A Repeatable Photodynamic Therapy with Triggered Signaling

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A Repeatable Photodynamic Therapy with Triggered Signaling Pathways of Fibroblast Cell Proliferation and Differentiation to Promote Bacteria-Accompanied Wound Healing Congyang Mao, Yiming Xiang, Xiangmei Liu, Zhenduo Cui, Xianjin Yang, Zhaoyang Li, Shengli Zhu, Yufeng Zheng, Kelvin Wai Kwok Yeung, and Shuilin Wu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b08500 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 27, 2018

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A Repeatable Photodynamic Therapy with Triggered Signaling Pathways of Fibroblast Cell Proliferation and Differentiation to Promote Bacteria-Accompanied Wound Healing Congyang Maoa,b, Yiming Xiang,a,b, Xiangmei Liub, Zhenduo Cuia, Xianjin Yanga, Zhaoyang Lia, Shengli Zhua, Yufeng Zhengc, Kelvin Wai Kwok Yeungd, Shuilin Wua,b* a

School of Materials Science & Engineering, Tianjin University, Tianjin 300072, China

b

Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials,

Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei Key Laboratory of Polymer 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)

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ABSTRACT Despite the development of advanced antibacterial materials, bacterial infection is still a serious problem for the wound healing because it usually induces severe complications and cannot be eradicated completely. Most current materials cannot simultaneously provide antibacterial activity, reusability, and biocompatibility as well as participate in stimulating cell behaviors to promote bacteria-accompanied wound healing. This work fabricated a hybrid hydrogel embedded with two-dimensional (2D) few-layer black phosphorus nanosheets (BPs) via simple electrostatic interaction. Within 10 min, 98.90% Escherichia coli (E. coli) and 99.51% Staphylococcus aureus (S. aureus) can be killed rapidly by this hybrid, due to its powerful ability to produce singlet oxygen (1O2) under simulated visible light. In addition, this hydrogel also shows a high repeatability, i.e., the antibacterial efficacy can still reach up to 95.6% and 94.58% against E. coli and S. aureus, respectively, even after challenging bacteria up to four times repeatedly. In vitro and in vivo results reveal that BPs in this hybrid hydrogel can promote the formation of the fibrinogen at the early stages during the tissue reconstruction process for accelerated incrustation. In addition, BPs can also trigger phosphoinositide 3-kinase (PI3K), phosphorylation of protein kinase B (Akt), and extracellular signal-regulated kinases (ERK1/2) signaling pathways for enhanced cellular proliferation and differentiation. Moreover, the hydrogel causes no appreciable abnormalities or damage to major organs (heart, liver, spleen, lung, and kidney) in rats during the wound healing process. Therefore, this BPs-based hydrogel will have a great potential as a safe multimodal therapeutic system for active wound healing and sterilization. Keywords: black phosphorus, photodynamic therapy, hydrogel, multimodal therapeutic system, wound healing 2


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Bacterial infections occur in tissues during the wound repair process after exposure to any injury, and serious inflammatory reactions always result in unsuccessful wound healing. Using an effective antimicrobial wound dressing to combat bacterial infections would minimize inflammatory reactions.1-5 Hydrogels are hydrophilic, environmentally-friendly polymers with three-dimensional polymeric networks and are widely used as wound dressings.6-10 Earlier generations of dressings were modified with drug or inorganic antimicrobial agents, but they still could induce systemic and organ toxicity to some extent, as well as microbe resistance.11-16 The development of a highly effective antibacterial strategy is urgent. An increasing interest in biological studies of two-dimensional (2D) nanomaterials with extraordinary photocatalytic properties has led to a burst of activity in the development of 2D nanomaterials for antimicrobial agents, such as graphene, MoS2, and MXenes. The proposed mechanism of action for these 2D nanomaterials is their ability to produce reactive oxygen species (ROS).17-22 ROS, especially for singlet oxygen (1O2), are best known for their ability to cause damage in cellular membranes, proteins, and even DNA.23 The 1O2 has been studied in a wide range of applications for photooxidation catalysis and photodynamic therapy (PDT) due to its strong oxidizing ability.24-30 Recently, 2D few-layer black phosphorus nanosheets (BPs) have emerged because of their fascinating electrical, optical, and thermal properties.31-35 Black phosphorus (BP) can be exfoliated easily into ultrathin 2D nanosheets because weak van der Waals forces result in puckered phosphorus layers.36 As a metal-free layered semiconductor, BPs differ from graphene, in that they exhibit a thickness-dependent band gap, which varies from about 0.3 eV for bulk to 2.0 eV for a single layer, indicating broad absorption across the entire visible light region.37-40 Considering its unique electronic 3


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structure, the few-layer BPs have also been used to generate 1O2, which can be further studied in PDT applications.41,42 However, the studies of BP mainly focus on its physical and transport properties, and using BPs to produce 1O2 that will kill bacteria under visible light has not been explored. It is necessary for wound healing to minimize infections and increasing tissue rehydration at the wound site. Furthermore, in order to accelerate wound healing, it is quite important to motivate endogenous cells to promote skin cells to participate in skin regeneration actively.43 Wound healing is a complex, dynamic and sequential process associated with coagulation, inflammation, angiogenesis, tissue formation and tissue remodeling.44 And fibrinogen has a double functions: yielding monomers that polymerize into fibrin, and acting as a cofactor in platelet aggregation, which can accelerate incrustation and wound healing.45-47 Moreover, the proliferation and differentiation of the fibroblasts at the wound site play a key role during skin regeneration, and fibroblasts proliferate and contribute to the synthesis of extracellular matrix.48 Among the inside-out signalings, phosphoinositide 3-kinase (PI3K), phosphorylation of protein kinase B (Akt), and extracellular signal-regulated kinases (ERK1/2) signaling pathways are involved in biomaterials induced cellular proliferation and differentiation.49,50 Herein, this article outlines the concept of using BPs-based hydrogel for photodynamic therapy integrated with stimulating skin cell behaviors that can promote the regenerative activities of the skin cells and actively participate in skin regeneration to accelerate bacteria-accompanied wound healing. This hybrid hydrogel is fabricated via a simple electrostatic interaction between the BPs and chitosan (CS) hydrogel. As illustrated in Scheme 1, this hydrogel system not only possesses a broad and reusable antibacterial efficacy against both Gram-positive and Gram-negative 4


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bacteria through the generation of 1O2 under visible light irradiation, but also enhances the formation of the fibrinogen at the early stages of the tissue repair process for accelerated incrustation, as well as triggers PI3K/Akt and ERK1/2 signaling pathways for enhanced cellular proliferation and differentiation to promote wound healing accompanied with bacteria-infections.

RESULTS AND DISCUSSION Synthesis and characterizations of the CS-BP hydrogel The BPs show typical 2D structures as indicated in the low-resolution transmission electron microscopy (TEM) image (Figure 1a). In addition, the high-resolution TEM (HRTEM) image demonstrates the crystallinity of the BPs (Figure 1b), and the lattice plane spacing is measured to be 0.166 nm corresponding to the (200) lattice planes. Typical network structures of chitosan (CS) are shown in the scanning electron microscopy (SEM) image (Figure 1c). Figure 1d showed shows the SEM image of BPs loaded with CS hydrogel. BPs were incorporated into the bulk of CS hydrogel uniformly, which can be observed by surface morphology and cross-section image as indicated in Figure S1a and Figure S1b, respectively. In addition, energy dispersive spectroscopy (EDS) analysis further confirms uniformly distributed BPs on the surface and cross-section as shown in Figure S1c and Figure S1d, respectively. The original brown BP aqueous solution was changed into colorless transparent solution after the most of BPs were loaded by the CS hydrogel through electrostatic absorption in the solution, and then this BP-loaded CS hydrogel was taken out from the solution shown in Figure 1e. The positively charged CS hydrogel (43.1 mV) (Figure 1f) had the ability to adsorb negatively charged BPs (-25.6 mV) to form CS-BP hydrogel. The 5


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zeta potential measurements also show that the surface potential of CS hydrogel was decreased from 43.1 to 17.3 mV after BPs loading (Figure 1f), due to the negatively charged BPs were bonded to the positively charged CS hydrogel through electrostatic interaction. Moreover, there was a slight effect on the mechanical property of CS hydrogel after BPs loading, CS-BP hydrogel displayed larger strain when applied the same pressure stress (Figure S2). This may be attributed to the fact that the positive charges were partly neutralized by the BPs, resulting in the reduction of cationic-cationic repulsive forces, which can resist to part of pressure stress in the networks of CS hydrogel, thus inducing the final increase of the strain. In addition, both CS and CS-BP hydrogel can be well adhered to the skin (Figure S3).

Identification of singlet oxygen (1O2) and evaluation of antibacterial activity The electron spin resonance spectrometer (ESR) spectra obtained from irradiated solutions containing spin traps is indicated in Figure 2a. The control group of tissue culture plate (TCP, in the dark or irradiated for 10 min) and CS hydrogel (in the dark or irradiated for 10 min) as well as CS-BP hydrogel (in the dark for 10 min) were ESR silent, while the typical ESR spectrum of three lines with relative intensities of 1:1:1 was observed after being irradiated for 10 min in the presence of CS-BP hydrogel. The three-lines-spectrum with relative intensities of 1:1:1 was the characteristic spectrum for the adduct formed between 2,2,6,6-tetramethylpiperidine (TEMP) and 1

O2,51 which indicates that the 1O2 was generated by BPs in CS hydrogel during

visible light irradiation. Moreover, the optical property of the BPs was analyzed by UV−vis spectra (Figure S4). BPs exhibit a very broad light absorption across the entire visible light regions, indicating that BPs can be more likely irradiated by visible 6


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light. Moreover, the group with BPs alone (the amount of BPs was same with the one in CS-BP hydrogel) in the dark was also ESR silent, while the typical ESR spectrum of three lines was observed after being irradiated for 10 min (Figure S5). And the relative intensity of ESR spectrum was the same to the CS-BP hydrogel. The antibacterial activity of the hydrogels was investigated in two different bacterial strains, namely E. coli (Gram-negative) and S. aureus (Gram-positive), because they are responsible for most bacterial infections among the most common species.52 As shown in Figure 2b, viable colonies of both E. coli and S. aureus grew well on LB agar plates in the absence of hydrogels and irradiation (no light for TCP), while a very small decrease in bacterial survival occurred after illumination with simulated sunlight (light for TCP), which should be attributed to the small amount of ultraviolet light in simulated sunlight. CS hydrogel exhibited moderate antibacterial efficacy against E. coli (70%, Figure 2c) and S. aureus (50%, Figure 2d), either irradiated or in the dark for 10 min. Moreover, the antibacterial ratio reached ca. 85% when the bacteria were treated with CS hydrogel for 1 h (Figure S6 and Figure S7). The antibacterial mechanism of pure CS hydrogel is bacterial membrane disruption and then microbe death as sections of anionic microbial membrane are attracted to the cationic CS hydrogel.1 And the antibacterial efficacy of CS hydrogel increased as the extension of culturing time in the dark (Figure S8). However, the antibacterial ratio of CS hydrogel could only reach to 93.58% when the bacteria were treated for 4 h (Figure S8a). With the doping of BPs, CS-BP hydrogel also displayed moderate antibacterial efficacy against both E. coli and S. aureus in the darkness for 10 min, which may be attributed to the fact that the surface potential of CS-BP hydrogel was kept positively charged (17.3 mV, Figure 1f). And there was no significant difference for antibacterial activity between CS hydrogel and CS-BP hydrogel in the dark for 10 7


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min when the surface potential of CS hydrogel was decreased from 43.1 to 17.3 mV after BPs loading (Figure 2c and Figure 2d), while antibacterial ratio exhibited significant difference (82.67% against E. coli and 82.61% against S. aureus for CS hydrogel while 71.78% against E. coli and 44.93% against S. aureus for CS-BP hydrogel) when the bacteria were treated for 1 h in the dark (Figure S6 and Figure S7), which indicated that it had to take more time to achieve better antibacterial efficacy for contacting bacteria-killing of CS hydrogel through electrostatic absorption. However, a significant enhancement in antibacterial efficacy (98.90% against E. coli and 99.51% against S. aureus) was observed when the CS-BP hydrogel was illuminated by simulated sunlight for only 10 min, which is predominantly ascribed to the rapid production of intensive ROS from BPs after irradiation (Figure 2a). Moreover, antibacterial efficacy also reached over 99% (Figure S6 and Figure S7) when the CS-BP hydrogel was illuminated by simulated sunlight for 10 min and then incubate in the dark for 50 min (total 1 h). The morphologies and membrane integrity of bacteria on samples were examined by SEM. As indicated in Figure S9, the typical morphologies of E. coli with a smooth surface and rod shape, and S. aureus with a smooth surface and spherical shape, were observed on the surfaces of the TCP. The distorted and wrinkled membranes of E. coli (red arrows in Figure S9a) together with the lesions and holes of S. aureus (red arrows in Figure S9b) were observed on the surfaces of the CS hydrogel. The bacteria, especially S. aureus, were more seriously damaged when treated with CS hydrogel for 1 h than for 10 minutes (bacterial debris was observed in Figure S9b marked by green arrows), indicating that longer electrostatic interactions between bacteria and CS hydrogel provide better antibacterial effects,1 which is consistent with the results of spread plate. The intact bacteria were also observed near the BPs (black arrows in 8


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Figure S9) when the bacteria were treated with CS-BP hydrogel in the darkness, indicating the non-toxicity of BPs, while bacterial debris (green arrows in Figure S9b) and lesions of S. aureus (red arrows in Figure S9b) were also observed far away from the BPs on the surfaces of CS-BP hydrogel. However, the bacteria near the BPs were seriously damaged only after irradiation with simulated sunlight for 10 min due to the action of produced 1O2, which is in good agreement with spread plate results (Figure 2). In addition, once the positive charges of the CS hydrogel were partially neutralized by attached bacteria, the antibacterial efficacy of the CS hydrogel would be weakened accordingly. The CS hydrogel and CS-BP hydrogel were repeatedly challenged with S. aureus in the darkness for 1 h and under simulated sunlight for 10 min, respectively, up to four times each. As shown in Figure 3a and Figure 3b, even after three previous challenges with high concentrations of bacteria, CS-BP hydrogel still maintained 94.58% antibacterial efficacy against S. aureus in the final (fourth) challenge, which should be attributed to the 1O2 generated from BPs quickly and effectively sterilizing bacteria by damaging cellular membranes, proteins, and DNA.22 However, in the case of pure CS hydrogel, the antibacterial activity decreased rapidly with increasing challenges, due to positive charges on the CS hydrogel surface were masked and partially neutralized by adsorbed bacterial debris (green arrows in Figure S9).1 The evolution of CS hydrogel surface potential with each challenge by S. aureus is indicated in Figure 3c. The potential decreased with each challenge, from 43.1 mV with the first to 18.9 mV after three challenges. Besides S. aureus, the CS-BP hydrogel also still kept a reusable antibacterial efficacy against E. coli as indicated in Figure S10, maintaining a high efficacy of 95.6% in the fourth challenge.

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In vitro cytotoxicity and skin cell modulation Prior to in vivo animal experiments, in vitro cytotoxicity of both pure CS hydrogel and CS-BP hydrogel was evaluated as shown in Figure 4a and Figure 4b. The CS hydrogel exhibited cytotoxic effects on dermal fibroblasts compared to TCP either initially irradiated for 10 min or in the dark, indicating that positive charges on the CS hydrogel can also cause cellular damage. After seven days of co-culture, the number of living cells increased to a certain extent, due to adsorbed cell debris masking positive charges on the surface of CS hydrogel, resulting in alleviation of cytotoxic effects. However, after doping with BPs, the CS-BP hydrogel became very friendly to NIH-3T3 cells in the dark (Figure 4a), which can be also attributed to the positive charges of the CS hydrogel were partially neutralized by BPs and the excellent biocompatibility of BPs.53 Moreover, the CS-BP hydrogel exhibited cytotoxic effects after 1 day of co-culture once initially irradiated for 10 min compared to the CS-BP hydrogel in the dark (Figure 4b), indicating that 1O2 can also cause cellular damage. However, as shown in Figure 4b, NIH-3T3 cells can continue to proliferate after 3 and 7 days of co-culture, which can be also attributed to the neutralization of the positive charges and the excellent biocompatibility of BPs. More importantly, ROS including 1O2 are metabolic byproducts of aerobic respiration and responsible for maintaining redox homeostasis in cells, and that maintaining a basal level of ROS in cells can support cellular proliferation, physiological function, and viability,54-56 which may reduce the damage of ROS to cells. However, in general, a large number of ROS can damage cells to a certain extent in a short time. In addition, NIH-3T3 cells cultured on CS-BP hydrogel showed enhanced expression of smooth muscle alpha-actin (α-actin) compared to the cells exposed on TCP or on CS hydrogel (Figure 4c). The α-actin resulted in fibroblast proliferation and 10


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differentiation into myofibroblasts, which are particularly important for wound healing. Additionally, collagen, especially collagen type III (COL III), is vital for granulation tissue reorganization and basement membrane regeneration during wound healing.57,58 In this work, NIH-3T3 cells growth on CS-BP hydrogel exhibited significantly enhanced expression of COL III compared to cells cultured on TCP or CS hydrogel (Figure 4d).

In vivo evaluation of wound healing The photodynamic therapeutic efficacies of the different samples for wound healing were evaluated in animal models. As indicated in Figure 5a and Figure 5b, the traumas in a control and 3M group (dark and light) as well as a CS-BP group (dark) showed serious bacterial infection with ichor after two-day treatment, which is consistent with in vitro antimicrobial activity investigation (Figure 2d). After treatment for 14 days, the CS-BP group (light) was significantly healed, while other groups, including the CS-BP group (dark), were not well healed, indicating that rapid sterilization by light irradiated 1O2 generated from BPs played a crucial role in wound healing. Hematoxylin and eosin (H&E) and Giemsa staining of the mid-portion of the tissues were performed. As indicated in Figure S11 (dark) and Figure S12 (light), local tissue necrosis and accompanied by a large number of inflammatory cells (red arrows) can be observed in all groups after 2 days of treatment, and tissue edema with cell protein degradation and accompanied by a large number of inflammatory cells occurs in all groups after 4 days treatment. After 8 days treatment, tissue edema with a small number of inflammatory cells can be still observed in all groups except CS-BP group 11


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under irradiation (Figure S12, only a small number of inflammatory cells). Moreover, after the 14-days treatment, cell vacuolization (black arrows) of each skin layers (stratum basale, stratum spinosum, granular layer, stratum lucidum, stratum corneum) were observed in the control, CS, and 3M groups (dark and light), and inflammatory cells (red arrows) were also observed in the control, 3M (dark and light), and CS-BP groups (dark) as shown in Figure 5c. Monolayer keratinocytes in the epidermis (blue arrows) appeared in the CS-BP group (dark), suggesting that the wounds were still not completely healed under these conditions. In contrast, the CS-BP group (light) showed normal tissues without any cell vacuolization, inflammatory cells and monolayer keratinocytes. In addition, Giemsa staining was utilized to identify the bacterial residue. As indicated in Figure 5d, significant bacteria (green arrows) remained in the control group, 3M group (dark and light) and CS-BP group (dark) after two days of treatment. In comparison, bacteria were visibly diminished in the CS group (dark and light). In contrast, there were no detectable signs of infection or bacteria in the CS-BP group (light).

Enhanced wound healing process and signaling pathways by BPs Next, the therapeutic efficacies of the samples under normal conditions were evaluated in order to investigate the mechanism for accelerating wound healing and skin regeneration by BPs. As indicated in Figure 6a, after treatment for 14 days, the n-CS-BP group has almost healed completely, while other groups, including the n-Control, n-3M, and n-CS groups, does not well heal, indicating that wound closure and skin regeneration can be promoted by the BPs on the CS hydrogel. And cell vacuolization of each skin layers can be observed in all groups, but inflammatory cells 12


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(red arrows) are also observed in n-Control, n-3M, and n-CS groups (Figure 6b). Moreover, the obvious incrustation (red rectangle in Figure 6a) is observed in n-CS-BP group after 4 days of treatment. The incrustation is formed by the coagulation of platelets and fibrin, and fibrinogen has a double function, i.e., yielding monomers that polymerize into fibrin, and acting as a cofactor in platelet aggregation.45-47 The immunohistochemical staining (black arrows in Figure S13) for fibrinogen, along with the quantification (Figure 7a) demonstrates that n-CS-BP treatments significantly enhance the formation of the fibrinogen compared to the n-Control and n-CS groups, especially for 4-days treatment, indicating that BPs can enhance the formation of the fibrinogen at the early stages of the tissue repair process to accelerate the formation of incrustation and wound healing. As demonstrated by the western blot analysis data (Figure 7b and Figure 7c), CS-BP treatments can activate signaling pathways of phosphoinositide 3-kinase (PI3K), phosphorylation of protein kinase B (Akt), and extracellular signal-regulated kinases (ERK1/2) that direct cells proliferation and differentiation.

The proliferation and

differentiation of the fibroblasts at the wound site play a critical role during skin regeneration, and fibroblasts contribute to the synthesis of extracellular matrix.48-50 Figure 7d summarizes the previous findings of how BPs induce signaling pathways to direct cell proliferation and differentiation, as well as enhance the expression of fibrinogen to speed up the formation of incrustation during wound healing.

In addition, as shown in Figure S14, histological analysis of the major organs (heart, liver, spleen, lung, and kidney) of all groups displayed no appreciable abnormalities or damage after 14 days of treatment, indicating that CS-BP hydrogel can be a safe therapeutic material for wound healing. 13


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CONCLUSION In summary, we reported a therapeutic system for wound healing based on BPs, using CS hydrogel as the carrier. The BPs in this hydrogel system were able to rapidly generate 1O2 under the illumination of visible light to enhance the antibacterial activity against both Gram-positive and Gram-negative bacteria. Importantly, the intrinsic properties of BPs allowed them to simultaneously fulfill the requirements of the excellent antibacterial activity, reusability, and biocompatibility as well as participate in stimulating cell behaviors of enhancing the formation of the fibrinogen for accelerated incrustation and triggering PI3K/Akt and ERK1/2 signaling pathways for

enhanced

cellular

proliferation

and

differentiation

to

promote

bacteria-accompanied wound healing. In vivo wound therapeutic outcome was realized effectively after BPs was loaded onto the CS hydrogel. This was a safe strategy for rapid clinical sterilization and reconstruction of damaged tissues through the combination of common hydrogels with 2D BPs.

EXPERIMENTAL SECTION Preparation of chitosan hydrogel. To prepare chitosan (CS) hydrogel, 2.5 g of CS (viscosity: 100-200, 179.17 MW) was dissolved in 100 mL of acetic acid solution (2 wt.%) with continuous mechanical stirring for 1 h to obtain a homogenous viscous mixture. Then 2 mL glycerinum and 2 g polyethylene glycol (2000 MW) were added at 50 oC for 10 min until a homogenous mixture was obtained, which can used as a transdermal agent to provide a hydrophilic and emulsified environment for the skin.59 Subsequently, 20 mL of 2% glutaraldehyde as the crosslinking agent was added with continuous mechanical stirring for 10 s.60 Finally, the mixture was poured into a glass 14


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petri dish immediately and placed into the oven at 50 oC for 8 h. All the prepared samples were washed several times with hot distilled water in order to remove residual glutaraldehyde and freeze-dried for 12 h. Preparation of black phosphorus nanosheets. The black phosphorus nanosheets (BPs) were prepared using a simple ultrasonic exfoliation technique. Specifically, 50 mg BP powder was dispersed in 80 mL distilled water. The mixture solution was bubbled with argon or nitrogen at an ice-cooled temperature to prevent BP oxidation. Then, the mixture solution was sonicated for 8 h. Next, the resulting brown suspension was centrifuged at 2000 rpm for 15 min to remove residual unexfoliated particles, and the supernatant was collected and dried for further use. Fabrication of the CS-BP hydrogel. The obtained CS hydrogel was cut to obtain a series of wafers with regular shape and uniform size of φ 6 mm × 2 mm . The wafer was immersed into 2 mL of BPs aqueous solution (200 µg mL-1) for 10 min to load the BPs adequately. Afterward, the obtained BPs loaded with CS hydrogel (CS-BP hydrogel) was freeze-dried and stored in argon. Characterization of hydrogels. The morphologies were observed by scanning electron microscopy (SEM, JSM7100F and JSM6510LV) and transmission electron microscope (Tecnai G2 20 U-Twin and Titan G2 60-300). The zeta potentials of the CS hydrogel, BPs, and CS-BP hydrogel were measured by ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd., Malvern) at room temperature. Electron spin resonance spectroscopy. All the reactive oxygen species (ROS) measurements were carried out by an electron spin resonance spectrometer (ESR, JES-FA200) at room temperature. In these ESR tests, 2,2,6,6-tetramethylpiperidine (TEMP, 50 mM) was used as a spin trap for the detection of singlet oxygen (1O2) 15


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during irradiation of samples by simulated sunlight. Spin traps were ESR silent but formed stable radicals with an ESR signal after donating electrons. The ESR measurements were as follows: micro frequency, 8.93 GHz; micro power, 3 mW. The samples were immersed in 200 µL of spin traps and irradiated under a 300 W xenon lamp (PLS-SXE300, Beijing Changming Technology Co., Ltd., China) for 10 min. Afterward, the correct amount of irradiated solution was put into quartz capillary tubes and sealed. The sealed capillary tubes were inserted in the ESR cavity, and the spectra were recorded. For comparison, the control group (TCP) without samples was also recorded. In vitro antibacterial activity assay. The samples were challenged with E. coli (Gram-negative) and S. aureus (Gram-positive) bacteria at a concentration of 1.0×107 CFU/mL. The antibacterial activity was first studied using spread plate, evenly seeding 20 µL of bacterial stock suspension onto hydrogels ( φ 6mm × 2mm ), which were placed in 48-well plates, and the tissue culture plate (TCP) in the darkness was served as the control (n =3). For the 10 min model, the 48-well plates containing bacteria and hydrogels were then irradiated under a xenon lamp (PLS-SXE300, Beijing Changming Technology Co. Ltd., China, 200 W, 1 m high) for 10 min, and the dark groups were placed in the dark for 10 min. And then the bacteria on the hydrogels and TCP were diluted 50-fold using Luria-Bertani (LB) broth. Afterward, the surviving bacteria were separated from the hydrogels and TCP using low-power ultrasound, and 20 µl of diluent was then collected and spread on an LB agar plate and incubated at 37 oC for 24 h to form viable colony units. The antibacterial ratio was calculated as follows in Equation (1).

16


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Antibacterial ratio (%) =

CFU0 (cell) − CFU (cell + gel + rad) *100% (1) CFU0 (cell)

Where CFU (cell + gel + rad) is the area of colony forming units measured in the presence of hydrogels, and CFUo (cell) is the area of colony forming units measured in the absence of both hydrogels and simulated sunlight. And for the 1 h model, bacteria on the samples were irradiated for 10 min and then placed in the dark for 50 min (total 1 h), and the dark groups were placed in the dark for 1 h. The following processing is the same as the 10 min model. Moreover, reusable antimicrobial assays of samples were applied by using the 10 min model with light of the CS-BP hydrogel and the 1 h model in the dark of the CS hydrogel. The same samples were treated repeatedly up to four times. In addition, for the long time antibacterial assays of CS hydrogel, bacteria were treated by the CS hydrogel for 10 min, 1 h, 2 h, and 4 h, respectively. For SEM (SEM, JSM6510LV) observation, the samples were processed in the same way as the spread plate test. After irradiation, the hydrogels and TCP were washed three times with PBS to remove non-adherent bacteria. The adherent bacteria were fixed with 2.5% glutaraldehyde (25% glutaraldehyde: distilled water: PBS= 1:4:5) for 2 h, and then dehydrated by alcohol with different concentrations of 30, 50, 70, 90 and 100% orderly for 15 min and finally freeze-dried overnight for SEM observation. Cell culture. NIH-3T3 (mouse embryonic fibroblast cell line) cells were cultured in MEM/EBSS medium (HyClone, contained 2 mM L-Glutamine

and Earle’s

Balanced Salts) and supplemented 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution (HyClone) at 37o C in an incubator of 5% CO2 and 95% humidity. The complete medium was replaced every three days, and confluent 17


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flasks were subcultured using trypsin. Mitochondrial metabolic activity assay. Mitochondrial metabolic activity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay (n=3). Particularly, the NIH-3T3 cells were seeded onto the hydrogels ( φ 6mm × 2mm ) and TCP in 48-well plates with 350 µL of medium in the dark. For the MTT assay irradiated with light, NIH-3T3 cells with the samples were irradiated for 10 min after cells were seeded onto the samples for 12 h, and then continuous co-cultured in the dark. And TCP was served as the control, the medium was refreshed every three days. After days 1, 3, and 7, the culture medium was removed from each plate, and 350 µL of the MTT solution (5 mg/mL in PBS) was added into each plate with continuous culture at 37o C for 4 h. Afterward, the MTT solution was replaced with 350 µL of dimethyl sulfoxide (DMSO), followed by shaking at a shaking table for 15 min to dissolve the formazan completely. After that, the absorbance at 570 nm was measured using a microplate reader (SpectraMax i3, Molecular Devices). Total RNA extraction, cDNA synthesis, and qRT-PCR. qRT-PCR was used to quantify the relative gene expression levels of smooth muscle alpha-actin (SM α-actin) and collagen type III (COL III) (n=3). NIH-3T3 cells were seeded onto hydrogels ( φ10mm × 2mm ) and TCP in 12-well plates with 2 mL of medium, the medium was refreshed every 3 days, and TCP was served as the control. After days 3 and 7, the culture medium was removed from each plate, and the cells were rinsed with ice-cold PBS twice. Afterward, total RNA was extracted using Total RNA Kit I (50) (OMEGA R6834-01), according to the manufacturer’s instructions. Total RNA was quantified by NanoDrop 2000 spectrophotometer (Thermo Scientific), and the RNA samples (500 ng each) were used for reverse transcription by using PrimeScript™ RT Master 18


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Mix (Perfect Real Time) (Takara), according to the manufacturer’s instructions. For qRT-PCR, SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara) and the CFX Connect™ Real-Time PCR Detection System (Bio-Rad) were used. In vivo animal experiment. In vivo animal experiments were approved by Wuhan Bioyear Biological technology CO., Ltd., China. Male Wistar rats (200−220 g body weight) were obtained from Wuhan Centers for Disease Prevention & Control. The rats were individually raised in cages under standardized temperature (24±2 oC) and humidity (60-70%) for 3 days and randomly divided into four groups (each group contained eight): Control group, CS group, the traditional treatment group (3M wound dressing, Minnesota Mining & Manufacturing Medical Equipment Shanghai Co., Ltd., 3M group), and CS-BP group. On the day of wounding, the rats were anaesthetized by 4 wt.% pentobarbital sodium salt (1 mL/kg), and then a partial thickness wound on the rats’ backbones were created with a disposable 6 mm skin biopsy punch. Afterward, the prepared wounds were treated with 10 µL S. aureus (1.0*107 CFU/mL) and covered with samples. One half of each group was irradiated under a xenon lamp (200 W, 1 m high) for 10 min, and the other half was treated without irradiation. All rats were individually raised in cages under standardized temperature (24±2 oC) and humidity (60-70%) on a 12:12 L/D cycle (lights on at 8 a.m.). The samples were changed every 2 days and continued for 14 days. After days 2, 4, 8 and 14, the area of the wounds were observed and photographed, and then the skin tissue samples were excised and fixed with 10% formalin for pathological slides. After staining with hematoxylin and eosin (H&E) and Giemsa, the histological images were observed by an optical microscope (NIKON Eclipse Ci). Finally, the major organs (heart, liver, spleen, lung, and kidney) were harvested and stained with H&E after 14 days of treatment. And in order to investigate therapeutic efficiencies and mechanisms of the 19


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samples under normal conditions, the rats were randomly divided into three groups (each group contained four rates): n-Control group, n-CS group, and n-CS-BP group. The next treatment was same with the above process but without S. aureus and irradiation.

Immunohistochemistry test. The tissues of the wound regions under normal conditions were harvested after 2, 4, and 8 days of treatment, followed by freezing and slicing into thick sections (10 mm). Then the tissues were blocked with 5% BSA for 20 min and incubated with the primary antibody (Anti-Fibrinogen alpha chain, 1:50, Abcam, no. ab92572) overnight at 4 oC. And positive signals were visualized using REAL™EnVision+ /HRP RABBIT/MOUSE (Dako Denmark A/S, K5007) and DAB. Three different and random images from each group using 200 × magnifications were analyzed for immunohistochemical quantification.

Western Blot analysis. The tissues under normal conditions were obtained from the mouse wound regions and homogenized using a homogenizer at 4 oC in ice-cold lysis buffer. The protein concentration was determined using BCA Protein Quantification Kit (ASPEN, AS1086). Western blot analysis was performed using SDS-PAGE Gel Kit (ASPEN, AS1012). After the proteins were transferred onto PVAD membrane (Millipore, IPVH00010), they were probed with antibodies against Akt, pAkt, PI3K, pPI3K, ERK1/2, pERK1/2, and β-actin (all antibodies were purchased from Abcam) and incubated with a horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature. And the blots were developed using ECL Kit (ASPEN,

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AS1059). Finally, the bands were imaged and optical density values of bands were quantified by AlphaEaseFC. Statistical Analysis. All data were evaluated as mean values ± standard deviation of three tests at least and contrasted via Kruskal-Wallis one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison tests, with values of P < 0.05 considered significant. ASSOCIATED CONTENT Supporting Information Available: Supporting Information including Supplementary Figures (SEM images; UV-vis spectra; SEM images of bacteria; Reusable antimicrobial activities and panels of formed viable colony units of bacteria; The immunology of histological images; Immunohistochemical staining; H&E staining of heart, liver, spleen, lung, and kidney tissue 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)

ACKNOWLEDGMENTS This work is jointly supported by the National Natural Science Foundation of China, Nos. 51671081 and 51422102, and the National Key Research and Development Program of China No. 2016YFC1100600 (sub-project 2016YFC1100604).

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Figures

Scheme 1. The schematic illustration of the sterilization under visible light irradiation and the process of stimulating skin cell behaviors that can promote the regenerative activities of the skin cells and actively participate in skin regeneration to accelerate bacteria-accompanied wound healing using black phosphorus nanosheets-based hydrogel.

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Figure 1. Characterizations of the CS-BP hydrogel. (a) TEM image of the BPs. Scale bar, 200 nm. (b) High-resolution TEM image of the BPs. The (200) crystal spacing is marked. Scale bar, 1 nm. (c) Low-magnification SEM image of the CS-BP hydrogel. Scale bar, 100 µm. (d) High-magnification SEM image of the BPs on the CS-BP hydrogel. Scale bar, 100 nm. (e) Optical images of left: the BPs aqueous solution; right: the remaining solution after the most of BPs were loaded by the CS hydrogel through electrostatic absorption in the solution, and then this BP-loaded CS hydrogel was taken out from the solution. (f) Surface zeta potential of the CS hydrogel, the BPs, and the CS-BP hydrogel.

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Figure 2. The identification of 1O2 was detected by ESR spectroscopy and the in vitro antibacterial activity. (a) The ESR spectra obtained from solutions containing 200 µL spin traps (50 mM TEMP) and samples (TCP, the CS hydrogel, and the CS-BP hydrogel) after being in the dark for 10 min (left) or irradiated with simulated sunlight for 10 min (right). (b) The formed viable colony units of E. coli (left) and S. aureus (right) after treated with samples (TCP, the CS hydrogel, and the CS-BP hydrogel) in the dark for 10 min or under simulated sunlight for 10 min, and then spread onto LB agar plate and incubated at 37 °C for 24 h. The corresponding abilities of the samples 32


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(TCP, the CS hydrogel, and the CS-BP hydrogel) in killing E. coli (c) and S. aureus (d), the control was in the TCP but in the absence of simulated sunlight. The experiment was performed in triplicate and independently (n=3, mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Reusable antimicrobial activities of samples (the CS hydrogel and the CS-BP hydrogel) against S. aureus. (a) The formed viable colony units of S. aureus 33


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after treated with the CS-BP hydrogel and CS hydrogel under simulated sunlight for 10 min and in the darkness for 1 h, respectively, and then spread onto LB agar plate and incubated at 37 °C for 24 h, repeatedly up to four times. (b) The corresponding abilities of the samples in killing S. aureus, the CS-BP hydrogel and CS hydrogel were repeatedly challenged with S. aureus under simulated sunlight for 10 min and in the darkness for 1 h, respectively, repeatedly up to four times. (c) The change of surface zeta potential of the CS hydrogel after repeatedly challenged with S. aureus for in the dark for 1 h. Data is represented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001 and n=3).

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Figure 4. In vitro cytotoxicity and skin cell modulation of samples (TCP, the CS hydrogel, and the CS-BP hydrogel). (a) MTT assay of the NIH-3T3 cells with the samples in the dark and co-cultured for 1, 3, and 7 days. (b) Cell viability of the NIH-3T3 cells with the samples and initially irradiated for 10 min, and then continuous co-cultured in the dark for 1, 3, and 7 days. Relative gene expression of (c) smooth muscle alpha-actin (α-actin) and (d) collagen type III (COL III) of the NIH-3T3 cells as evaluated by qRT-PCR after co-cultured with samples for 3 and 7 days. Data is represented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001 and n=3).

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Figure 5. In vivo assessments of photodynamic therapy of the samples (the Control, the CS hydrogel, 3M wound dressing, and the CS-BP hydrogel) for bacteria-accompanied wound healing. In vivo study on the effects of treatment of S. aureus-accompanied wound infections (a) in the dark and (b) under simulated sunlight for 10 min at the beginning by samples and the corresponding wound photographs of the rats’ wounds at day 0, 2, 4, 8, and 14. (c) The immunology of histological images of the skin tissue samples on rats’ wounds after treating with samples for 14 days and staining with hematoxylin and eosin (H&E). Scale bar, 100 µm. Black arrows: cell vacuolization; Red arrows: inflammatory cells; Blue arrows: monolayer keratinocytes. (d) Giemsa staining of the mid-portion of the tissues was performed on 2 days. Scale bar, 50 µm. Green arrows: bacteria.

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Figure 6. In vivo assessments of the samples (the n-Control, the n-CS hydrogel, and the n-CS-BP hydrogel) under normal conditions for wound healing. (a) In vivo study on the effects of treatment of by samples under normal conditions and the corresponding wound photographs of the rats’ wounds at day 0, 2, 4, 8, and 14. (b) The immunology of histological images of the skin tissue samples on rats’ wounds after treating with samples for 14 days and staining with hematoxylin and eosin (H&E). Scale bar, 100 µm. Red arrows: inflammatory cells.

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Figure 7. Wound healing process enhanced by BPs and the intracellular signaling pathways for cell proliferation and differentiation during wound healing. (a) Quantification of fibrinogen positive of the samples (the n-Control, the n-CS hydrogel, and the n-CS-BP hydrogel) at the wound healing region after treatment for 2, 4, and 8 days. (b) Western blot analysis and (c) quantification for the molecules involved in the intracellular signaling pathways for wound healing regarding in vivo tissue 40


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samples after treatment for 14 days. (d) Schematic illustration showing BP induced signaling pathways direct cell proliferation and differentiation, as well as enhanced the expression of fibrinogen to speed up the formation of incrustation during wound healing. Data is represented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001 and n=3). For Table of Contents Only

Schematic diagram of hydrogel system not only possesses a broad and reusable antibacterial efficacy against both Gram-positive and Gram-negative bacteria through the generation of 1O2 under visible light irradiation, but also enhances the formation of the fibrinogen at the early stages of the tissue repair process for accelerated incrustation, as well as triggers PI3K/Akt and ERK1/2 signaling pathways for enhanced cellular proliferation and differentiation to promote wound healing accompanied with bacteria-infections. 41


A Repeatable Photodynamic Therapy with Triggered Signaling

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