Photodynamic Synergistic Therapy by Disrupting

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Applications of Polymer, Composite, and Coating Materials

Local Photothermal/Photodynamic Synergistic Therapy by Disrupting Bacterial Membrane to Accelerate Reactive Oxygen Species Permeation and Protein Leakage Congyang Mao, Yiming Xiang, Xiangmei Liu, Yufeng Zheng, Kelvin Wai Kwok Yeung, Zhenduo Cui, Xianjin Yang, Zhaoyang Li, Yanqin Liang, Shengli Zhu, and Shuilin Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05787 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019

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ACS Applied Materials & Interfaces

Local Photothermal/Photodynamic Synergistic Therapy by Disrupting Bacterial Membrane to Accelerate Reactive Oxygen Species Permeation and Protein Leakage Congyang Maoa,b, Yiming Xiang,a,b Xiangmei Liub, Yufeng Zhengc, Kelvin Wai Kwok Yeungd, Zhenduo Cuia, Xianjin Yanga, Zhaoyang Lia, Yanqin Lianga, Shengli Zhua, Shuilin Wua,b*

a

The Key Laboratory of Advanced Ceramics and Machining Technology by the

Ministry of Education of China, School of Materials Science & Engineering, Tianjin University, Tianjin 300072, China b

Hubei Key Laboratory of Polymer Materials, Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, School of Materials Science & Engineering, Hubei University, Wuhan 430062, China c

State Key Laboratory for Turbulence and Complex System and Department of

Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China d

Department of Orthopaedics& Traumatology, Li KaShing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong 999077, China

* To whom correspondence should be addressed: E-mail: [email protected]; [email protected] (S.L. Wu)

Keywords: Synergistic therapy; Bacterial membrane permeation; Sterilization; Biosafety; Guiding significance; Phototherapy 1

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ABSTRACT Bacterial infection is still a ticklish clinical challenge even though some advanced antibacterial materials and techniques have been put forwardd. This work reports that rapid and effective antibacterial performance is achieved by the synergistic local photothermal and photodynamic therapy (PTDT). Within 10 min light irradiation, both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are almost completely eliminated by the action of photothermy (52.1 oC) and limited reactive oxygen species (ROS).The corresponding bacteria-killing efficiencies are 99.91% and 99.97%, severally, which are far higher than single modal therapy, i.e., photothermal therapy (PTT) or photodynamic therapy (PDT) with antibacterial efficacy of 50% and 70%, respectively. The mechanism is that bacterial membrane permeation is increased by PTDT because photothermy shows more severe impact only on E. coli by destroying the outmost bacterial panniculus while the inner panniculus of the two kinds of bacteria is more sensitive to ROS. Hence, ROS is more easily to penetrate the bacterial membrane, and meanwhile, the proteins in the bacteria are severely lost after the bacterial membrane disruption, which leads to the bacterial death. In vivo results reveal that rapid and effective sterilization is an important process to accelerate wound healing, and the traumas on the rats’ backbones heal well within 12 days by PTDT. Furthermore, the PTDT is friendly to major organs of rats during the therapeutic process. Therefore, the synergistic therapy system can be a safe therapeutic system for the sterilization in clinical with a great potential. More importantly, the antibacterial mechanism presented in this work has the great guiding significance for the design of other advanced antibacterial systems and techniques.

2


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1. INTRODUCTION Despite the remarkable advances of artificial antimicrobial techniques and materials recently, infection induced by bacteria is still one of the most ticklish clinical diseases that can threaten human health.1-3 The traditional therapeutic methods including antibiotics and some inorganic antimicrobial agents could cause microbe resistance and serious systemic toxicity,4-6 respectively. Recently, because of their high therapeutic efficiency and noninvasive advantage compared to the traditional therapeutic systems, phototherapy systems including photothermal therapy (PTT) and photodynamic therapy (PDT) have attracted wide attention.7-12 For PTT, by converting the light energy, the heat generated from photothermal agents can cause the photothermal ablation of cell.13-16 However, the higher temperature (over 70 oC) is required to achieve effective antibacterial efficacy for single PTT,17-19 which would scald the normal tissues and cause serious side effects.20 Differently, for PDT, the generation of reactive oxygen species (ROS) after irradiating photosensitizers destroyed the normal cells by disrupting cellular membranes, proteins, and even DNA.21-25 As the two major kinds of ROS with strong oxidizing ability, the singlet oxygen (1O2) and hydroxyl radical (•OH) have been studied in PDT studies.26-29 Practically, in order to kill most of bacteria through single PDT, a large number of ROS is needed. However, excessive ROS also caused damage to the normal tissues during this process, including inflammation, fibrosis, and even necrosis.30-33 Therefore, it is urgent to develop much safer phototherapy strategies for rapid disinfection, i.e., PTT with lower temperature (around 50 oC) and moderate PDT with limited ROS during therapeutic process. To date, compared with various photothermal agents used in PTT, including 3



novel metals,34-36 carbon-based nanomaterials,37 and inorganic nanoparticles,38,39 melanin-like polydopamine (PDA) exhibits good biocompatibility and excellent photothermal conservation property,40,41 suggesting its potential for PTT.42-44 In addition, a stable photocatalytic system of Ag@AgCl nanostructure which was triggered by visible light has been proposed.45-47 And the strong coupling of the surface plasmon resonance (SPR) impact of Ag is responsible for the photocatalysis of Ag@AgCl nanostructures in the visible range. However, this photocatalytic system only showed limited photocatalytic performance with a small content of Ag presented in nanostructured [email protected] Considering that the similar light triggering conditions for PTT and PDT, the photothermal/photodynamic synergistic therapy (PTDT) is developed in this work, which is achieved by relatively low temperature (52.1 oC) and moderate ROS generation. However, the PTDT system is rarely reported in the antibacterial field. The PDA is responsible for the PTT while Ag@AgCl nanostructures generate limited ROS under visible light because there is only a small amount of metallic Ag reduced from AgCl by PDA. In addition, the rapid and effective antibacterial performance has been achieved by PTDT while single PTT or PDT only provided moderate antibacterial efficiency. More importantly, PTDT with low temperature and limited ROS promotes tissue repair but cause no damage to primary organs of rats, indicating that this treatment method can be safe and efficient. As illustrated in Scheme 1, PTDT efficiently enhances bacterial membrane permeation because for E. coli, the outmost panniculus is more sensitive to photothermal response while the ROS displays the more significant effects on the intima of two kinds of bacteria. Furthermore, after disrupting the bacterial outer and inner membrane by heat and ROS, respectively, the ROS is more easily to enter into the bacteria to cause the outbreak of oxidative stress 4


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in bacteria and the severe leakage of protein from the bacteria, which should be responsible for the rapid and effective antibacterial activity.

Scheme 1. The illustration showing the antibacterial mechanism and the process of disrupting bacterial membrane, which accelerates reactive oxygen species permeation and protein leakage under visible light irradiation.

2. EXPERIMENTAL PROCEDURES 2.1. The detection of reactive oxygen species (ROS). The electron spin resonance spectrometer (ESR, JES-FA200) was used to detect ROS. Singlet oxygen (1O2) was detected by 2,2,6,6-tetramethylpiperidine (TEMP, 50 mM) while hydroxyl radicals

(•OH)

was

trapped

by

the

spin

trap

of

0.1

M

5,5-dimethyl-1-pyrroline-N-oxide (DMPO) during the exposure of samples to visible light. The following was measurement parameters of ESR tests: micro frequency, 8.93 GHz; micro power, 3 mW. In the typical tests,29,49 200 μL of spin traps containing the dried samples 5



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(  6mm  2mm ) in the 48-well plates were illuminated for 10 min using a 0.2 W/cm2 300 W xenon lamp (1 m high). After that, quartz capillary tubes were used to withdraw the irradiated spin traps and then sealed. Finally, the ESR was used to record the ESR spectra of the sealed capillary tubes. Additionally, 1O2 was further evaluated by ADMA with full name of 9, 10-anthracenediyl-bis

(methylene)

dimalonic

acid.

Briefly,

the

samples

(  6mm  2mm ) were immersed in 200 μL of ADMA with the concentration of 10 μM in PBS with pH value of 7.4. Then the mixtures were irradiated for 10 min by a xenon lamp (0.2 W/cm2, 1 m high). Finally, the absorption intensities (OD400) of irradiated ADMA were recorded at the setting time (0, 2, 4, 6, 8, and 10 min) using a Multiscan Spectrum made by the company of Molecular Devices. 2.2. In vitro antibacterial activity tests. E. coli and S. aureus were used in the test. The antibacterial test of Ag+ was firstly evaluated using optical density (OD600) by a microplate reader. More specifically, the samples (  6mm  2mm ) were placed in the bottom of 96-well plates and then 150 μL of the bacterial suspensions (1.0*107 CFU/mL) were added into the wells and fostered in an incubator for 1 day, and PAM hydrogel was served as the control. The antibacterial ratio was calculated by Equation (1).

Antibacterial ratio (%) 

ODcontrol group - ODexperimental group ODcontrol group

100%

(1)

The antimicrobial activity was then evaluated using the spread plating method. The samples (  6mm  2mm ) were placed in the bottom of 96-well plates and then 150 μL of the bacterial stock suspensions (1.0*107 CFU/mL) were added into the wells with samples, and the PAM hydrogel without irradiation was served as the control. 6


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Meanwhile, the PAM-PDA and PAM-PDA/Ag@AgCl hydrogels were named as photothermal therapy (PTT) group and PTDT group, respectively, while the 96-well plates containing PAM-PDA/Ag@AgCl hydrogel immersed in ice bath without heat effect was labeled as PDT group. The 96-well plates containing bacterial stock suspensions and hydrogel samples were then irradiated for 10 min under the xenon lamp (0.2 W/cm2, 1 m high). Afterward, spread plating method was used to conduct antibacterial test. Finally, the viable colony units were formed on the surface after culturing for 1 d at 37 oC. The antibacterial ratio was determined by Equation (2).

Antibacterial ratio (%) 

CFU0 (cell)  CFU (cell + gel + rad) *100% CFU0 (cell)

(2)

Where CFU0 (cell) refers to the number of viable colonies formed after treated with PAM hydrogel without irradiation, while CFU (cell + gel + rad) means the number of viable colonies formed with visible light or composite hydrogels without irradiation. After the evaluation of spread plating method, the treated bacteria adhered on the surface of samples were fixed for 2 h with 2.5% glutaraldehyde, and then orderly dehydrated for 15 min by 30, 50, 70, 90 and 100% of alcohol and finally freeze-dried for SEM (JSM6510LV) tests.29 And the irradiated bacterial solution was withdrawn and then centrifuged (6,000 rpm) for 5 min by refrigerated centrifuge. The centrifuged bacteria were withdrawn and placed into glutaraldehyde solution (2.5%) for 2 h and then fixed by OsO4 aqueous solution (1%) for 2 h. Afterwards, washing those samples three times with PBS and then orderly dehydrating them with 30, 50, 70, 90 and 100% of alcohol for 15 min. The samples were then orderly placed in the 50% Epon 812 (SPI 90529-77-4) 7



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(diluted by acetone) and pure Epon 812 for 12 h. After that, embedding them with Epon/Araldite resin (60 oC, 48 h). Next, a diamond knife fixed in a microtome (Leica UC7) was used to prepare bacterial sections with thickness of 60-80 nm, which were then stained using uranylacetate and finally transferred on copper grids for TEM (Titan G2 60-300 and Tecnai G20) observation.49 2.3. Bacterial membrane permeability test. The permeability tests of E. coli bacterial outer panniculus were evaluated using 8-anilinonapthalene-1-sulfonic acid (ANS). Firstly, E. coli bacteria were washed twice with PBS (pH=7.4) by refrigerated centrifuge (6,000 rpm) for 5 min and diluted back in PBS with 10 μM ANS to the desired working concentration at an OD600 of 0.05-0.1. And 150 μL diluted bacterial samples were then equilibrated in 96-well plates at 37 °C in the darkness for 20 min. Afterwards, samples (  6mm  2mm ) were added into the 96-well plates containing equilibrated bacterial solutions and then irradiated for 10 min. Finally, 100 μL treated bacteria were withdrawn in 96-well plates and the changes in the fluorescence emission (450-600 nm) were measured (excitation: 380 nm) by a microplate reader and photographed by a fluorescence microscope (IFM, Olympus, IX73, Japan). For comparison, the untreated equilibrated bacterial solutions were also recorded. And the impact of samples on the permeability of inner membrane (cytoplasmic panniculus)

of

the

above

two

kinds

of

bacteria

was

performed

by

o-Nitrophenyl-β-D-Galactopyranoside (ONPG). The bacteria were cultured in LB medium with isopropyl β-D-1-thiogalactopyranoside (IPTG, 10 μg/mL) (37 oC, 12 h). The media was then removed using refrigerated centrifuge at 6,000 rpm for 5 min. Then washing the resulting bacteria twice using PBS (pH=7.4) by refrigerated centrifuge (6,000 rpm) for 5 min. Then diluting it back in PBS to obtain the desired 8


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working concentration at an OD600 of 0.05-0.1. Afterwards, samples (  6mm  2mm ) were added to the 150 μL diluted bacterial solutions in 96-well plates and irradiated for 10 min. Finally, absorbance at OD420 was recorded using a microplate reader in 96-well chambers with 15 μL treated bacterial solutions, 15 μL ONPG (12.5 mM), 10 μL DMSO (7%), and 110 μL PBS. 2.4.

Detection

of

total

ROS

within

the

bacteria.

2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay (Reactive Oxygen Species Assay Kit, cat#S0033; Beyotime) was used to evaluate the level of ROS within the bacteria induced by samples after irradiation. To load probe, the two kinds of bacteria were washed twice by PBS (pH=7.4) by refrigerated centrifuge (6,000 rpm) for 5 min with followed culturing with DCFH-DA (10 μM) in PBS (37 oC, 30 min). After removing the unloaded DCFH-DA by refrigerated centrifuge (6,000 rpm) for 5 min, the bacteria were diluted back in PBS to the desired working concentration at an OD600 of 0.05-0.1. Afterwards, samples (  6mm  2mm ) were added to the 150 μL diluted bacterial solutions in 96-well plates and irradiated for 10 min. Finally, 100 μL treated bacteria were withdrawn in 96-well plates and the relative fluorescence intensity was detected at 525 nm with excitation at 488 nm using a microplate reader and photographed using a fluorescence microscope. 2.5. Evaluation of protein leakage. The BCA Protein Assay Kit (cat#PC0020; Beyotime) was used to detect protein leakage from bacteria induced by samples after irradiation. In brief, E. coli and S. aureus were washed twice with PBS (pH=7.4) by refrigerated centrifuge (6,000 rpm) for 5 min and diluted back in PBS to the desired working concentration at an OD600 of 0.05-0.1. Afterwards, samples (  6mm  2mm ) were added to the 150 μL diluted bacterial solutions in 96-well plates and irradiated 9



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for 10 min. And the 100 μL treated bacteria were further withdrawn and diluted to 500 μL. The diluted bacterial solutions were then centrifuged (6,000 rpm) for 5 min by refrigerated centrifuge. Finally, the supernatant liquid was immediately withdrawn and the relative protein leakage of each sample was determined by a microplate reader at OD562. 2.6. In vivo antibacterial tests and would healing evaluation. All the animal experiments and Sprague Dawley rats (200-220 g body weight) were supported by Hubei Provincial Centers for Disease Prevention & Control. In brief,29 there were four groups:

Ctrl

(PAM

hydrogel),

PTT

(PAM-PDA

hydrogel),

PTDT

(PAM-PDA/Ag@AgCl hydrogel) and 3M group (3M wound dressing for the traditional treatment group), and each group contained eight rats. Before the surgery of creating the round wounds on the backbones of rats using a skin biopsy punch (diameter: 6 mm), anaesthetizing these rats using 4 wt.% pentobarbital sodium salt (1 mL/kg). Afterward, 10 μL bacteria (S. aureus, 1.0*107 CFU/mL) were added into the traumas of four groups and the samples were covered on the traumas. Then the traumas covered with different samples were further treated for 10 min by the xenon lamp (0.2 W/cm2, 1 m high). After the surgery, all of the rats were dressed using sterile medical tapes and individually raised in cages. Every 2 days, the samples on the traumas were changed with corresponding dressings and continued to 12 days. And the wounds with samples were further irradiated for 10 min under a xenon lamp every 2 days. After treated for 2, 4, 8 and 12 days, the traumas were photographed and the skin tissues of traumas were then obtained for histological analysis. And routine analysis of blood was also evaluated. Finally, after 12 days of treatment, H&E staining of major organs was used to test the biosecurity of the samples.

10


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3. RESULTS AND DISCUSSION 3.1. Synthesis and characterizations of the hydrogels. As indicated in Figure 1A, the polyacrylamide (PAM) hydrogel exhibited the sparsely macroporous sponge-like structures. As a contrast, after composited with dopamine, the PAM-PDA hydrogel showed the compactly and uniformly macroporous sponge-like structures (Figure 1B), which can be attributed to that PDA chains mutually crosslinked with the PAM network via noncovalent interaction during this course.50 And Ag+ was easily to combine with Cl- in Tris-HCl buffer solution to form AgCl. Meanwhile, partially formed AgCl was reduced to metallic Ag through reduction by catechol groups in the PDA chains,51 and consequently to form the Ag@AgCl nanostructures embedded in hydrogel (the inset image in Figure 1C). In addition, Figure 1C shows the less compactly network structures of the PAM-PDA/Ag@AgCl hydrogel compared to the PAM-PDA hydrogel because of the partial consumption of catechol groups in the PDA chains when reducing AgCl, which led to the weakness of interaction between the PAM network and PDA chains. Moreover, the reduced metallic Ag was evidenced by the distinct diffraction peaks (2θ) at 38.1o, 44.2o which are indexed to the (1 1 1) and (2 0 0) diffractions,52 respectively, while the concerted peaks at 2θ = 27.7°, 32.1°, 46.2°, 54.7°, 57.4°, and 67.4° was clearly detected, which were indexed to the (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), and (4 0 0) diffractions of AgCl,53 respectively shown in Figure 1D. The PAM-PDA hydrogel exhibited the maximum tensile strain of ca. 250%, which was higher than that of the PAM-PDA/Ag@AgCl hydrogel (ca. 200%) and the PAM hydrogel (ca. 100%) as indicated in Figure 1E. And the PAM-PDA hydrogel was stretched from 20 mm to 72 mm while the PAM hydrogel and the PAM-PDA/Ag@AgCl hydrogel was only stretched from 20 mm to 38 and 59 mm, 11



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respectively as shown in Figure S1. Furthermore, the pressure strength of the PAM hydrogel with a cylindrical shape was ca. 2.2 MPa at the compression strain of ca. 80%, while the pressure strengths for the PAM-PDA hydrogel and the PAM-PDA/Ag@AgCl hydrogel were only ca. 0.4 and 0.6 MPa, respectively, with the same compression strain. After removed the compression force, the PAM hydrogel broke severely while the PAM-PDA and PAM-PDA/Ag@AgCl hydrogels automatically and rapidly recovered to their original cylindrical shapes as shown in Figure

S2.

These

results

of

mechanical

properties

indicated

that

the

synergistic contribution of the noncovalent interaction in the PDA chains and covalent bonds in PAM network led to the high stretch-ability and toughness of this composite hydrogel.

Figure 1. Characterizations of the hydrogels. (A)-(C) SEM images of the PAM hydrogel, PAM-PDA hydrogel, and PAM-PDA/Ag@AgCl hydrogel. Scale bar, 100 μm. The inset image shows the Ag@AgCl nanostructures embedded in hydrogel. Scale bars, 100 nm. (D) XRD patterns of the hydrogels. The inset shows the magnifying diffraction peaks of metallic Ag. (E)-(F) The typical tensile stress-strain 12


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and compressive stress-strain curves of the hydrogels. 3.2.

Evaluation

of

photothermal

and

photodynamic

properties.

Polydopamine (PDA) can show excellent photothermal property due to its broad absorption from the visible to NIR wavelengths (300-1000 nm).20 As shown in Figure 2A, with the same amount of PAM, the PAM-PDA and PAM-PDA/Ag@AgCl hydrogels exhibited an obviously enhanced absorption compared to the PAM hydrogel, which was attributed to the combination of PDA. To evaluate the photothermal effect of the hydrogels, the samples were irradiated in 150 μL PBS solution. As indicated in Figure 2B, the solution temperatures of the PAM, PAM-PDA, and PAM-PDA/Ag@AgCl hydrogels reached 35.7, 51.3 and 52.1 oC

after 600 s irradiation, respectively, which was fully consistent with the results of

UV-Vis-NIR absorption. Herein, the PAM, PAM-PDA and PAM-PDA/Ag@AgCl hydrogel was named as Ctrl group, photothermal therapy (PTT) group, and PTDT group. In order to evaluate the effects of ROS generated from PAM-PDA/Ag@AgCl hydrogel on bacteria alone, the 96-well plates containing the PAM-PDA/Ag@AgCl hydrogel and 150 μL PBS solution were immersed into the ice bath during the irradiation, and the solution temperature only reached 32.8 oC after 600 s. This group with PAM-PDA/Ag@AgCl hydrogel immersed in ice bath but without heat effect was labeled as PDT group. As shown in Figure 2C, the electron spin resonance (ESR) spectra were recorded by irradiating spin traps containing samples. Firstly, singlet oxygen (1O2) produced by samples was detected by 2,2,6,6-tetramethylpiperidine (TEMP). The pure TEMP, Ctrl group, and PTT group were ESR silent, while the PDT and PTDT groups showed the typical three lines (1:1:1) of ESR spectra after irradiation for 10 min, which should be the characteristic spectra for the adduct formed between TEMP 13



and

1O .54 2

And

hydroxyl

radical

(•OH)

was

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perceived

by

the

5,5-Dimethyl-1-pyrroline-N-oxide (DMPO). Similarly, the pure DMPO, Ctrl group, and PTT group showed no ESR signal, while the ESR spectra of four lines (1:2:2:1) were observed after PAM-PDA/Ag@AgCl hydrogel was irradiated either in the air or in the ice bath, which was the typical characteristic for the reaction between DMPO and •OH.55 These results indicated that the 1O2 and •OH were generated from the irradiation of Ag@AgCl nanostructures in the hydrogel. In addition, the relavive intensity of ESR signal produced in PDT group was almost the same as that of in PTDT group for the same recording time, indicating that PDT group and PTDT group showed almost the same 1O2 and •OH generation ability, which was attributed to the same hydrogels (PAM-PDA/Ag@AgCl hydrogels) in the two groups. And the difference between the two groups was only the presence or absence of ice during the irradiation under the visible light. And the photothermal effect has little effect on the production of ROS in this photosensitized oxidation reaction system. As shown in Figure S3, after irradiation for 10 min, the PAM-PDA/Ag@AgCl hydrogel immersed in the pure 2,2,6,6-tetramethylpiperidine (TEMP) solution showed ESR silent. However, the typical three lines (1:1:1) of ESR spectra were observed after added deionized water into the pure TEMP solution (the final concentration is 50 mM) containing PAM-PDA/Ag@AgCl hydrogel and then irradiated for 10 min, which indicated that the oxygen in deionized water was involved in the photosensitized oxidation reactions. Type 1 and 2 reactions are rational for being oxygen-dependent, which means that type 1 and 2 photosensitized oxidation reactions require oxygen as a reagent.56,57 Therefore, we suspected that the process of ROS production from PAM-PDA/Ag@AgCl hydrogel was the type 1 and 2 reactions. Since the ESR characterization merely provided qualitative information 14


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regarding the generation of 1O2, in order to provide quantitative information, the production of 1O2 was further evaluated by ADMA, which showed a decrease in the absorption intensity at 400 nm after reacted irreversibly with 1O2.57 Figure S4 showed that the change in the absorption intensity was negligible in the both of Ctrl and PTT groups, while the PDT and PTDT groups showed a significant decrease absorption intensity at 400 nm with the irradiation time increasing, which indicated the generation of 1O2. However, compared to those reported highly efficient generation of 1O

2

systems with the decrease in absorption intensity of more than 50% within 10

min,58,59 this system only decreased from 0.086 to 0.059 (31.40% decline), suggesting a moderate efficiency in generation of 1O2.

Figure 2. The PTT and PDT performance of different samples. (A) Visible-NIR absorption of PAM, PAM-PDA, and PAM-PDA/Ag@AgCl hydrogels. (B) Temperature changes of PAM hydrogel (Ctrl), PAM-PDA hydrogel (PTT), 15



PAM-PDA/Ag@AgCl hydrogel in the ice bath (PDT) and PAM-PDA/Ag@AgCl hydrogel (PTDT) with irradiation (0.2 W/cm2, 1 m high) in the 150 μL PBS solution. (C) The ESR spectra of three lines (1:1:1, 1O2) and four lines (1:2:2:1, •OH) induced by samples (Ctrl, PTT, PDT, and PTDT) and trapped by TEMP (50 mM) and DMPO (0.1 M), respectively, after irradiated the spin traps with samples for 10 min (0.2 W/cm2, 1 m high). The pure TEMP and DMPO were also recorded for comparison. 3.3. In vitro antibacterial activity. As shown in Figure 3A, a large number of viable colonies grew well on LB agar plates in Ctrl group (either no light or under light) and PTT group without light, while a small decrease of viable colony units occurred after illumination with visible light in PTT group, which should be attributed to the photothermy on bacteria. And the PTT group showed antibacterial efficiencies of under 50% after 10 min of irradiation as shown in Figure 3B and Figure 3C. Also, the bacteria exposed to PDT and PTDT groups without irradiation showed a decreased bacterial survival, which was attributed to the release of Ag+ (Figure S5) from the PAM-PDA/Ag@AgCl hydrogel (PDT and PTDT groups) during the co-culture process. The PAM-PDA/Ag@AgCl hydrogel exhibited antibacterial efficacy of only 89.85% and 81.25% for E. coli and S. aureus, respectively, in darkness for 24 h (Figure S6). Moreover, PDT group only killed ca. 70% of bacteria after 10 min of irradiation as shown in Figure 3B and Figure 3C, which was attributed to the generation of a small amount of 1O2 and •OH. The moderate antibacterial efficiency induced by PDT in this system was no match for other published PDT system,26-29 which may be owed to a small content of Ag presented in nanostructured Ag@AgCl that only generated moderate ROS. The photocatalytic activity of the Ag@AgCl nanostructures in the visible light region was induced by the strong coupling of the Surface Plasmon Resonance (SPR) effect of metallic Ag.48 As a 16


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contrast, for both E. coli and S. aureus, only few viable colonies were observed in PTDT group under visible light for 10 min (Figure 3A). And the antibacterial ratios for E. coli and S. aureus were 99.91% and 99.97%, respectively, as calculated in Figure 3B and Figure 3C, indicating that rapid and effective antibacterial performance was achieved by the synergistic photothermy and moderate ROS generation. In order to show the advantage of PTDT, this system was also challenged by multiple resistant Staphylococcus aureus (MRSA). Figure S7A showed that the viable colonies of MRSA in the PTDT group were significantly decreased compared to the Ctrl group after irradiation for 10 min. And the antibacterial efficiency of the PTDT group under visible light was 99.18% as calculated in Figure S7B, indicating that PTDT also showed excellent antibacterial effect against MRSA.

Figure 3. In vitro antimicrobial evaluation. (A) the viable colony units grew on the plates after treated with samples (Ctrl, PTT, PDT, and PTDT) without irradiation or 17



under visible light for 10 min. (left: E. coli; right: S. aureus) The corresponding antibacterial abilities for (B) E. coli and (C) S. aureus after treated by the samples (Ctrl, PTT, PDT, and PTDT). The independent tests had been conducted three time for each group (*P < 0.05, **P < 0.01, ***P < 0.001, n=3). The membrane integrity of treated bacteria was then investigated by SEM and TEM. As shown in Figure 4A and Figure 4B, the typical morphologies with the smooth surface of E. coli (rod shape) and S. aureus (spherical shape) were observed in Ctrl group (either no light or under light) and PTT group without light. However, E. coli bacterial membranes were distorted and wrinkled with the varying degrees (red arrows in Figure 4A) and S. aureus bacterial membranes appeared lesions and holes (red arrows in Figure 4B) in other groups. Moreover, the bacterial membranes in PTDT group displayed the most serious destruction after irradiation with visible light for 10 min. In addition, TEM and the corresponding EDS analysis were further utilized to understand the damage to bacterial membranes and change in the intracellular components of bacteria. The two kinds of bacteria showed bacterial membrane integrity and compact intracellular substrates in Ctrl group after irradiation for 10 min. However, both S. aureus and E. coli in other groups showed different degrees of distortion. For E. coli, the bacterial membranes appeared to be disorganized (black arrows) and damaged (green arrows), while for S. aureus, the edge of the bacterial membranes became obscure (blue arrows), which was observed in the PTT, PDT, and PTDT groups. Furthermore, the corresponding EDS analysis showed that the contents of main components of intracellular substrates (C and O)60,61 sharply dropped in PTT, PDT, and PTDT groups compared to the Ctrl group, indicating the loss of some intracellular substrates. And the bacteria in PTDT group showed the largest amount of leakage of carbon and oxygen after irradiation for 10 18


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min. For E. coli, the carbon content was reduced from 44.6% to 8.6% and the content of oxygen was reduced from 5.8% to 0.8%, while for S. aureus, the amount of carbon was reduced from 46.8% to 7.9% and the content of oxygen was decreased from 5.5% to 1.1%. These results revealed the serious disruption of bacterial membranes during the irradiation

Figure 4. The evaluation of bacterial morphologies. SEM and TEM images as well as EDS analysis of (A) E. coli and (B) S. aureus after induced by samples (Ctrl, PTT, PDT, and PTDT) in the darkness for 10 min or under visible light for 10 min. 500 nm scale bars for SEM images and TEM images in E. coli; 200 nm scale bars for TEM images in S. aureus. (Red arrows: distorted membranes; Black arrows: disorganized E. coli bacterial membranes; Green arrows: damaged E. coli bacterial membranes; Blue arrows: obscured and damaged S. aureus bacterial membranes) 3.4. Investigation of the bacterial membrane disrupting mechanism and subsequent changes in the interior of the bacteria. The severe morphology disruption (Figure 4) caused by the synergistic action of PTT and PDT indicated that the bacterial membrane was indispensable for the survival of bacteria. Herein, the 19



bacterial membrane permeation, ROS within the bacteria and protein leakage from bacteria were systematically evaluated. Firstly, the 8-anilinonapthalene-1-sulfonic acid (ANS) was used to detect the outer membrane permeability of E. coli because there was the bacterial outer membrane only in Gram-negative bacteria.62 ANS was a probe, which showed the increased fluorescence by binding to hydrophobic region of outer membrane,63 which indicated enhanced bacterial outer membrane permeation. Figure 5A shows fluorescence images of treated E. coli after co-cultured with ANS. The Ctrl and PDT groups displayed almost no fluorescence after treated for 10 min under visible light, while the intense blue fluorescence was observed in both the PTT and PTDT groups, indicating significantly enhanced outer membrane permeation of E. coli. And the corresponding fluorescence intensities of treated E. coli were measured and shown in Figure 5B. The PTT group showed similar fluorescence intensity compared to PTDT group but much higher fluorescence intensity than that of in the Ctrl and PDT groups. However, the gap of fluorescence intensity between the PTT group and PDT (or Ctrl) group was narrowed after treated for less than 4 min as indicated in Figure S8, which was ascribed to the lower temperature within the first 4 minutes as shown in Figure 2B. And this gap was widened when the temperature increased to 50 oC after treated for more than 4 min. These results indicated that PTT enhanced bacterial outer membrane permeation, namely the outer membrane of the E. coli was more sensitive to heat. Moreover, an outer panniculus, a peptidoglycan cell wall, and a cytoplasmic panniculus made up the complex cell envelope of Gram-negative bacteria, and the outer panniculus was responsible for an essential load-bearing element in Gram-negative bacteria because it was stiffer than the cell wall.62 Therefore, the E. coli bacterial death was accelerated by the disruption of the outer membrane.62 20


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As shown in Figure S9, with the increase of treating time, the fluorescence intensity increased in the PTDT group, suggesting that the outer panniculus permeability of E. coli was also enhanced with the increase of treating time. And the corresponding fluorescence images in Figure S10 also showed enhanced blue fluorescence with the increase of treating time in the PTDT group. The PTT group displayed the similar trends compared to the PTDT group shown in Figure S11. However, the fluorescence intensity remained basically stable with the increase of treating time in the Ctrl and PDT groups, indicating that the moderate 1O2 and •OH had little effect on the permeability of E. coli bacterial outer membrane. In addition, the inner membrane (cytoplasmic membrane) permeability of bacteria was investigated by ONPG,64,65 and the high value of OD420 indicated enhanced bacterial inner membrane permeability. And the PTDT group showed the highest value of OD420 (Figure 5C and Figure 5D), indicating that the synergistic action of PTT and PDT also effectively increased the inner membrane permeation of bacteria. Moreover, as the treating time increased, the OD420 value obtained from the Ctrl group remained stable while the one from other groups gradually rose. In addition, the OD420 value in the PDT group was always higher than that of in the PTT group, suggesting that the bacterial inner membrane was more sensitive to the 1O2 and •OH than to heat. The aforementioned bacterial membrane permeation results indicated that the synergistic action of photothermy and moderate ROS efficiently enhanced the outer and inner membrane permeation of bacteria. Furthermore, the E. coli bacterial outer membrane was more sensitive to photothermy but the 1O2 and •OH exhibited more significant effects on the inner panniculus of two kinds of bacteria.

21



Figure 5. The analysis of bacterial membrane permeability. (A) The changes of outer membrane permeation of E. coli induced by samples (Ctrl, PTT, PDT, and PTDT) after irradiation for 10 min. Blue fluorescence of the ANS probe indicates enhanced E. coli bacterial outer membrane permeability. Scale bar, 50 μm. (B) The corresponding fluorescence intensities of treated E. coli were measured between 450-600 nm with excitation at 380 nm. And the effects on the inner panniculus permeability of (C) E. coli and (D) S. aureus after induced by the samples (Ctrl, PTT, PDT, and PTDT) under visible light for 0, 1, 2, 4, 6, 8, and 10 min orderly. The high value of OD420 indicates enhanced bacterial inner membrane permeability. The independent experiments were conducted in triplicate (n=3). The subsequent changes in the interior of the bacteria after the bacterial membrane disruption were further detected to better shed light on the antibacterial mechanism induced by the synergistic action of PTT and PDT. And DCFH-DA was used to detect the level of ROS within the bacteria induced by samples after irradiation for 10 min. According to the ROS Assay Kit, DCFH-DA was non-fluorescence, which freely permeated the cell membrane and enter into the 22


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bacteria, and it was hydrolyzed to form DCFH by the esterase in bacteria. But DCFH cannot permeate the cell membrane, so that the probe was easy to be loaded into the bacteria. Then ROS in bacteria can oxidize DCFH to form the fluorescent DCF. Detecting the fluorescence of DCF directly reflected the level of ROS within the bacteria. The strongest green fluorescence was observed in the PTDT group shown in fluorescence images of treated bacteria (Figure 6A), which should be owed to the entering of ROS into the bacteria after the bacterial membrane disruption during the irradiation. According to Figure 2C, the PDT groups

generated almost the same

amount of ROS as the PTDT group under the same condition. However, the green fluorescence in the PDT group was weaker than that of in the PTDT group (Figure 6A), indicating that the ROS was more easily to enter into the bacteria after efficiently enhancing the membrane permeation of bacteria by the synergistic action of PTT and PDT. Moreover, both the Ctrl and PTT groups without the generation of ROS also displayed little fluorescence after irradiation for 10 min, which was attributed to the very low level of ROS existed in bacteria during the normal aerobic respiration.66 And the corresponding fluorescence intensities of treated E. coli and S. aureus were measured at 525 nm with excitation at 488 nm as shown in Figure 6B and Figure 6C, respectively. The higher value of fluorescence intensity directly reflected the higher amount of ROS in the bacteria induced by samples. Compared to the Ctrl group, the PTT group showed almost the same fluorescence intensity, which indicated that almost the same amount of ROS in the bacteria was induced by these two groups. However, the amount of ROS in the E. coli and S. aureus induced by the PDT group was 4-fold and 2-fold of that caused by the Ctrl group, respectively. In contrast, the PTDT group induced about 10-fold and 4-fold of ROS in the E. coli and S. aureus triggered by PAM hydrogel, respectively, which was fully consistent with the 23



fluorescence images. In addition, the obvious cytoplasm leakage detected by TEM (Figure 4) was further evidenced by the protein leakage analysis, which was taken as the representative indicator of cytoplasm leakage,67 and the high value of OD562 indicated the enhanced protein leakage. The PTDT group showed the highest value of OD562 (Figure 6D and Figure 6E), indicating that the synergistic action of photothermy and moderate ROS efficiently promoted protein leakage of bacteria, which should be ascribed to the most severe disruption of the bacterial membrane in PTDT group after the irradiation (Figure 5). These results suggested that the synergistic PTT and PDT accelerated bacterial death by the outbreak of oxidative stress in bacteria and the protein leakage after the bacterial membrane damage. 3.5. In vivo test of S. aureus-accompanied wound healing. The photo-assisted therapeutic abilities for wound healing of different groups (Ctrl: the group without antibacterial efficacy; 3M: traditional treatment group; PTT: the group with moderate antibacterial efficacy; PTDT: the group with highly efficient antibacterial efficacy) were investigated by animal models. Ctrl and 3M groups showed serious bacterial infection with ichor in the traumas after treatment for 2 days (Figure 7A), while PTT group displayed less ichor in traumas and no obvious infection was observed in PTDT group. After treated for 12 days, the Ctrl, 3M, and PTT groups were not well healed while the PTDT group was almost healed, which suggested that rapid bacterial killing by the synergistic action of PTT and PDT played an important role in wound healing.

24


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Figure 6. The determination of ROS in the bacteria and protein leakage of bacteria. (A) The ROS in the bacteria induced by samples (Ctrl, PTT, PDT, and PTDT) under visible light for 10 min. Green fluorescence of the DCF indicates enhanced generation of ROS in the interior of bacteria. Scale bar, 50 μm. And the corresponding fluorescence intensities of treated (B) E. coli and (C) S. aureus were measured at 525 nm with excitation at 488 nm. The protein leakage from (D) E. coli 25



and (E) S. aureus bacteria induced by samples (Ctrl, PTT, PDT, and PTDT) after irradiation for 10 min. The high value of OD562 indicates enhanced protein leakage. Hematoxylin and eosin (H&E) and Giemsa staining were further performed to evaluate the bacterial infection of the mid-portion of the tissues. It was observed from Figure S12, all groups played disordered skin epidermal layers and massive inflammatory cells (red arrows) after treated for 2 and 4 days. After treated for 8 days, inflammatory cells decreased in all groups, especially in PTDT group. After treatment for 12 days, inflammatory cells (red arrows), cell vacuolization (blue arrows) in granular layer and monolayer keratinocytes (black arrows) in the epidermis were observed in the Ctrl group (Figure 7B). And 3M and PTT groups also appeared monolayer keratinocytes (black arrows) and inflammatory cells (red arrows), suggesting that the wounds in the Ctrl, 3M, and PTT groups were still not completely healed after 12 days of treatment. In contrast, normal tissues were observed in the PTDT group after treatment for 12 days. Furthermore, the bacterial residue was identified by Giemsa staining. As shown in Figure 7B, significant bacterial residue (green arrows) was observed in the Ctrl, 3M, and PTT groups after two days of treatment. As a contrast, there was almost no detectable sign of bacteria in the PTDT group, which was in good agreement with the optical photographs of the traumas in 2 days.

26


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Figure 7. In vivo investigation. (A) The therapeutic results by different dressings (Ctrl, 3M wound dressing, PTT, and PTDT) and the corresponding photographs of the rats’ traumas at specific times. (B) The histological analysis of the reconstructed skin tissues. Scale bar, 100 μm for H&E staining; 50 μm for Giemsa staining. (Blue arrows: cell vacuolization; Black arrows: monolayer keratinocytes; Red arrows: inflammatory 27



cells; Green arrows: bacteria) It is well-known that massive white blood cells (WBC), including neutrophils and lymphocyte could be produced by the immune system after bacterial infection in vivo, which can provide limited antibacterial activity.68,69 As shown in Figure 8, the number of WBC, neutrophils, and lymphocyte in the Ctrl, 3M, and PTT groups was much high than that of in PTDT group after 2 days of treatment, which was attributed to the serious bacterial infections in Ctrl, 3M, and PTT groups shown in Figure 7. Moreover, the number of WBC, neutrophils, and lymphocyte in the Ctrl, 3M, and PTT groups gradually reduced with the increase in time, which was owed to the reduction of infection with the treatment. Interestingly, the number of WBC, neutrophils, and lymphocyte in the PTDT group increased at the mid-stage of the treatment, which should be attributed to the release of Ag+ promoted the immune function and produced a large number of WBC, neutrophils, and lymphocyte for synergistic antibacterial activity. After 12 days of treatment, the number of three kinds of immune cells decreased in all groups, especially for PTDT group with the most extensive degree, which was in good agreement with wound healing (Figure 7A) and immunohistological analysis (Figure 7B and Figure S12) In addition, as shown in Figure 9, after 12 days of treatment, no appreciable damage was observed in major organs including liver, kidney, spleen, heart, and lung according to the H&E staining image in all groups. Additionally, EDS detection of above organs showed that there was no significant silver signal detected in either the Ctrl or PTDT groups (Figure S13), which suggested that Ag@AgCl nanostructures hardly accumulate in vivo and the prepared PAM-PDA/Ag@AgCl hydrogel was a safe phototherapeutic dressing for wound healing. 28


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Figure 8. Blood analysis in vivo. The routine analysis of blood of the (A) WBC, (B) neutrophils, and (C) lymphocyte in the whole blood of the rats after treatment with samples (Ctrl, 3M wound dressing, PTT, and PTDT) for 2, 4, 8, and 12 days. 4. CONCLUSION To summarize, we have developed a novel phototherapeutic system for rapid wound healing with serious bacterial infection under relatively low temperature (52.1 oC)

and moderate ROS generation. This photothermal/photodynamic synergistic

therapy (PTDT) efficiently enhanced bacterial membrane permeation by the effect of photothermy on the outer membrane and the ROS on the inner membrane. As a result, the outbreak of oxidative stress in bacteria and severe protein leakage from the bacteria were occurred after the bacterial membrane disruption, which led to the bacterial death. Importantly, this phototherapeutic system with rapidly bacterial killing ability has a great potential for clinical sterilization through the combination of PTT and PDT due to the relatively low temperature and moderate ROS. It is worth noting that the antibacterial mechanism indicated in this work has the great guiding significance for the design of other advanced antibacterial materials and techniques, such as photo/drug synergistic therapy and photo/inorganic antimicrobial agent synergistic therapy, which may achieve satisfactory antibacterial efficiency with a small number of drugs or inorganic antimicrobial agents.

29



Figure 9. The assessment of biological security. H&E staining of major organs after treated with samples (Ctrl, 3M wound dressing, PTT, and PTDT) for 12 days. Scale bars, 100 μm.

ASSOCIATED CONTENT Supporting Information Available: Supporting Information including Experimental section (Fabrication of hydrogels; Characterization of hydrogels; Tensile and pressure tests) and Supplementary Figures (Digital photos of stretched and compressed hydrogels; ESR spectra; Decay curves of the absorption of ADMA; Silver ions release profiles; Antimicrobial 30


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activities of silver ions; The abilities in killing MRSA; The fluorescence intensity curves of outer membrane permeability; The fluorescence intensity images of outer membrane permeability; The immunology of histological images; The EDS analysis of the H&E staining glass slices). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] (S.L. Wu)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is jointly supported by the National Natural Science Foundation of China, Nos. 51871162, 51671081, 51801056 and 51422102, and the National Key Research and

Development

Program

of

China

No.

2016YFC1100600

(sub-project

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Photodynamic Synergistic Therapy by Disrupting

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