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

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Biological and Medical Applications of Materials and Interfaces

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, Just Accepted Manuscript • DOI: 10.1021/acsami.8b12873 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 8, 2018

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ACS Applied Materials & Interfaces

Surface-Adaptive and Initiator-Loaded Graphene as a Light-Induced Generator with Free Radicals for Drug-Resistant Bacteria Eradication Xunzhou Yu1, Danfeng He1, Ximu Zhang2*, Hongmei Zhang2, Jinlin Song2, Dezhi Shi3, Yahan Fan4, Gaoxing Luo1*, Jun Deng1* 1. Institute of Burn Research, Southwest Hospital, State Key Lab of Trauma, Burn and Combined Injury, Army Medical University, Chongqing 400038, China. 2. 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. 3. Key Laboratory of Three Gorges Reservoir Region's Eco-Environment, Ministry of Education, Faculty of Urban Construction and Environmental Engineering, Chongqing University, Chongqing 40005, PR China. 4. Department of Blood Transfusion, Southwest Hospital, Army Medical University, Chongqing 400038, China. * Corresponding authors. E-mail

addresses:

[email protected]

(X.

Zhang),

[email protected] (J. Deng)

1

ACS Paragon Plus Environment

[email protected]

(G.

Luo),

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Since generating toxic reactive oxygen (ROS) is largely dependent on oxygen, bacteria infected wounds hypoxia significantly inhibits photodynamic therapy’s (PDT) 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 (NIR) irradiation. As a result, oxidative-stress (OS) can be elevated, DNA damages in bacteria can be caused, and finally the bacteria even the 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, the researcher proves that combination oxygen-independent free radical based therapy along with a stimulus-responsiveness moiety not only can be used as an effective treatment of the multidrug-resistant bacteria infection, but also creates a use of a variety of free radicals for multidrug-resistant bacteria infection wounds treatment. Keywords: Hypoxia; Free radicals; pH-responsive; Drug-resistant bacteria; Graphene.

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1 Introduction Bacterial infectious disease, in particular multi-drug resistance, has turned into the world's big health problem and attracted people's wide concern1, 2. Recently, One study suggests that failure to control drug-resistant bacterial infections can cause the death of more than 10 million patients a year by 2050 and cause losses of up to $100 trillion3. However, at a time of slow antimicrobial development, the world could one day face a pandemic of incurable bacterial infections that could be catastrophic in severity

4, 5, 6

. Such worry boosts the improvement of alternative efficient therapeutic

approaches to treat drug-resistant bacteria infections. Free radicals are mostly high reactive molecular or molecules fragments with valence electrons unpaired7. Generally, free radicals, especially the oxygen related radicals, have wide existence and keep homeostasis in living organisms8. Free radicals are positive for cell metabolism during the normal state; yet the excessive of free radicals can have a direct interaction/reaction with lipids, proteins, and DNA to induce cell dysfunction8. Thus, the free radicals have been applied in cancer therapy9, 10 and bacteria eradication11, 12, 13. Photodynamic therapy (PDT) is recognized the best strategy for the current free radical treatment model due to its tumor that is invasive

14, 15

or bacteria localized

features11, 12, 13. In general, light energy in PDT can undergo the transfer by photo-sensitizers (PSs) to produce reactive oxygen species (ROS) (e.g., 1O2, ·OH) for ablating of tumor or bacteria16. Nevertheless, ROS generation in PDT largely depends on oxygen, significantly limiting the therapeutic effects in hypoxic bacteria infected wounds17, 18, 19. Thus, many efforts have been tried to overcome these problems, such as self-generation of O2 and combining PSs with other therapeutic agents20, 21, 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,

3


21, 22

.


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Carbon-based free radicals are also highly reactive and can be formed without oxygen. However, controlling these radicals’ generation at the bacteria infection sites still remains a great challenge. Thermal degradation azo initiator is a famous free radical generator, which can be used for free radical polymerization and to induce 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 the agents of photothermal conversion that have been broadly explored, showing excellent biocompatibility24, 25, 26. Additionally, as a class of two-dimensional (2D) nanomaterials, graphene has aroused wide concern in drug delivery fields due to its large specific surface area 24,

25, 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 Fig. 1, the 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 charge switchable (pKa ~ 6.5)27,

28,

29

.

Finally,

the

researcher

selected

an

initiator

of

2,

2-azobis(2-(2-imidazolin-2yl)propane)dihydrochloride (AIBI) as the source of radicals and loaded it on the GCS-PDA@CG for the formation of an 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 therapy mechanism of the generated free radicals were assessed. Based on this system, a novel

4


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oxygen-independent strategy is developed for multidrug-resistant ablation, and the potential of many free radicals for biomedical use is revealed. 2 Experimental sections 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 (C8H11O2N·HCl, DA,＞ 98%), glycol chitosan (GCS, ≥ 60% deacetylation), α-(4-Pyridyl N-oxide)-N-tert-butylnitrone (POBN, 99%).

Sigma-Aldrich (USA) 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). If not specially mentioned, all the rest of chemicals belonged to analytical grade and were applied without being purified. MilliQ water was applied during the whole experiments. Cy5 NHS Ester (Cy5-SE) was provided by MedChem Express Co., Ltd. (Shanghai, China). Multidrug-resistant S. aureus (MRSA, ATCC 43300) and Staphylococcus aureus (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.

5



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 disposals followed ethical principle of the Institutional Animal Care and Use Committee of Army Medical University. 2.2 Synthesize of AIBI-GCS-PDA@CG Synthesize of PDA modified CG (PDA@CG). Briefly, we introduced 2 mg of CG into dopamine solution in 20 mL (0.2 mg·mL-1, pH 8.5, 10 mM Tris-HCl). After 30 min sonication, we concentrated and collected PDA@CG by centrifugation (15000 rpm, 60 min). Preparation of GCS coated PDA@CG (GCS-PDA@CG). By the chemical reaction between quinone group in PDA and amine group in GCS, the GCS was coated on the PDA@CG. Typically, we dissolved GCS in 50 mg first in 10 mL of 0.1 M HCl. Subsequently, 0.4 mL of GCS (5 mg·mL -1) 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 oC. Then, the GCS-PDA@CG was obtained by centrifugation. Loading GCS-PDA@CG with AIBI (AIBI-GCS-PDA@CG). Briefly, 10 mg of AIBI (5 mg·mL-1) was mixed with the GCS-PDA@CG solutions concentrated under stirring for 3 h at 30 oC. After that, the free AIBI molecules were removed three times for 60 min by centrifugation at 15000 rpm and 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 microscopy (TEM-1400 PLUS, Japan). The hydrodynamic diameters of samples were measured by DLS

6


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(ZEN5600, Malvern Instruments, UK). The content of PDA, GCS and AIBI on AIBI-GCS-PDA@CG was acquired by using a thermogravimetric analysis (TGA, TG-Q500) in N2 environment. Under an IR Prestige-21 infrared spectrophotometer (Shimadzu, Japan), the researcher recorded Fourier transform infrared (FTIR) spectra . To analyze the pH-dependent changes of GCS-PDA@CG’s and AIBI-GCS-PDA@CG’s surface charges, the researcher dispersed test samples in phosphate buffer saline (PBS, pH 5.0-7.4, 100 μg/mL) and measured their zeta potentials with a Zetasizer (ZEN5600, Malvern Instruments, UK). With the use of a UV-vis-NIR spectrophotometer (UV-3600 SHIMADZU), the researcher recorded the UV-vis-NIR optical properties of CG, PDA@CG, GCS-PDA@CG and AIBI-GCS-PDA@CG in PBS at pH 6.3. To elucidate GCS-PDA@CG’s and AIBI-GCS-PDA@CG’s photothermal abilities, the researcher dispersed test samples in PBS at different concentrations (0-1 mg·mL-1) at pH 6.3 in a 96-well plate and subsequently under the irradiation with 808-nm NIR laser (VLSM-808-B, Connet). The temperature of the suspensions above was monitored by using a thermocouple thermometer (FLIR, Sweden). The NIRF imaging and biodistribution in vivo were visualized using IVIS Lumina II (Xenogen IVIS Spectrum, Perkin Elmer). Finally, the researcher calculated the photothermal conversion efficiency (η) with the method reported.30,

31

The Raman

spectra of graphene composites were recorded using a LabRAMHR800 spectrometer (Horiba JobinYvon,

France).

To

synthesis

the

fluorescence

labeled

AIBI-GCS-PDA@CG

(f-

AIBI-GCS-PDA@CG), the researcher dissolved 2 mg of Cy5-SE in dimethyl sulfoxide (DMSO, 4 mL). Subsequently, the researcher added the prepared Cy5-SE solution to 4 mL of AIBI-GCS-PDA@CG suspension. For the removal of the free Cy5-SE, after about 4 h stirring, the researcher centrifuged the mixture 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

7



spectrophotometer (RF-5310PC, Japan). 2.4 The electron spin resonance (ESR) measurements The ESR signal was detected by using an ESR spectrometer (Bruker X-band A200). In brief, the solution of AIBI-GCS-PDA@CG (0.2 mg·mL-1) 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. 200 μL of AIBI-GCS-PDA@CG (1 mg·mL-1) and POBN (100 mM) were 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 at a certain time, 200 μL of the solution underwent transfer to a quartz capillary tube and was detected by ESR spectrometer (Bruker X-band A200). Control experiments were conducted in parallel with PBS (pH 6.3, 0.2 mg·mL-1), AIBI (0.2 mg·mL-1) and GCS-PDA@CG (0.2 mg·mL-1), respectively. 2.5 Hemolysis Assay of AIBI-GCS-PDA@CG Resh human blood was used in the experiment, and the blood sample is from the Southwest Hospital, Third Military Medical University with the consent of patient. The erythrocytes were collected via centrifugation (1500 rpm, 15 min) and washed with saline three times. Next, centrifuged erythrocytes (3 mL) were added into saline (11 mL) to prepare the stock dispersion, and 200 μL of the stock dispersion to 1 mL of AIBI-GCS-PDA@CG dispersions. The final red blood cell (RBC) hematocrit level was nearly about 4%. We incubated the combined solutions for 3 hours at the temperature of 37 °C. Afterward, by UV−vis analysis of the supernatant (540 nm), the percentage of hemolysis was measured after centrifugation (12000 rpm, 15 min). Pure water was the positive control,

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and saline served as the opposite. In the same way, Hemolysis of AIBI-GCS-PDA@CG was assayed. Hemolysis percentage of was calculated by: hemolysis (%) = ( AS - 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 addition of AIBI-GCS-PDA@CG to the erythrocyte suspension. 2.6 In vitro experiments Bacteria culture. The culture of both the standard S.aureus and MRSA bacterial strains were in 4 mL of liquid Luria-Bertani (LB) medium and shaken at 200 rpm for 12-16 h at 37 oC. Subsequently, during the exponential growth phase of resultant bacteria, the researcher collected the resultant bacteria and rinsed them by using PBS buffer (pH 6.3 or 7.4) by centrifuging at 3500 rpm. In vitro GCS-PDA@CG’s and AIBI-GCS-PDA@CG’s specific targeting to bacteria. By incubating them together in the acidic environment (pH 6.3) and physiological environment (pH 7.4), respectively,

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-1) or AIBI-GCS-PDA@CG (1 mg·mL-1) and 800 μL of the test bacteria was 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 researcher suspended the sediment (bacteria together with nanomaterials) in 1 mL of the relative PBS buffer and recorded its zeta potentials with a Zetasizer Nano. Meanwhile, aggregated pellets’ morphology was detected under a scanning electron microscope (SEM, Inspect F, Philips, Netherlands).

9



The researcher co-incubated the model host cells NIH 3T3 fibroblasts with test nanomaterials (GCS-PDA@CG or AIBI-GCS-PDA@CG) at pH 7.4 for 24 h to verify if the GCS-PDA@CG or AIBI-GCS-PDA@CG will be interacted with the host cells in physiological cases. Then, the researcher washed the cells two times with PBS and made the observation under SEM (Inspect F, Philips, Netherlands) according to our previous methods. In vitro free radical generation analysis. The researcher performed in vitro free radical generation analysis following the previously report methods with little modifications32. Briefly, 200 μL of AIBI-GCS-PDA@CG (1 mg·mL-1) 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 underwent transfer to a quartz capillary tube immediately, and then its ESR spectra was monitored at room temperature. In vitro ROS generation assay. The intracellular ROS level was detected using DCFH-DA under 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 ℃ in the dark. Then the bacteria were cultured with 0.2 mg·mL-1 of AIBI-GCS-PDA@CG for 4 h, and the mixture was washed three times with PBS. Afterward, the researcher irradiated the bacteria 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-1) and GCS-PDA@CG (0.2 mg·mL-1), control experiments were conducted. In order to confirm whether 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. AIBI-GCS-PDA@CG’s in vitro antibacterial efficiency. With the standard plate count, the

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researcher assessed AIBI-GCS-PDA@CG’s in vitro antibacterial efficiency

. In brief, 200 μL of

33

AIBI-GCS-PDA@CG (1 mg·mL-1) solution was mixed with 800 μL of bacteria solution (pH 6.3, OD600 0.4-0.5) and shaken sufficiently for 30 min. Afterwards, we irradiated the solution by 808-nm NIR laser (0.5 W·cm-2, 7 min). The researcher fully shook the processed suspension of resultant bacteria and subsequently diluted and uniformly spread the suspension onto nutrient agars. After the culture at 37 oC 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, the researcher performed control experiments. 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, the researcher mixed Vitamin C solution (0.4 mg·mL-1, 0.8 mg·mL-1, 1.2 mg·mL-1) and 200 μL of AIBI-GCS-PDA@CG (1 mg·mL-1) solution with 800 μL of bacteria solution (pH 6.3, OD600 0.4-0.5). Then, the researcher shook the mixture sufficiently for 30 min. Afterwards, the solution was irradiated for 7 min with an 808 nm NIR laser (0.5 W·cm-2). The processed suspension of 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, the 50 oC 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 oC) under shaking for different times (8 min, 10 min and 12 min). Then, following the standard plate count method, the researcher ascertained the number of bacteria colonies. Control experiments were also conducted in parallel with AIBI and GCS-PDA@CG, respectively. In vitro Live/Dead bacteria staining assay. The researcher irradiated bacteria suspensions

11



cultured with AIBI-GCS-PDA@CG under NIR laser (0.5 W/cm2, 7 min) in normal or hypoxic condition. Then, the researcher stained the suspensions 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 PI dye only entered bacteria with damaged cell membrane or wall. Glutathione (GSH) assay. Bacteria cells were seeded in EP tube and were collected at the exponential growth phase (OD600 = 0.4, ～5×107 cells/mL). After incubating with AIBI (0.2 mg·mL-1), GCS-PDA@CG (0.2 mg·mL-1) and AIBI-GCS-PDA@CG (0.2 mg·mL-1) for 4 h, the bacteria were irradiatedfor 7 min with 808 nm NIR laser light (0.5 W/cm2). After the mixture was washed 3 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. Afterwards, supernatant in 50 μL was mixed with Ellman`s reagent in 200 μL (0.5 mM 5,5'-Dithiobis-2-nitrobenzoic (DTNB)), and the lysates were centrifuged for 5 min at 6000 r/min. In the end, with a microplate reader (Thermo Varioskan Flash, USA), we measured the absorbance (405 nm) of the samples. DNA damage assay. The DNA damage ability of the free radicals generated by AIBI-GCS-PDA@CG was studied using the method reported before34. Briefly, 200 ng of pUC 18 DNA was introduced to 1 mL of AIBI-GCS-PDA@CG (0.2 mg·mL-1) 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 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-EDTA (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).

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2.7 In vivo experiments Animal model: To study the antibacterial effect of AIBI-GCS-PDA@CG in vivo, the subcutaneous abscess on BALB/C mice (6-8 weeks, 20-25 g) was established. The researcher anesthetized mice with 1% pentobarbital to form subcutaneous abscesses. The researcher gave a 100 μL MRSA (5 × 107 CFU·mL-1) subcutaneous injection on each test mouse’s disinfected and shaved back and at the same time injected the left side as control with PBS. 24 h later, this paper formed a focal infection formed as a subcutaneous abscess.

In vivo NIRF and fluorescent imaging of bacterial infection. The infected mice were injected with AIBI-GCS-PDA@CG nanomaterials (200 μL, 1mg·mL-1) through tail vein for in vivo NIFR imaging. Afterward, the researcher anesthetized the infected mice with isoflurane (0.2-0.3 L/min) and recorded NIRF images with an IVIS Lumina II imaging system (Xenogen IVIS Spectrum, Perkin Elmer) at scheduled time points.

Under an IVIS Lumina imaging system, the researcher took near infrared fluorescence (NIRF) images 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 IVIS cavity imaging system. Each side of the injection side in the back of the mice was irradiated with 808nm near-infrared laser (0.5 W/cm2 , 7 min) at the pre-ascertained time intervals (0, 2, 4, 6 and 24 h), and the researcher, with an IR thermal camera (FLIR-E49001, Estonia), took thermographic images for demonstrating AIBI-GCS-PDA@CG’s in vivo targeting capacity. Additionally, to assess the capability of GCS-PDA@CG and AIBI-GCS-PDA@CG for the production of in vivo hyperthermia at abscess, we intravenously injected 100 µL PBS, GCS-PDA@CG (0.8 mg/Kg) or AIBI-GCS-PDA@CG (0.8

13



mg/Kg) to the mice infected (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).

In vivo antimicrobial activity evaluation. Our bactericides hybrid’s 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 (5 mice per group): Control (PBS), NIR (0.5 W·cm-2, 7 min), GCS-PDA@CG (200 μL, 1 mg·mL-1), AIBI-GCS-PDA@CG (200 μL, 1 mg·mL-1), GCS-PDA@CG (200 μL, 1 mg·mL-1) + NIR (0.5 W·cm-2, 7 min) and AIBI-GCS-PDA@CG (200 μL, 1 mg·mL-1) + NIR (0.5 W·cm-2, 7 min) groups. The mice were intravenously administration only at the first time. After 10 days treatment, all the mice were scratched and the infected tissue was collected and assessed via standard plate-counting approach and haematoxylin and eosin (H&E) staining. 2.8 In vitro and in vivo biocompability evaluation of AIBI-GCS-PDA@CG By a cell counting kit-8 (CCK-8) assay, AIBI-GCS-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 twenty-four hours. Next, we replaced the culture medium with 10% FBS/DMEM containing different AIBI-GCS-PDA@CG’s concentrations (0.1-1 mg·mL-1). After co-culture for twenty-four hours, the plates were washed with PBS 3 times. Next, we introduced the CCK-8 reagent into each well and cultured them for another 3 hours at 37 °C. Finally, with microplate reader (Thermo Varioskan Flash, USA), we measured the optical density (OD) value of the medium at 450 nm. We acquired the cell viability through: cell viability = (OD(samples) /OD(control)) ×100%, and OD(samples) and OD(control) denote the absorbance value at 450 nm in the absence and presence of AIBI-GCS-PDA@CG. Control 14


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experiments were also conducted in parallel with Hacat cells. With the use of the healthy BALB/c mice (20-25 g, 6-8 weeks, 5 mice each group), the in vivo biotoxicity

of

AIBI-GCS-PDA@CG

was

assessed.

After

injected

with

0.5

mL

of

AIBI-GCS-PDA@CG (0.2 mg·mL-1, 0.5 mg·mL-1) through the tail vein and without NIR irradiation, the mice were sacrificed for a certain time (1 or 30 days). The major organs, e.g., lung, heart, spleen, kidney, and liver were gathered and 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 AIBI-GCS-PDA@CG in depth, the blood of each test mouse was drown 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 as mean ± standard deviation. With the use of and one-way analysis of variance (ANOVA) (for two groups) and two-way ANOVA (for more than two groups) in the 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), aromatric C=C (1618 cm-1) and surface carboxylic groups C=O-O

15



(1726 cm-1) are shown in the spectrum of CG. Then, the hydrodynamic size and morphology of CG were respectively ascertained with the DLS and TEM. CG was shown structured to be sheet-like (Fig. S2) with a size of 122-220 nm (Fig. S2A). The size of ~164 nm represents the largest amount of sheets with this size. To assess GCS’s and CG’s thickness, the researcher employed AFM. As shown in Fig. S2A2, the thickness of CG was ~ 1.0 nm. Through dopamine self-polymerization in the alkaline case (pH, 8.5), the researcher deposited PDA coating on CG nanosheets, enabling further modification on CG35. PDA@CG nanosheets were analyzed by FTIR, AFM and Zeta potential for a reliable demonstration of finished synthesis of PDA@CG. The spectrum of PDA revealed peaks at 1512 cm-1and 1600 cm-1, following the indoline or indole structures in PDA36 (Fig. 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 PDA sample, giving an evidence of the successful deposition of PDA coating on CG (Fig. S1A). In comparison with the TEM image of CG, there were basically not any significant size and morphological change after PDA coating, suggesting that the PDA deposits uniformly on CG and its thickness is relatively thin (Fig. S2B1). The AFM images also supported this (Fig. S2B2). After PDA coating, the thickness was increased only 0.2 nm. After PDA coating, CG’s zeta potential was reduced from -38.1±3.5 mV to -44.9±1.1 mV (Fig. S3A). Zeta potential reduction was primarily attributed to the less negatively charged of the hydroxyl groups in PDA coating. GCS, a water-soluble chitosan derivative featuring pH-sensitive charge reversal37, has been reported capable of ameliorating the in vivo behavior of CG by our previous works26. Here, by the reaction between the quinone groups in PDA and the amine groups of GCS based on Schiff base

16


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reaction, GCS was transfected on PDA@CG

38, 39

. According to Fig. 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 (Fig. S2C1) in comparison with the TEM images of PDA@CG before and after the modification with GCS. While the AFM images showed that the thickness was increased up to 1.7 nm after the GCS grafting (Fig. SC2). These results certificated that the GCS molecules were successfully grafted on the PDA@CG. Using a Zetasizer (Fig. S3B), the zeta 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, GCS-PDA@CG’s zeta 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, the researcher loaded AIBI onto GCS-PDA@CG as a free radical generation agents through the coordination, hydrophobic and hydrogen bond interaction. For the selection of a reaction concentration of AIBI that is economic and efficient, the researcher studied the loading amount of AIBI with various concentrations using TGA (Fig. S4). With the increase in AIBI concentration, the loading amount of AIBI increased. Upon the concentration of AIBI increased to 0.3 mg/mL, 60 μg AIBI was loaded on 1 mg of AIBI-GCS-PDA@CG. Nevertheless, by further improving the concentration of AIBI, there existed very slight change in the loading amount of AIBI. Therein, 0.3 mg/mL was the optimal reaction concentration for AIBI loading. In AIBI-GCS-PDA@CG spectrum (Fig. S1C), we found a novel peak at 1576 cm-1 in comparison with GCS-PDA@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 the GCS-PDA@CG, the TEM and AFM

17



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images showed no obvious variation in the morphology and thickness of AIBI-GCS-PDA@CG nanosheets (Fig. 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 DLS analysis in depth. According to Fig. 2C, AIBI-GCS-PDA@CG exhibited a size of 164-295 nm. Moreover, the zeta potential results proved that the AIBI-GCS-PDA@CG possesses the ability of pH-triggered charge-reversible (Fig. 2D), indicating that the AIBI loading could not influence the pH-sensitive performance of our bactericidal depot. 3.2 Optical and photothermal properties of AIBI-GCS-PDA@CG Because of its low absorbance by water, blood and biological tissue 40, an NIR laser 808 nm in wavelength is considered to be an optimal light source for PTT, able to enter the skin 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 the CG, PDA@CG, GCS-PDA@CG and AIBI-GCS-PDA@CG at pH 6.3 (complying with that at the sites of skin abscesses) was studied. As shown in Fig. 2E, after the PDA coating, at the same concentration of nano-sheet, the absorption value at 808nm 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 electron activity range 41. Note that the GCS grafting could optimize the surface of our bactericidal depot and significantly improved absorption of NIR light (808 nm) possibly because of the hydrogen-bond interactions between the hydrogen-bond interactions between GCS and PDA, improving electrons’ motion

42

. The AIBI

loading had no obvious effects on the absorption at the 808 nm. To further ascertain its photothermal ability, a variety of concentrations of AIBI-GCS-PDA@CG

18


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in PBS at pH 6.3 was in exposure to 808 nm NIR laser; pure PBS (pH 6.3) served as 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 W·cm-2 and 0.75 W·cm-2 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 concentration-dependent temperature change (Fig. 2F, G and Fig. S5). The temperature of GCS-PDA@CG and AIBI-GCS-PDA@CG solution increased rapidly in the case of the NIR light (0.5 or 0.75 W·cm-2), achieving concentration-dependent plateaus in 7 min. However, the PBS slightly changed in temperature from 28.8 oC to 31.5 oC (0.5 W·cm-2) and 29 oC to 32.6 oC (0.75 W·cm-2) even after 10 min 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 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 previously31. 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 Fig. S6. Considering the thermal ablation of most microbes happens over 50 oC and the skin tolerance to the laser power, a 0.2 mg·mL-1 GCS-PDA@CG or AIBI-GCS-PDA@CG concentration with 0.5 W·cm-2 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 conducted before and after irradiation at 0.5 W/cm2 at the concentration of 0.2 mg/mL for 7 min to verify if heating could damage the morphology of our antibacterial depot. According to Fig. S7A, after 5 cycles of irradiation at 0.5 W·cm-2, AIBI-GCS-PDA@CG suspensions’ absorbance (808 nm) loss was below 5%,

19



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illustrating AIBI-GCS-PDA@CG had a prominent photothermal-stability. After five cycles of laser irradiation (808 nm, 0.5 W·cm-2; Fig. S7B), the AIBI-GCS-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 infection biological conditions (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, Fig. S8). Overall, these results demonstrated that AIBI-GCS-PDA@CG presented high photothermal conversion efficiencies and good stability even under the acidic condition, which render our bactericidal depot very promising as a photothermal therapeutic agent. Subsequently, the stability of the AIBI-GCS-PDA@CG in serum containing media was evaluated using DLS (Fig. S9) and RAMAN spectroscopy (Fig. S10). After being cultured in 10% serum containing PBS, the hydrodynamic diameter and of AIBI-GCS-PDA@CG showed negotiable change with the incubation time increasing, indicating the nanosheets in serum containing medium were very stable at least 7 days. And the RAMAN spectra of AIBI-GCS-PDA@CG also confirmed the presence of the graphene in the 7 days。 3.4 Identification of the Generated Free Radicals With electron spin resonance (ESR) technique, the researcher investigated free-radical generation ability

of

AIBI-GCS-PDA@CG.

To

capture

the

highly

free

radicals,

we

employed

5,5-Dimethyl-1-pyrroline N-oxide (DMPO), 2-Methyl-2-nitrosopropane dimmer (MNP) and α-(4-Pyridyl N-oxide)-N-tert-butylnitrone (POBN). We observed Particular alkyl radical (R·) signals with POBN (αN = 15.1 G and αβ-H = 2.8 G), as shown in Fig. 2I. This study plotted the ESR peak

20


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intensity to reflect the number of R· versus NIR irradiation time (Fig. 2J, K) for monitoring the R· release kinetics. In the initial 3 min, The ESR peak intensity showed a slow increase with irradiation, certificating the temperate rise at this time. Hereafter, ESR peak increased sharply in the following 6 minutes, 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 minutes. As expected, we did not observe the ESR signals in the group of GCS-PDA@CG or AIBI-GCS-PDA@CG without the NIR irradiation (Fig. S11). Comparatively, there was no ESR signals in AIBI and GCS-PDA@CG groups, suggesting that AIBI-GCS-PDA@CG’s free-radical generation ability results from the plasmonic heating of initiators (Fig. 2K, Fig. S11). 3.5 pH-dependent bacterium-specific interactions of AIBI-GCS-PDA@CG Due 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 bacteria-specific 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 staphylococcus here aureus were treated with synthetic nanomaterials at various pH values (7.4 or 6.3). Through incubating bacteria with GCS-PDA@CG or AIBI-GCS-PDA@CG at pH 6.3, mixture rapidly condenses and precipitates to tube bottom in 30 minutes (Fig. S12). Yet in the pH 7.4 treatment (Fig. S12), we saw no such phenomenon. The mentioned phenomena possibly resulted from the potent binding ability of GCS-PDA@CG or AIBI-GCS-PDA@CG for bacteria through electrostatic force in acidic condition. The zeta potential results also confirmed these. As presented in Fig. 3A and Fig. S13A, the

21



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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 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 AIBI-GCS-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 (Fig. 3B, Fig. S13B). 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 in exposure to 3T3 fibroblasts at 7.4, the pH of host cells under physiological conditions (Fig. 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 Fig. 4A, B, the materials showed no antibacterial effect. Under the 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

AIBI-GCS-PDA@CG group, in particualr at a concentration of 0.2 mg/mL; the antibacterial efficiency was ～25% for GCS-PDA@CG group, while it was about 100% for the AIBI-GCS-PDA@CG group, revealing that the bactericidal effect of AIBI-GCS-PDA@CG (< 0.2 mg/mL) primarily resulted from

22


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the generated free radicals (Fig. 4C, D). Furthermore, similar results could be observed in live/dead staining assay (Fig. 4E, F), the AIBI-GCS-PDA@CG group showed obvious bactericidal effect under both normoxia and hypoxia. To further confirm the role of alkyl free radicals, we examined the antibacterial effect of AIBI-GCS-PDA@CG after adding the free radicals scavenger (Vitamin C). As shown in Fig. S14, with the concentration of Vitamin C increasing, the sterilization efficiency was gradually reduced. This phenomenon proved that alkyl free radicals played a key role in the sterilization process. The mentioned results prove that the free radicals produced can effectively induce bacterial cell death, which is oxygen independent. In addition, after assessing the bactericidal effect against S.aureus, similar results were achieved, proving this method is general. Then, this paper explored therapeutic mechanism of the generated free radicals. It has been widely reported that under normoxia, the produced alkyl radicals (R·) can be converted into alkoxyl radicals (RO·) by cellular oxygen for inducing OS45. Nevertheless, this issue under hypoxia has been rarely discussed. Here, the free radicals generation ability of AIBI-GCS-PDA@CG was identified under normal oxygen condition (Air) and oxygen-free condition (N2). As shown in Fig. S15, three different spin traps α-(4-Pyridyl N-oxide)-N-tert-butylnitrone (POBN), 2-Methyl-2-nitrosopropane dimer (MNP) and 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) were used as the free radical trappers. In the condition of AIBI-GCS-PDA@CG with NIR irradiation, typical R· signals were found both under N2 and air atmosphere using POBN (Fig. S15A, αN = 15.104 G and αβ-H = 2.760 G) and MNP (Fig. S15B, αN = 17.020 G), indicating the successful generation of R· without the dependence of oxygen tensions. In the condition of DMPO (Fig. S15 C), oxygen seems to affect the form of generated radicals. Under N2 environment, R· signals can be observed with the ESR spectral pattern of

23



six lines of equal intensity (αN = 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 as DMPO compared with oxygen exhibits slower reaction rate with R·. And R· prefer reacts with oxygen to form RO·, then RO· is captured by DMPO under air environment. While, POBN and MNP react faster with R· than oxygen, 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 Fig. 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 (Fig. 4G4-6). When bacteria were further cultured for 4 h, green fluorescence was shown in the AIBI-GCS-PDA@CG group (Fig. 4G9), suggesting that the produced R· under hypoxia could induce cell OS as well but showing a distinct system. Antioxidant systems, e.g., 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 Fig. 4H, only AIBI-GCS-PDA@CG (0.2 mg/mL) under the laser irradiation group indicated remarkable ability of

24


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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 plasmid pUC 18 DNA assay, this paper also assessed the intracellular DNA-damaging activity of our bactericidal depot. After the single strands being cleared by free radicals, the plasmid pUC 18 DNA can be covered from supercoiled form (SC) to open circular form (OC), which can be distinguished using agarose gel electrophoresis. As shown in Fig. S16A and Fig. S16B, the SC form was effectively covered to OC form in AIBI-GCS-PDA@CG under the laser irradiation group, especially under hypoxic condition; while in the condition of AIBI and GCS-PDA@CG ((808 nm, 0.5 W·cm-2), the DNA still remained SC form, indicating the AIBI or GCS-PDA@CG alone could not induce the single strands breaking of DNA. These results indicated 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 As inspired by the significant therapeutic efficacy of AIBI-GCS-PDA@CG in vitro, the bactericidal effectiveness of AIBI-GCS-PDA@CG in vivo was verified with the use of Balb/c mice with MRSA-infected subcutaneous abscess. Subcutaneous abscesses refer to the localized skin infections resulting from pathogenic bacteria, e.g., Staphylococcus aureus. In clinical aspect, antibiotics alone are not enough to treat subcutaneous abscesses. The main treatment for this infection is incision/drainage, followed by stuffing to prevent the wound from collapsing. In addition, the process is painful and may need to be repeated and stop in the case of the elimination of the infection. Also, systemic antibiotic therapy is often followed by antibiotic resistance evolution in bacteria. It has been reported that the incidence of focal skin infections attributed to methicillin-resistant

25



staphylococcus aureus (MRSA) has risen sharply in recent years, posing a serious challenge to human health. We assumed that the use of our bactericidal library not only eliminates the need for incisions/drainage, but also avoids the use of antibiotics for skin abscesses, especially the ones attributed to drug-resistant strains. Before intravenous injection, we conduct AIBI-GCS-PDA@CG’s hemolytic property on human erythrocytes (Fig. S17). AIB-GCS-PDA@CG did not damage erythrocytes even nanosheets’ concentrations up to 1 mg·mL-1 (hemolysis rate: 2.19%). Therefore, AIBI-GCS-PDA@CG 0.2 mg·mL-1 in concentration is suitable for blood-contacting applications. The AIBI-GCS-PDA@CG nanosheets were labeled with CY5-SE fluorescence agents (f-AIBI-GCS-PDA@CG) to assess if the AIBI-GCS-PDA@CG can be accumulated in the bacterial infection site. The Cy5 molecules were conjugated on the nanosheets through the covalent bond, therefore Cy5 molecules could not dissociated with the complex, which was also confirmed by the fluorescence spectroscopy characterizations (Fig. S18). The Balb/c mice bearing MRSA-infected subcutaneous abscess were intravenously injected with f-AIBI-GCS-PDA@CG nanosheets and monitored by fluorescent signals of f-AIBI-GCS-PDA@CG in the abscess regions (at 650 nm), where emission wavelength falls within the “optical window” in biological tissue. Many fluorescence could be observed at 2h after injection, and it gradually reached the maximum at 6h after injection, indicating that NPs accumulated continuously at the abscess site and decreased gradually at 24h after injection (Fig. 5A). The thermographic images also supported these. Ex vivo fluorescence images of 24 h post-injection (Fig. 5D) further demonstrated enrichment of nanosheets in abscess and some metabolic organs like liver owing to the reticuloendothelial system (RES). Our antibacterial depot’s capacity of producing in vivo hyperthermia at abscess was further evaluated, we injected intravenously 100 μL of PBS,

26


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GCS-PDA@CG (8 mg/Kg) and AIBI-GCS-PDA@CG (8 mg/Kg) to mice infected, then let them undergo NIR irradiation under 808 nm (10 min, 0.5 W cm−2) at 6 h post-injection. As expected, the temperature rises with the time exposure to the abscess increasing, and reached a plateau within the 8 min. The temperature elevated to 50.3 °C and 50.5 °C for GCS-PDA@CG and AIBI-GCS-PDA@CG group; at the same itme, temperature increased only 3.2 °C in the PBS group (Fig. 5F), indicating that the GCS-PDA@CG and the AIBI-GCS-PDA@CG nanosheets possessed remarkable photothermal conversion capacity in vivo. The assessment the hyperthermia-induced alkane free radical treatment on MRSA-infected mice in vivo was inspired by additional bactericidal effects of the AIBI-GCS-PDA@CG nanosheets in vitro experiments and the high abscess accumulation of those nanosheets in the imaging experiments here. After the formation of abscess in the subcutaneous tissue on the back of the mouse, the BALB/c mice were divided into the following different groups: (1) PBS + NIR, (2) AIBI + NIR, (3) GCS-PDA@CG, (4) AIBI-GCS-PDA@CG, (5) GCS-PDA@CG + NIR, (6) AIBI-GCS-PDA@CG + NIR. After 10-day treatment, only the mice in AIBI-GCS-PDA@CG + NIR group indicated no remarkable abscess and inflammation, while red swelling of the skin and abscess were still exited in other groups. In addition, the infected tissue was homogenized and the colony count was performed by standard plate counting method. According to Fig. 6B, the CFU counts in the groups receiving PBS + NIR, AIBI + NIR, GCS-PDA@CG, AIBI-GCS-PDA@CG treatment were similar to that in the control group (p > 0.05). On the contrary, the GCS-PDA@CG + NIR group and AIBI-GCS-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

27



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 significnat infected skin lesion with a huge amount of aggregated inflammatory cells compared with the healthy skin, as shown in Fig. 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 can serve as a good alternative therapeutic agent for treatment in vivo. 3.8 In vitro and In vivo biocompatibility evaluation Above results overall proved the prominent antibacterial effects of AIBI-GCS-PDA@CG depot, the in vivo and in vitro biosafety of the bactericidal system should also be taken into account. Firstly, to assess AIBI-GCS-PDA@CG’s cytotoxicity in vitro, the 3T3 fibroblasts were co-cultured with AIBI-GCS-PDA@CG (0.05 -1.0 mg/mL) for 24 h in exposure to NIR light (0.5 W·cm-2, 7 min). According to the results, the cells viability were all above 95% with NIR irradiation, indicating that AIBI-GCS-PDA@CG showed ignorable toxicity to 3T3 fibroblasts (Fig. S19), which was possibly because of the weak interactions between AIBI-GCS-PDA@CG and the cells. Additionally, no damages or appreciable abnormalities of main organs (e.g., kidney, lung, spleen, liver and heart) from the healthy mice (without infection) were observed 1 days and even 30 days after AIBI-GCS-PDA@CG injection with an applied concentration of 0.2 mg/mL as well as even at a high concentration of 0.5 mg/mL (Fig. 7A and Fig. 7B). Thus, this antibacterial agent caused side effects to the mice that can be neglected in the process of the bactericidal therapy. Meanwhile, blood analysis was also carried out to evaluate the long-term toxicity of AIBI-GCS-PDA@CG in healthy mice. The

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blood routine index, liver function markers and kidney function markers were in reasonable range (Fig. 8), suggesting no obvious hematological system, hepatic or kidney damages caused by AIBI-GCS-PDA@CG in mice. Overall, the excellent biocompatibility of the AIBI-GCS-PDA@CG makes it as promising antibacterial agents 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, 47, 48

. For example,

Methicillin-resistant staphylococcus aureus (MRSA) is one of the most feared pathogenic bacteria (superbugs) clinically, which can lead to life-threatening diseases, e.g., septicemia and acute endocarditis, with high morbidity and mortality clinically49, 50, 51, 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 multi-drug resistant bacteria therapy in recent years53. 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 infection particularly the inner organ infections (i.e., Lung, Liver) located a few millimeters below the human skin. Due 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. Considering these issues together, we developed a NIR light-activatable bactericidal depots as well as bacterial-specific targeting and assessed their efficacy to eradicate the subcutaneous, MRSA

29



infection in the mouse model in Fig. 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 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 light-activated AIBI-GCS-PDA@CG was significantly higher than that of light-activated GCS-PDA@CG (Fig. S20) or heat-activated AIBI alone (Fig. S21). This enhancement effect is partly due to the synergistic effect of photothermal and free radical sterilization. As shown in Fig. 4 and Fig. S16, the main bactericidal effect for MRSA is caused by generated free radicals, while the antibacterial effect of the photothermal action (GCS-PDA@CG + NIR) alone was very weak (Bactericidal efficiency 24.3%). Likely, compared to the heat-activated 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 lived56, which possibly possesses no enough time to reach the bacteria, resulting in significantly bactericidal efficiency reduction. More importantly, the strong antibacterial effect caused by our bactericidal depots was similar under the normal oxygen and

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hypoxia conditions, indicating that our bactericidal depots could overcome the hypothermia of the subcutaneous. Compared to previously strategies that 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 has not change the hypothermia environment of the subcutaneous, which is beneficial to wound healing. 5 Conclusions To sum up, a novel therapeutic strategy according to free radicals’ light-induced generation is proposed for drug-resistant bacteria therapy. Combining the photothermal effect of our bactericidal depot with thermally decomposability of AIBI, AIBI-GCS-PDA@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. In exposure to normoxia, the generated alkyl radicals (R·) are able to undergo the conversion into alkoxyl radicals (RO·) by oxygen, as shown in Fig. 1C and Fig. 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 the subsequent research. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (51673171, 31500789),the

Science

and

Technology

Innovation

Plan

of

Southwest

Hospital

(No.

SWH2016ZDCX2014, SWH2016JCYB-04 and SWH2017ZDCX1001), the Third Military Medical University (Grant No. 2016XPY12), The Chongqing Yuzhong District science and technology plan

31



project grants (20170124), Nature Science Foundation of Chongqing, China (cstc2018jcyjAX0807,No. cstc2017jcyjAX0020 and No.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. Conflict of interest The authors declare no conflict of interest. Associated Content Supporting Information. FTIR , TEM ,AFM,SEM, zeta potential, thermogravimetric analysis, photothermal effect,hydrodynamicdiameter, RAMAN spectra, EPR analysis, DNA damaging activity and the fluorescence hemolysis activity of the material assessment. References (1) Levin, B. R.; Antia, R., Why We Don't Get Sick: The Within-Host Population Dynamics of Bacterial Infections. Science 2001, 292, 1112-1125. (2) Dan, I. A.; 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. 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.; 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. 2009, 58, 725-725. (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. Edit. 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. Edit. 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 Aggregation‐ Induced Emission Characteristics for Selective Fluorescence 32


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Figure captions 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. Figure 2 Characterization of AIBI-GCS-PDA@CG. (A) TEM and (B) AFM image of AIBI-GCS-PDA@CG nanosheets. (C) Hydrodynamic diameter of AIBI-GCS-PDA@CG after 10 h ultrasound bath. (D) Zeta potential of AIBI-GCS-PDA@CG under different pH values (5.0, 5.5, 6.0, 6.3, 6.5, 7.0, 7.4). (E) UV-vis-NIR spectra of PBS, CG, PDA@CG, GCS-PDA@CG, AIBI-GCS-PDA@CG. (F) Photothermal effect of AIBI-GCS-PDA@CG under NIR (808 nm) irradiation with different powers (0.5 W/cm2, 0.75 W/cm2, 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 the NIR irradiation (808 nm, 0.5 W/cm2) for different time (4 min, 6 min, 8 min, 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. Figure 3 (A) Zeta 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 & 7.4) and (C) 3T3 fibroblasts (pH 7.4) before and after incubation with AIBI-GCS-PDA@CG. Figure 4 Images of bacterial CFUs of AIBI (0.10 mg/mL), GCS-PDA@CG (0.10 mg/mL),

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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, respectively. The images of Live/Dead staining assay of AIBI (0.20 mg/mL), GCS-PDA@CG (0.20 mg/mL), AIBI-GCS-PDA@CG (0.10 mg/mL) under (E) normoxic and (F) hypoxic conditions, separately. The bacteria were irradiated with a 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 (0.20 mg/mL), (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), AIBI-GCS-PDA@CG (0.20 mg/mL) (808 nm, 0.5 W/cm2, 7 min), respectively. Figure 5 (A) In vivo NIRF images and (B) thermographic images of the mice bearing abscess intravenously injected with f-AIBI-GCS-PDA@CG at a dose of 0.8 mg/Kg at 0 h, 2 h, 4 h, 6 h and 24 h post-injection, respectively. For the thermographic images, the test mice were irradiated with NIR laser light (808 nm, 0.5 W/cm2) for 10 min at different time points after intravenous injection. (C) Corresponding NIRF intensities determined from Figure 5A. (D) NIRF images of heart, liver, spleen, lung,

kidney

and

abscess

extracted

from

the

test

mice

intravenously

injected

with

f-AIBI-GCS-PDA@CG at 24 h post-injection. (E) Infrared thermography and (F) temperature elevations of the mice bearing abscess intravenously injected with AIBI-GCS-PDA@CG at a dose of 0.8 mg/Kg at 6 h post-injection under the laser irradiation (808 nm, 0.5 W/cm2) for 12 min. * and ** present p < 0.05 and p < 0.01, respectively.

37



Figure 6 (A) Photographs of subcutaneous abscess on the dorsal surface of mice after 10 days with various treatments. (B) Photographs and C) corresponding quantitative analysis of bacterial CFUs under various treatments. (D) Representative H&E staining images of infected skins that received various treatments. The blue circles indicate the subcutaneous abscess. Scale bar: 200 μm. * and ** present p < 0.05 and p < 0.01, respectively. Figure 7 In vivo biocompatibility evaluation of AIBI-GCS-PDA@CG. H&E staining images of the major organs (heart, liver, spleen, lung and kidney) from mice at (A) day(s) 1 and (B) 30 days postintravenous injection with different concentrations of AIBI-GCS-PDA@CG (0.20 mg/mL, 0.50 mg/mL). Figure 8 Hematology and blood biochemistry analysis 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 (BUN), (D) platelets, (E) hemoglobin (HGB), (F) creatinine (Cr), (G) alanine aminotransferase (ALT), (H) aspartate aminotransferase (AST), (I) albumin, (J) lactate dehydrogenase (LDH), (K) α-HBDH and (L) CK-MB. NS denotes no significant difference (p>0.05).

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A

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Target PDT Ablation of MRSA

AIBI Figure 1

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MRSA S.aureus

pH 6.3

pH 6.3

5 μm

10 μm

5 μm

5 μm

10 μm

5 μm

MRSA

15

Bacteria + AIBI-GCS-PDA@CG

S.aureus

pH 7.4

Zeta potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


pH 7.4

50 μm

50 μm

Figure 3

41



AIBI-GCSGCSPDA@CG PDA@CG

1% O2

E AIBI+NIR

D

21% O2

AIBI GCS-PDA@CG AIBI-GCS-PDA@CG 0

0.05

0. 1

0.2

0.5

1% O2

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5

AIBI GCS-PDA@CG AIBI-GCS-PDA@CG 0.05

0

F

GCSAIBI-GCSPDA@CG+NIR PDA@CG+NIR

AIBI-GCSGCSPDA@CG PDA@CG

AIBI

MRSA (108cfu/mL)

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5

MRSA (108 cfu/mL)

C

B With NIR Without NIR

AIBI

With NIR Without NIR

AIBI+NIR

0.1

0.2

0.5

GCSAIBI-GCSPDA@CG+NIR PDA@CG+NIR

1% O2

21% O2

PI

G AIBI+NIR G1 G

G4

GCSAIBI-GCSPDA@CG+NIR PDA@CG+NIR G2

H

G3

120

G5

GSH Level (%)

21% O2

A

STYO 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 47

G6

100

21% O2

1% O2

80 60 40 20 0

G7

G8

G9

Figure 4

42


Page 43 of 47

A

0h

4h

2h

6h

24 h Max

Min

Fluorescence intensity P/s/cm2/sr,109

Abscess

Normal Skin

B

C

2.5

Normal skin ** Abscess **

2.0 1.5

**

**

1.0

Heart

Liver

Spleen

Lung

Kidney

Abscess

0.5 0.0

0

2

4

6

Time (h)

PBS

E

D

GCS-PDA@CG

24

AIBI-GCSPDA@CG

F

180 s

Temperature (oC)

0s

55

PBS GCS-PDA@CG AIBI-GCS-PDA@CG

50 45 40 35

0

200

400

600

800

Time (s)

600 s

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5

43



A

AIBI+NIR

PBS+NIR

GCS-PDA@CG

AIBI-GCSPDA@CG

AIBI-GCSGCSPDA@CG+NIR PDA@CG+NIR

B

C

Survival bacteria (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 44 of 47

140 120 100 80

D

**

Normal Skin

Before Treatment

PBS+NIR

AIBI-GCSPDA@CG

GCS-PDA@CG

GCSPDA@CG+NIR

AIBI+NIR

** **

60 40 20

AIBI-GCSPDA@CG+NIR

0 200 μm

Figure 6

44


Page 45 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


45


ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

46


Page 46 of 47

Page 47 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


TOC

47


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

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