial Agents. Trends Microbiol. 2001, 9, 34−39. (7) Jenkins, R. R. Free Radical Chemistry. Sports Med. 1988, 5, 156− 170. (8) Land, E. T. Free Radicals In Biology and Medicine. Int. J. Radiat. Biol. 1990, 58, 725. (9) Wang, X. Q.; Gao, F.; Zhang, X. Z. Initiator Loaded Gold Nanocages As Light-Induced Free Radical Generator For Cancer Therapy. Angew. Chem., Int. Ed. 2017, 56, 9029−9033. (10) Shen, S.; Zhu, C.; Huo, D.; Yang, M.; Xue, J.; Xia, Y. A Hybrid Nanomaterial For the Controlled Generation of Free Radicals and Oxidative Destruction of Hypoxic Cancer Cells. Angew. Chem., Int. Ed. 2017, 56, 8801−8804. (11) Huang, X.; Chen, G.; Pan, J.; Chen, X.; Huang, N.; Wang, X.; Liu, J. Effective PDT/PTT Dual-Modal Phototherapeutic Killing of Pathogenic Bacteria by Using Ruthenium Nanoparticles. J. Mater. Chem. B 2016, 4, 6258−6270. (12) Gao, M.; Hu, Q.; Feng, G.; Tomczak, N.; Liu, R.; Xing, B.; Tang, B. Z.; Liu, B. A Multifunctional Probe with AggregationInduced Emission Characteristics for Selective Fluorescence Imaging and Photodynamic Killing of Bacteria Over Mammalian Cells. Adv. Healthcare Mater. 2015, 4, 659−663. (13) Feng, G.; Yuan, Y.; Fang, H.; Zhang, R.; Xing, B.; Zhang, G.; Zhang, D.; Liu, B. A Light-Up Probe with Aggregation-Induced Emission Characteristics (AIE) for Selective Imaging, Naked-Eye Detection and Photodynamic Killing of Gram-Positive Bacteria. Chem. Commun. 2015, 51, 12490−12493. (14) Chen, H.; Tian, J.; He, W.; Guo, Z. H2O2-Activatable and O2Evolving Nanoparticles for Highly Efficient and Selective Photodynamic Therapy Against Hypoxic Tumor Cells. J. Am. Chem. Soc. 2015, 137, 1539−1547. (15) Gao, S.; Wang, G.; Qin, Z.; Wang, X.; Zhao, G.; Ma, Q.; Zhu, L. Oxygen-Generating Hybrid Nanoparticles to Enhance Fluorescent/ Photoacoustic/Ultrasound Imaging Guided Tumor Photodynamic Therapy. Biomaterials 2017, 112, 324−335. (16) Dolmans, D. E. J. G. J.; Dai, F.; Jain, R. K. Photodynamic Therapy for Cancer. Nat. Rev. Cancer 2003, 3, 380−387. (17) Pabst, B.; Pitts, B.; Lauchnor, E.; Stewart, P. S. Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit

5. CONCLUSIONS To sum up, a novel therapeutic strategy based on free radicals’ light-induced generation is proposed for drug-resistant bacteria therapy. Combining the photothermal effect of our bactericidal depot with the thermal decomposability of AIBI, AIBI-GCSPDA@CG could produce free radicals in the case of NIR light. The produced free radicals show equivalent therapeutic efficacy under hypoxia and normoxia yet with various mechanisms. On exposure to normoxia, the generated alkyl radicals (R•) are able to undergo conversion into alkoxyl radicals (RO•) by oxygen, as shown in Figures 1C and S15, directly causing bacteria damage. Under hypoxia, the produced alkyl radicals (R•) also show DNA-damaging activity. This is a cause of bacterial death. Besides, the AIBI-GCS-PDA@CG has a significant therapeutic effect on drug-resistant bacteria in vivo. The results show that this method provides the ability and possibility for the application of phototherapy under more complex conditions and is of great value for subsequent research.

■

Research Article

FTIR, TEM, AFM, SEM, ζ-potential, thermogravimetric analysis, photothermal effect, hydrodynamic diameter, Raman spectra, EPR analysis, DNA-damaging activity, fluorescence hemolysis activity of the material assessment (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.Z.). *E-mail: [email protected] (G.L.). *E-mail: [email protected] (J.D.). ORCID

Dezhi Shi: 0000-0003-4452-1855 Jun Deng: 0000-0002-9951-2393 Notes

The authors declare no competing financial interest. 1779


Research Article


2,2′-Azobis(2-Amidinopropane) Hydrochloride (AAPH). Free Radical Res. Commun. 1993, 19, 323−332. (35) Hong, S.; Kim, K. Y.; Wook, H. J.; Park, S. Y.; Kang, D. L.; Dong, Y. L.; Lee, H. Attenuation of the In Vivo Toxicity of Biomaterials by Polydopamine Surface Modification. Nanomedicine 2011, 6, 793−801. (36) Dreyer, D. R.; Miller, D. J.; Freeman, B. D.; Paul, D. R.; Bielawski, C. W. Elucidating the Structure of Poly(dopamine). Langmuir 2012, 28, 6428−6435. (37) Korupalli, C.; Huang, C. C.; Lin, W. C.; Pan, W. Y.; Lin, P. Y.; Wan, W. L.; Li, M. J.; Chang, Y.; Sung, H. W. Acidity-Triggered Charge-Convertible Nanoparticles that can Cause Bacterium-Specific Aggregation In Situ to Enhance Photothermal Ablation of Focal Infection. Biomaterials 2017, 116, 1−9. (38) Liu, Y.; Ai, K.; Lu, L. Polydopamine and Its Derivative Materials: Synthesis and Promising Applications In Energy, Environmental, and Biomedical Fields. Chem. Rev. 2014, 114, 5057−5115. (39) Lynge, M. E.; Schattling, P.; Städler, B. Recent Developments In Poly(dopamine)-Based Coatings for Biomedical Applications. Nanomedicine 2015, 10, 2725−2742. (40) Lapotko, D. O. Nanophotonics and Theranostics: Will Light Do the Magic? Theranostics 2013, 3, 138−140. (41) Matsui, M.; Yamamoto, T.; Kakitani, K.; Biradar, S.; Kubota, Y.; Funabiki, K. UV−vis Absorption and Fluorescence Spectra, Solvatochromism, and Application to pH Sensors of Novel Xanthene Dyes Having Thienyl and Thieno[3,2- b ]thienyl Rings As Auxochrome. Dyes Pigm. 2017, 139, 533−540. (42) Marcasuzaa, P.; Reynaud, S.; Ehrenfeld, F.; Khoukh, A.; Desbrieres, J. Chitosan-Graft-Polyaniline-Based Hydrogels: Elaboration and Properties. Biomacromolecules 2010, 11, 1684−1691. (43) Qian, W.; Yan, C.; He, D.; Yu, Y.; Yuan, L.; Liu, M.; Luo, G.; Deng, J. pH-triggered Charge-Reversible of Glycol Chitosan Conjugated Carboxyl Graphene for Enhancing Photothermal Ablation of Focal Infection. Acta Biomater. 2018, 69, 256−264. (44) Korupalli, C.; Huang, C. C.; Lin, W. C.; Pan, W. Y.; Lin, P. Y.; Wan, W. L.; Li, M. J.; Chang, Y.; Sung, H. W. Acidity-Triggered Charge-Convertible Nanoparticles that can Cause Bacterium-Specific Aggregation In Situ to Enhance Photothermal Ablation of Focal Infection. Biomaterials 2017, 116, 1−9. (45) Wahl, R. U. R.; Zeng, L.; Madison, S. A.; Depinto, R. L.; Shay, B. J. Mechanistic Studies on the Decomposition of Water Soluble Azo-Radical-Initiators. J. Chem. Soc., Perkin Trans. 2 1998, 1998, 2009−2018. (46) Smith, P. A.; Romesberg, F. E. Combating Bacteria and Drug Resistance by Inhibiting Mechanisms of Persistence and Adaptation. Nat. Chem. Biol. 2007, 3, 549−556. (47) Zhang, Q.; Austin, R. H.; et al. Acceleration of Emergence of Bacterial Antibiotic Resistance In Connected Microenvironments. Science 2011, 333, 1764−1767. (48) Chait, R.; Craney, A.; Kishony, R. Antibiotic Interactions that Select Against Resistance. Nature 2007, 446, 668−671. (49) Koch, G.; Yepes, A.; Förstner, K. U.; Wermser, C.; Stengel, S. T.; Modamio, J.; Ohlsen, K.; Foster, K. R.; Lopez, D. Evolution of Resistance to A Last-Resort Antibiotic In Staphyloccocus aureus via Bacterial Competition. Cell 2014, 158, 1060−1071. (50) Li, X.; Robinson, S. M.; Gupta, A.; Saha, K.; Jiang, Z.; Moyano, D. F.; Sahar, A.; Riley, M. A.; Rotello, V. M. Functional Gold Nanoparticles As Potent Antimicrobial Agents Against Multi-DrugResistant Bacteria. ACS Nano 2014, 8, 10682−10686. (51) Levy, S. B.; Marshall, B. Antibacterial Resistance Worldwide: Causes, Challenges and Responses. Nat. Med. 2004, 10, S122. (52) Cha, S. H.; Hong, J.; Mcguffie, M.; Yeom, B.; Vanepps, J. S.; Kotov, N. A. Shape-Dependent Biomimetic Inhibition of Enzyme by Nanoparticles and Their Antibacterial Activity. ACS Nano 2015, 9, 9097−9105. (53) Dolmans, D. E. J. G. J.; Dai, F.; Jain, R. K. TIMELINE: Photodynamic Therapy for Cancer. Nat. Rev. Cancer 2003, 3, 380− 387.

Growth In Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression. Antimicrob. Agents Chemother. 2016, 60, 6294−6301. (18) Colmone, A. Hypoxic Conditioning of Immune Cells. Science 2017, 355, 706. (19) James, G. A.; Ge, Z. A.; Usui, M.; Underwood, R. A.; Nguyen, H.; Beyenal, H.; Delancey, P. E.; Agostinho, H. A.; Bernstein, H. C.; Fleckman, P.; et al. Microsensor and Transcriptomic Signatures of Oxygen Depletion In Biofilms Associated with Chronic Wounds. Wound Repair Regener. 2016, 24, 373−383. (20) Mao, C.; Xiang, Y.; Liu, X.; Cui, Z.; Yang, X.; Li, Z.; Zhu, S.; Zheng, Y.; Yeung, K. W. K.; Wu, S. Repeatable Photodynamic Therapy with Triggered Signaling Pathways of Fibroblast Cell Proliferation and Differentiation to Promote Bacteria-Accompanied Wound Healing. ACS Nano 2018, 12, 1747−1759. (21) Jeong, S.; Lee, J.; Im, B. N.; Park, H.; Na, K. Combined Photodynamic and Antibiotic Therapy for Skin Disorder via LipaseSensitive Liposomes with Enhanced Antimicrobial Performance. Biomaterials 2017, 141, 243−250. (22) Cao, F.; Ju, E.; Yan, Z.; Wang, Z.; Liu, C.; Wei, L.; Huang, Y.; Kai, D.; Ren, J.; Qu, X. An Efficient and Benign Antimicrobial Depot Based On Silver-Infused MoS2. ACS Nano 2017, 11, 4651−4659. (23) Liao, W.; Ning, Z.; Chen, L.; Wei, Q.; Yuan, E.; Yang, J.; Ren, J. Intracellular Antioxidant Detoxifying Effects of Diosmetin on 2,2Azobis(2-Amidinopropane) Dihydrochloride (AAPH)-Induced Oxidative Stress Through Inhibition of Reactive Oxygen Species Generation. J. Agric. Food Chem. 2014, 62, 8648−8654. (24) Goenka, S.; Sant, V.; Sant, S. Graphene-Based Nanomaterials for Drug Delivery and Tissue Engineering. J. Controlled Release 2014, 173, 75−88. (25) Yang, K.; Feng, L.; Liu, Z. Stimuli Responsive Drug Delivery Systems Based On Nano-Graphene for Cancer Therapy. Adv. Drug Delivery Rev. 2016, 105, 228−241. (26) Qian, W.; Yan, C.; He, D.; Yu, Y.; Yuan, L.; Liu, M.; Luo, G.; Deng, J. pH-Triggered Charge-Reversible of Glycol Chitosan Conjugated Carboxyl Graphene for Enhancing Photothermal Ablation of Focal Infection. Acta Biomater. 2018, 69, 256−264. (27) Lee, Y. D.; Lim, C. K.; Kim, S.; Kwon, I. C.; Kim, J. SquaraineDoped Functional Nanoprobes: Lipophilically Protected NearInfrared Fluorescence for Bioimaging. Adv. Funct. Mater. 2010, 20, 2786−2793. (28) Wu, W.; Lee, S. Y.; Wu, X.; Tyler, J. Y.; Wang, H.; Ouyang, Z.; Park, K.; Xu, X. M.; Cheng, J. X. Neuroprotective Ferulic Acid (FA)Glycol Chitosan (GC) Nanoparticles for Functional Restoration of Traumatically Injured Dpinal Cord. Biomaterials 2014, 35, 2355− 2364. (29) Yan, L.; Crayton, S. H.; Thawani, J. P.; Amirshaghaghi, A.; Tsourkas, A.; Cheng, Z. A pH-Responsive Drug-Delivery Platform Based on Glycol Chitosan-Coated Liposomes. Small 2015, 11, 4870− 4874. (30) Li, B.; Wang, Q.; Zou, R.; Liu, X.; Xu, K.; Li, W.; Hu, J. Cu7.2S4 Nanocrystals: A Novel Photothermal Agent With A 56.7% Photothermal Conversion Efficiency for Photothermal Therapy of Cancer Cells. Nanoscale 2014, 6, 3274−3282. (31) Tian, Q.; Jiang, F.; Zou, R.; Liu, Q.; Chen, Z.; Zhu, M.; Yang, S.; Wang, J.; Wang, J.; Hu, J. Hydrophilic Cu9S5 Nanocrystals: A Photothermal Agent with A 25.7% Heat Conversion Efficiency for Photothermal Ablation of Cancer Cells In Vivo. ACS Nano 2011, 5, 9761−9771. (32) Cowan, K. H.; Batist, G.; Tulpule, A.; Sinha, B. K.; Myers, C. E. Similar Biochemical Changes Associated with Multidrug Resistance In Human Breast Cancer Cells and Carcinogen-Induced Resistance to Xenobiotics In Rats. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 9328− 9332. (33) Hammond, S. M. An Overview of microRNAs. Adv. Drug Delivery Rev. 2015, 87, 3−14. (34) Hiramoto, K.; Johkoh, H.; Sako, K.; Kikugawa, K. DNA Breaking Activity of the Carbon-Centered Radical Generated From 1780


Research Article

ACS Applied Materials & Interfaces (54) Zhang, M.; Cui, Z.; Song, R.; Lv, B.; Tang, Z.; Meng, X.; Chen, X.; Zheng, X.; Zhang, J.; Yao, Z.; Bu, W. SnWO4-Based Nanohybrids with Full Energy Transfer for Largely Enhanced Photodynamic Therapy and Radiotherapy. Biomaterials 2018, 155, 135−144. (55) Allen, D. B.; Maguire, J. J.; Mahdavian, M.; Wicke, C.; Marcocci, L.; Scheuenstuhl, H.; Chang, M.; Le, A. X.; Hopf, H. W.; Hunt, T. K. Wound Hypoxia and Acidosis Limit Neutrophil Bacterial Killing Mechanisms. Arch. Surg. 1997, 132, 991−996. (56) Akaike, T.; Sato, K.; Ijiri, S.; Miyamoto, Y.; Kohno, M.; Ando, M.; Maeda, H. Bactericidal Activity of Alkyl Peroxyl Radicals Generated by Heme-Iron-Catalyzed Decomposition of Organic Peroxides. Arch. Biochem. Biophys. 1992, 294, 55−63. (57) Gao, L.; Liu, Y.; Kim, D.; Li, Y.; Hwang, G.; Naha, P. C.; Cormode, D. P.; Koo, H. Nanocatalysts Promote Streptococcus mutans Biofilm Matrix Degradation and Enhance Bacterial Killing to Suppress Dental Caries In Vivo. Biomaterials 2016, 101, 272−284. (58) Wang, P.; Li, X.; Yao, C.; Wang, W.; Zhao, M.; Eltoni, A. M.; Zhang, F. Orthogonal Near-Infrared Upconversion Co-Regulated Site-Specific O2 Delivery and Photodynamic Therapy for Hypoxia Tumor by Using Red Blood Cell Microcarriers. Biomaterials 2017, 125, 90−100.

1781


Surface-Adaptive and Initiator-Loaded Graphene as a Light-Induced

Recommend Documents