Bioinspired multifunctional biomaterials with hierarchical microstructure for wound dressing

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Bioinspired Multifunctional Biomaterials with Hierarchical Microstructure for Wound Dressing Jianmin Xue , Xiaocheng Wang , Endian Wang , Tian Li , Jiang Chang , Chengtie Wu PII: DOI: Reference:

S1742-7061(19)30688-9 https://doi.org/10.1016/j.actbio.2019.10.012 ACTBIO 6397

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Acta Biomaterialia

Received date: Revised date: Accepted date:

17 May 2019 24 September 2019 4 October 2019

Please cite this article as: Jianmin Xue , Xiaocheng Wang , Endian Wang , Tian Li , Jiang Chang , Chengtie Wu , Bioinspired Multifunctional Biomaterials with Hierarchical Microstructure for Wound Dressing, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.10.012

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Bioinspired Multifunctional Biomaterials with Hierarchical Microstructure for Wound Dressing

Jianmin Xue1,2, Xiaocheng Wang1,2, Endian Wang1,2, Tian Li1,2, Jiang Chang1,2, Chengtie Wu1,2,*

1. State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, No.1295 Dingxi Road, Shanghai 200050, People’s Republic of China.

2. Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, People’s Republic of China.

* Corresponding author: E-mail: [email protected] (C. Wu) Statement of Significance Although it is an effective strategy to prepare high-performance materials by mimicking hierarchical microstructure of nacre, the preparation of nacre-inspired materials in tissue engineering fields still needs to be explored. In this work, nacre-inspired multifunctional graphene oxide-chitosan-calcium silicate (GO-CTS-CS) biomaterial with hierarchical microstructure has been prepared. The hierarchical microstructure endows the biomaterials with desired properties of strength, breathability and water absorption. Meanwhile, the hierarchical GO-CTS-CS biomaterial showed good photothermal antibacterial/antitumor and wound healing effects. This work may provide a way to combine the preparation of multifunctional biomaterials with bioinspired engineering via constructing hierarchical 1

microstructure, indicating that the assembling hierarchical microstructure in biomaterials is of great importance for tissue engineering and regenerative medicine.

ABSTRACT Developing multifunctional wound dressing with desired mechanical strength is of great significance for the treatment of different types of skin wounds. Inspired by the close relationship between strength and hierarchical structure of nacre, hierarchical and porous graphene oxide-chitosan-calcium silicate (GO-CTS-CS) film biomaterials are fabricated in combination of vacuum filtration-assisted assembly and freeze-drying methods. The bioinspired hierarchical materials emulate orderly porous lamellar micron-scale structure and the “brick-and-mortar” layered nanostructure. The hierarchical microstructure endows the GO-CTS-CS biomaterials with good tensile strength, compatible breathability and water absorption.

Furthermore,

the

hierarchical

GO-CTS-CS

biomaterials

exhibit

ideal

photothermal performance, leading to significant photothermal antibacterial and antitumor efficiency. Meanwhile, the hierarchical GO-CTS-CS biomaterials show stimulatory effect on in vivo chronic wound healing. Therefore, such a high performance and multifunctional biomaterial is believed to offer a promising alternative to traditional wound dressing in future.

KEYWORDS: hierarchical structure, bioinspired materials, multifunctional materials, photothermal antibacterial and antitumor, wound healing

2

1. INTRODUCTION Multifunctional materials, one of developing trends of advanced materials, have received great applications in areas of energy, electronics and tissue engineering [1-5]. Wound dressings, typical skin tissue engineering materials, have been widely used to promote the healing of serious skin wounds. However, traditional wound dressing (e.g. gauze) hardly fulfill the diverse requirements of wounds in complex conditions, such as infected wounds, chronic wounds (e.g. diabetic foot wounds) or operative wounds after cutaneous tumor resection [6-11]. Moreover, sufficient mechanical strength should be provided by wound dressings to cover the wounds, especially for the application of wound dressings under high stress [12]. Although lots of hydrogels or electrospun nanofibers have been developed for wound dressing, the insufficient tensile strength or single function of wound healing limited their further application in complex skin wounds [6,13-17]. In the past decades, many researchers have focused on finding inspiration to design and prepare high-performance materials from natural materials [5,18]. As an example of high strength of natural materials, nacre possesses hierarchical “brick-and-mortar” layered architectures and different interface interactions between organic chitin/protein and aragonite platelets [19,20]. The hierarchically ordered structure plays an important role in its superior mechanical properties [19-21]. In addition, the nacre-inspired artificial materials could easily acquire the desired properties by adjusting the material compositions [22-25]. Apart from strength and multifunction, desirable oxygen permeation and absorbing tissue exudates are of equal importance for wound dressing during the tissue ingrowth of wound healing process [11]. However, most nacre-inspired materials with dense hierarchical layered structures have high strength, but lack satisfying breathability and hydrophilicity [25,26], leading to insufficient oxygen supply and excess tissue exudates around the wound when directly used for skin wound healing. Therefore, it is necessary to prepare a high performance of multifunctional biomaterial with desired mechanical strength to fulfil various requirements of wound dressing. 3

Herein, inspired by the relationship between hierarchical structure and strength of nacre, hierarchical and porous graphene oxide-chitosan-calcium silicate (GO-CTS-CS) film biomaterials are fabricated. The hierarchical biomaterials are constructed with the “brick-andmortar” layered nanostructure and orderly porous lamellar micron-scale structure by combining bottom-up self-assembly and vacuum freeze-drying methods. The hierarchical GO-CTS-CS biomaterials have good tensile strength, breathability and water absorption. Meanwhile, the hierarchical GO-CTS-CS biomaterials exhibit satisfactory photothermal performance for in vitro photothermal antibacterial and in vivo photothermal antitumor due to the addition of GO. Furthermore, hierarchical GO-CTS-CS biomaterials possess good cytocompatibility and could help heal wounds of diabetic mice.

2. Materials and Methods 2.1． Preparation of Hierarchical and Porous GO-CTS-CS Film Biomaterials. GO suspension (1 mg/mL) was obtain from graphite oxide (Shanghai Haoye Electronic Technology Co., Ltd., China) in deionized water by ultrasonic method. Then, GO suspension was mixed with CTS acid solution (5 mg/mL). Next, Na2SiO3·9H2O and Ca(NO3)2•4H2O (Sinopharm Chemical Reagent Co., Ltd., China) were added into the mixed solution of CTS and GO and the pH of mixture solution was adjusted to 10 [26]. Then, the mixture solution was vacuum filtered to obtain the damp GO-CTS-CS composite films. After that, the damp films were further processed to construct porous structure by vacuum freeze-drying. Then, the films were moistened with deionized water spray for several times. The theoretical component of CS in all GO-CTS-CS films was 20 wt %. The GO-CTS-CS films containing 10, 20, 30 and 40 wt % GO were prepared and named as 10GO-CTS-CS, 20GO-CTS-CS, 30GO-CTSCS and 40GO-CTS-CS, respectively. 30GO-CTS-CS-Dense film was fabricated without vacuum freeze-drying process and used for a Control. 30GO-CTS-CS-Disorder film was fabricated by vacuum freeze-drying heavy bodied slurry. 2.2． Characterization of Hierarchical GO-CTS-CS Biomaterials. 4

The fracture section structure at different magnification of films was characterized by fieldemission scanning electron microscopy (SEM, S4800, Hitachi, Japan). The tensile strength of films was determined by using mechanical testing machine (Shimadzu, Japan). The breathability of different films was determined according to the previously reported method [27]. 80g of allochroic silicagel was dried at 110 ℃ and then sealed in wide mouth bottle with films. The allochroic silicagel could absorb water vapor in air through the films. The weight change of allochroic silicagel at different time intervals was determined. The water absorption of materials was determined as follows. The films were sectioned into square (25 mm × 25 mm) and immersed in deionized water for 2 minutes. The weight of films was recorded after removal of the water from the film surface. The water absorption was defined as: Water absorption (g/g) = (m1-m0)/m0, where m0 is the weight of films before immersed into deionized water, m1 is the weight of films after taken out from deionized water. The commercial film dressing is 3M TegadermTM Film. 2.3． Photothermal Performance of Hierarchical GO-CTS-CS Biomaterials. Different films were sliced into rounds (Φ = 10 mm) and put in 48-well culture plates. Then, the films were exposed to laser (808 nm, 0.40 W/cm2) in dry state (air) for 5 minutes and in the wet state (500 μL deionized water) for 10 minutes, respectively. The IR thermal imaging system (PM100D, Thorlabs GmbH，Germany) was applied to record the temperature and infrared thermal images of films under laser. In order to investigate the influence of GO content and laser power density on photothermal performance, a series of GO-CTS-CS materials with different content of GO were irradiated by the 808 nm laser with varied laser power densities (i.e. 0.27, 0.31, 0.36, 0.40 and 0.44 W/cm2). To investigate the photothermal stability, the 30GO-CTS-CS films was treated by repeating laser irradiation for 5 cycles under same condition (808 nm, 0.40 W/cm2, 5 min). 2.4． In Vitro Photothermal Antibacterial Performance. Plate-counting method was used to evaluate in vitro photothermal antibacterial 5

performance of 30GO-CTS-CS. Escherichia coli (E. coli) suspension with 0.01 of optical density (OD, 600nm) was prepared in advance. CTS, CTS-CS and 30GO-CTS-CS were cut into rounds (Φ = 10 mm) and immersed into the bacterial suspension. For CTS+laser, CTSCS+laser and 30GO-CTS-CS+laser groups, the suspension was irradiated by laser (808 nm, 1.06 W/cm2) for 30 minutes. The groups without irradiation were used as control. Then, the bacterial suspension containing films was transfer into tubes and cultured in constant temperature shaking incubator (37 ℃, 120 rpm). After 12 hours, the original bacterial suspension of each groups was diluted 1×105 times. 25 μL of dilute solutions was plated onto agar plate in dish and cultured in incubator (37 °C) for 12 hours. For the antibacterial experiment of Staphylococcus aureus (S. aureus), the agar plates with dilute bacterial solutions were cultured in incubator (37 °C) for 15 hours. Other experimental conditions are the same as those of E. coli. Finally, the numbers of colony forming in each dish were counted. 2.5． In Vitro Photothermal Antitumor Efficiency. B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 10% fetal bovine serum, 1% IU/mL penicillin and 1μg/mL streptomycin) in humidified incubator (37 °C, 5% CO2). To observe the tumor cells morphology on films after photothermal therapy, B16F10 cells (3×104) were cultured on CTS, CTS-CS and 30GO-CTS-CS films for overnight and then exposed to laser irradiation (808nm, 0.37 W/cm2, 15 min). As controls, B16F10 cells were cultured on CTS, CTS-CS and 30GO-CTS-CS films without laser irradiation. After cultured for 24 hours, the cellular samples were sequential processed by 2.5% glutaraldehyde, graded ethanol solution (40, 50, 60, 70, 80, 90, 95, 100 v/v %) and hexamethyldisilazane (HMDS). The cells morphologies was characterized by SEM (S4800, Hitachi, Japan). Live/Dead cell assay was applied to investigate photothermal antitumor effect of 30GO-CTSCS on surrounding tumor cells. Typical tumor cells (B16F10 cells) were seeded on glass slides for 24 hours. After that, the different films were added on the glass slides and treaded 6

by laser irradiation (808nm, 0.37 W/cm2, 15 min). Then, tumor cells were stained with Ethidium homodimer-1 and Calcein AM. The live/dead tumor cells were observed by confocal laser scanning microscope (Leica, Germany). In vitro photothermal antitumor efficiency of 30GO-CTS-CS on surrounding tumor cells was quantitatively determined by CCK-8 assay.[10] B16F10 cells were treated with laser irradiation (808nm, 0.37 W/cm2) for 15 minutes or without laser irradiation. Then, the tumor cell viability of different groups was determined by CCK-8 assay after 24 hours. The influences of irradiation duration, irradiation times and temperature on in vitro photothermal antitumor efficiency of 30GO-CTS-CS were further explored. For different irradiation duration, B16F10 cells were cultured with 30GOCTS-CS in 48-well culture plates and kept at same photothermal temperature (approximately 50℃) for 0, 5, 10, 15 and 20 min. For irradiation times, tumor cells being cultured with 30GO-CTS-CS were irradiated by laser (50℃ ， 15 min) for 0, 1, 2 and 3 times. The irradiation interval was 6 hours. As to temperature, the tumor cells were irradiated under different photothermal temperature (37, 41, 45, 49, 53 and 57 °C) for 15 min by real-time adjusting the laser power densities. After 12 hours, the tumor cell viability was determined by CCK-8 assay. 2.6． In Vivo Photothermal Antitumor Efficiency. Balb/c nude mice (6-8 weeks) were purchased from Shanghai Rat & Mouse Biotech Co., Ltd, China. All animal protocols were approved by Institutional Animal Care and Use Committee of Nanjing First Hospital, Nanjing Medical University. B16F10 tumor-induced mouse wound models were established though subcutaneous injecting B16F10 cells (5 × 105 cells) into the flank of mice [10,28]. The in vivo photothermal antitumor experiment was carried out when tumors grow with the diameter of 3-5 mm. A round wound (Φ = 10 mm) was cut at tumor site on the right flank of each mouse. The materials were cut into circle (Φ = 10 mm) and completely covered the visible tumor in the skin wounds. To systematically evaluate the photothermal antitumor effect of materials, the mice were randomly divided into 7

7 groups (n=8): Control, CTS, CTS-CS, 30GO-CTS-CS, CTS+laser, CTS-CS+laser and 30GO-CTS-CS+laser groups. For the laser-irradiation groups, the tumor-induced wounds were irradiated by laser (808 nm, 0.35 W/cm2, 15 min) for 4 consecutive days. The infrared thermal images and surface temperature of tumor-induced wounds were recorded by an IR thermal imaging system (PM100D, Thorlabs GmbH ， Germany). The tumor sizes were measured at day 0, 1, 3, 5, 8, 11 and 14, respectively. After 14 days, the mice were sacrificed and tumors were taken out from mice. The mouse was sacrificed in advance for animal welfare if the length of the tumor exceeds 20 mm before day 14. The volume of tumor was calculated as following: The volume of tumor = (L × W2)/2, where L is the length of tumor, W is the width of tumor. Four mice in each group were used to count the change of tumor volume over time. The tumors with surrounding skin were fixed in 4 v/v % paraformaldehyde, embedded in paraffin and then stained with H&E for histological analysis. 2.7． In Vitro Biocompatibility. In order to observe cell morphologies of human umbilical vein endothelial cells (HUVECs) on different films, HUVECs were seeded on films in endothelial cell medium in 48-well culture plates. After 24 hours, cellular samples were was sequential processed by 2.5% glutaraldehyde, graded ethanol solution (40, 50, 60, 70, 80, 90, 95, 100 v/v %) and HMDS. The cell morphology was observed by SEM. For cell proliferation, HUVECs were cultured in ECM containing different films for 1, 3 and 5 days. The cell viability was determined by CCK-8 assay. For the cell morphology and cell proliferation of human dermal fibroblasts (HDFs), HDFs were cultured in DMEM at 37 °C and 5% CO2. After 24 hours, the morphologies of HDFs on different films were observed by SEM. The cell viability of HDFs was determined by CCK-8 assay at day 1, 3 and 5. 2.8． In Vivo Wound Healing. All animal protocols were approved by Institutional Animal Care and Use Committee of Nanjing First Hospital, Nanjing Medical University. C57BL/6 mice were selected to establish 8

the diabetic mouse models. The diabetic mouse models were induced by intraperitoneal injection of streptozocin in 0.1 mol/L citrate buffer according to our previous report [10,28]. A skin wound (Φ = 10 mm) was created on the back of the diabetic mouse. The CTS, CTS-CS and 30GO-CTS-CS films were sliced into rounds (Φ = 10 mm) and covered the wounds of diabetic mice. The wounds without treated were set as control group. There are six mice in each group. The wounds of different groups were photographed at day 0, 3, 6, 9, 12 and 14. The relative area of wounds was calculated as following：Relative wound area (%) = Sx/S0 × 100%, where S0 is wound area at day 0, Sx is wound area at day x (x = 3, 6, 9, 12, 14). Four mice in each group were used to count the relative skin wound area at different time. After 14 days, the mice were sacrificed and the skin with wound sites were stained with H&E for histological analysis. 2.9．Statistical Analysis The data were expressed as the means ± standard deviation (SD). Student’s t-test were applied to evaluate significant difference between two groups. p < 0.05 indicated significant difference. *p < 0.05, **p < 0.01 and ***p < 0.001.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Hierarchical and Porous GO-CTS-CS Film Biomaterials The fabrication process of hierarchical and porous GO-CTS-CS biomaterials was shown in Scheme 1. Firstly, damp GO-CTS-CS composite films were fabricated through vacuum filtration-assisted self-assembly. After that, the damp films were further processed to construct porous structure by vacuum freeze-drying and thus the hierarchical GO-CTS-CS films with porous lamellar structure were prepared. Here, GO was used as template to induce layered structure [20]. CTS was widely used in wound healing due to the functions of accelerating wound healing and antibacterial viability [29]. Previous studies demonstrated that Si ions play

9

a key role in angiogenesis [30-32]. The GO-CTS-CS films containing 10, 20, 30 and 40 wt % GO were defined as 10GO-CTS-CS, 20GO-CTS-CS, 30GO-CTS-CS and 40GO-CTS-CS, respectively. Subsequently, the fracture section morphologies of different materials were observed by field-emission scanning electron microscopy (SEM). 20GO-CTS-CS, 30GOCTS-CS and 40GO-CTS-CS showed the “brick-and-mortar” layered structure ranging from 300 nm to 800 nm (Figure 1c). However, CTS, CTS-CS and 10GO-CTS-CS did not assemble the ordered nanostructure, because there were no graphene oxide or few graphene oxide to assist the formation of layered nanostructure. Moreover, owing to orderly layered nanostructure,

20GO-CTS-CS,

30GO-CTS-CS

and

40GO-CTS-CS

formed

orderly

interconnected lamellar structure while other groups exhibited disordered porous structure at microscale, in which vacuum freeze-drying and GO play a key role to induce porous and uniformly lamellar microstructure. The tensile strength of hierarchical GO-CTS-CS materials (20GO-CTS-CS, 30GO-CTS-CS and 40GO-CTS-CS) was further investigated (Figure 2a,b). The tensile strength of CTS and CTS-CS was less than 5.50 MPa. After being mixed with GO, the tensile strength obviously increased, especially for 30GO-CTS-CS and 40GO-CTS-CS. The tensile strength of 30GOCTS-CS and 40GO-CTS-CS reached 10.10 MPa and 9.76 MPa, respectively. Meanwhile, hierarchical and porous GO-CTS-CS films possessed the properties of good flexibility, light weight and high strength (Figure 2c). Moreover, 30GO-CTS-CS showed higher tensile strength as compared to CTS-based or other wound healing materials (Figure 2d).[17,33-38] However, it is also observed the elongation at break of GO-CTS-CS films reduced dramatically although their tensile strength was improved, which may be due to the addition of inorganic GO and CS. To investigate the influence of uniform nanostructure on the mechanical strength of films, we further fabricated 30GO-CTS-CS films with disordered structure at nanoscale (named as 30GO-CTS-CS-Disorder, Figure 1c) by direct mixing and vacuum freeze-drying without vacuum filtration. It is found that the tensile strength of 30GO10

CTS-CS-Disorder was 1.25 MPa, which was significantly lower than 30GO-CTS-CS (10.10 MPa), indicating that the nacre-inspired uniformly layered structure played a critical role in contributing to the mechanical strength of hierarchical 30GO-CTS-CS films. Considering the importance of breathability for wound healing, the breathability of different films was tested as previously reported [27]. The allochroic silicagel was sealed with different films and used to absorb water vapor in air through the films. The weight change of allochroic silicagel at different times was determined. The slopes of fitting lines could reflect the breathability of different films. As shown in Figure 2e, CTS, CTS-CS and 30GO-CTS-CS showed better breathability than commercial film dressing and GO-CTS-CS film without freeze-drying process (named as 30GO-CTS-CS-Dense, Figure S1, Supporting Information). An ideal wound dressing should absorb excess tissue exudates, because excess tissue exudates could cause wound infection and affect wound healing [6,39,40]. Therefore, the water absorption of materials was subsequently determined. As shown in Figure 2f, the water absorption capacities of CTS-CS and hierarchical 30GO-CTS-CS were significantly higher than that of 30GO-CTS-CS-Dense films. Considering the different microstructure of 30GOCTS-CS-Dense and hierarchical 30GO-CTS-CS, the porous microstructure could be responsible for the improvement of breathability and water absorption of hierarchical 30GOCTS-CS films. 3.2. Photothermal Performance of Hierarchical and Porous GO-CTS-CS Film Biomaterials GO and GO-based composites are typically photothermal materials with high photothermal conversion efficiency and have attracted much attention in photothermal therapy [41-44]. The hierarchical 30GO-CTS-CS films not only possess uniform porous lamellar structure, enhanced mechanical strength, breathability and water absorption, but also exhibit good photothermal performance under near-infrared (NIR) irradiation owing to the addition of GO. When irradiated under laser (808 nm, 0.40 W/cm2) for 5 minutes, the surface temperature of 11

20GO-CTS-CS, 30GO-CTS-CS and 40GO-CTS-CS rapidly increased and stably maintained above 120 ℃ in air (Figure 3a). The photothermal performance of different materials in wet condition (with 500 μL deionized water) was recorded (Figure 3b). The temperature of 30GOCTS-CS and 40GO-CTS-CS films could be over 54 ℃ in 10 minutes in wet condition. On the contrary ， the temperature of CTS and CTS-CS was hardly changed under the same irradiation condition either in air or wet conditions. Furthermore, the highest temperature of GO-CTS-CS under NIR irradiation could be effectively regulated by changing GO content and laser power density in composite films (Figure 3c). Moreover, there was no decrease in the highest temperature of 30GO-CTS-CS films over five Laser ON/OFF cycles, showing good photothermal stability of 30GO-CTS-CS (Figure S3, Supporting Information). Considering both tensile strength and photothermal performance, 30GO-CTS-CS hierarchical biomaterials were selected for further exploring in vitro and in vivo biological performances. 3.3. In Vitro Photothermal Antibacterial Performance Skin tissue, the first barrier of human body, plays key role in defending external invasion [12,15]. Skin wound sites is vulnerable to the attack from bacterial and cause infections, which leads to more complex process of wound healing, especially for the chronic wounds [6,12,45]. Thus, developing dressing with antibacterial ability is necessary for skin wound healing. Recently, photothermal therapy has become a potential strategy to antibacterial field, particularly in inhibiting drug-resistant microbes growth [46,47]. Encouraged by the superior photothermal performance, we further evaluated the in vitro photothermal antibacterial effect of 30GO-CTS-CS hierarchical biomaterials. Gram-negative E. coli and gram-positive S. aureus were used to test antibacterial effect. For the group of 30GO-CTS-CS+laser, the temperature of E. coli solutions maintained above 60 ℃ for 20 minutes (Figure S4). After 12 hours, the colony forming unit ratio of E. coli in 30GO-CTS-CS+laser group was decreased to 18.07 % (Figure 3d,e). In remarkable contrast, the colony forming unit ratios of E. coli in the CTS+Laser and CTS-CS+laser groups were over 76.70 % and 47.38 % under the same 12

irradiation conditions, respectively. Meanwhile, there were no significant antibacterial effects after being incubated with E. coli for 12 hours in the groups of CTS, CTS-CS and 30GOCTS-CS without laser irradiation. It is reported that CTS shows strong antibacterial effect only in acidic medium due to its poor solubility at neutral and alkaline conditions [48]. In this study, CTS and CTS-matrix composites were incubated with bacterial suspension in neutral pH environment, which may lead to their weak antibacterial ability. Furthermore, compared with antibacterial experiment of E. coli, 30GO-CTS-CS showed better photothermal antibacterial effect on S. aureus under the same irradiation conditions. There were few bacterial colony in 30GO-CTS-CS+laser group (Figure 3f,g). All these results indicated good in vitro photothermal antibacterial effect of hierarchical 30GO-CTS-CS film biomaterials. 3.4. Photothermal Antitumor Efficiency For the operative wounds after cutaneous tumor resection, ideal functional wound dressing is required to kill the residual asymptomatic tumor before healing skin wounds [10,28]. In this study, hierarchical 30GO-CTS-CS film biomaterials were utilized to investigate in vitro and in vivo photothermal antitumor effects. Typical skin tumor cells (B16F10 cells) were cultured on the different films under different irradiation conditions, respectively. It was obviously that tumor cells cultured on 30GO-CTS-CS films with laser irradiation had abnormal cellular morphology without pseudopodia, indicating that the activity of tumor cells was greatly reduced. On the contrary, tumor cells on other groups spread well with obvious pseudopodia (Figure S5, Supporting Information). Furthermore, tumor cells were seeded on glass slides and then cultured with films. For 30GO-CTS-CS+laser group, the temperature was controlled at approximately 52 ℃ by maintaining a certain power density of laser (808nm, 0.37 W/cm2). Almost all of skin tumor cells died in 30GO-CTS-CS+laser group, while the tumor cells of other groups had good cytological bioactivity. CCK-8 assays showed that the cell viability of surrounding tumor cells was only 2.03% in 30GO-CTS-CS+laser group while the tumor cell viability of other groups was close to 100% (Figure 3i), indicating the significant 13

photothermal antitumor effect of 30GO-CTS-CS on surrounding tumor cells in vitro. Furthermore, the in vitro antitumor efficiency could be further improved by increasing photothermal temperature, irradiation duration and irradiation times (Figure S6, Supporting Information). Subsequently, B16F10 tumor-induced mouse wound model was established to imitate operative wounds with remaining tumor cells after cutaneous tumor resection. The subcutaneous B16F10 tumor model has been applied for evaluating the in vivo antitumor efficacy of materials in many previous studies [49,50]. Furthermore, in clinical treatment, surgical excision of skin cancers will leave the asymptomatic tumor tissues and simultaneously result in cutaneous defects. Therefore, in order to imitate operative wounds after cutaneous tumor resection as much as possible, a round wound (Φ = 10 mm) was cut at tumor site on the flank of mouse in our tumor-induced wound model. To systematically evaluate in vivo photothermal antitumor effect, skin tumor-induced mice were divided into 7 groups as follows: Control, CTS, CTS-CS, 30GO-CTS-CS, CTS+laser, CTS-CS+laser and 30GO-CTS-CS+laser groups. The materials were cut into circle and completely covered visible tumor at the wounds. When exposed to laser (808 nm, 0.35 W/cm2), the surface temperature of 30GO-CTS-CS had increased sharply while the temperature of CTS+laser and CTS-CS+laser groups hardly changed (Figure 4a,b). The temperature at tumor sites in 30GOCTS-CS+laser group was maintained at approximately 52~54 ℃ for 15 minutes. Same NIR irradiation was performed from day 0 to day 3 in groups of CTS+laser, CTS-CS+laser and 30GO-CTS-CS+laser. The evolution of tumor volumes were recorded at regular intervals. After 4 days of treatment, the growth of tumors in 30GO-CTS-CS+laser group was obviously inhibited, while the tumors in other groups gradually grew up. For 30GO-CTS-CS+laser group, 14 days later, tumor did not recur and only a little tumor tissue could be observed in some cases (Figure 4c,e). In contrast, tumor in other groups uncontrolledly increased. It was found that the skin wounds gradually healed in 30GO-CTS-CS+laser group, while most of skin wounds in the other groups were not healed with the obstruction of the growing tumor 14

(Figure 4d). Furthermore, hexatoxylin and eosin (H&E) staining images of tumor-induced wounds were photographed and shown in Figure 4f. Unlike subcutaneous tissues with numerous tumor cells in other groups, the 30GO-CTS-CS+laser group showed regenerated tissues with normal skin tissue architectures. All the results suggested the significant photothermal antitumor effects of hierarchical 30GO-CTS-CS biomaterials. However, it was not enough to directly prove the possible photothermal effect on wound healing in this special wound model. Proper experiments need to be further designed for evaluating the efficiency and mechanism of photothermal effect on wound healing. Moreover, B16F10 tumor model on C57BL/6 mouse is also commonly used in skin tumor research. We will consider to apply B16F10 tumor model on C57BL/6 mouse to test our materials in the future research. 3.5. In Vitro Biocompatibility and In Vivo Wound Healing The tissue regeneration of skin itself is hard to heal wounds while the skin has suffered the extensive or deep wounds, which requires functional biomaterial to assist wound healing. As skin tissue engineering material, good compatibility and bioactivity were essential properties for functional wound dressing [12]. Therefore, the cell proliferation and cell attachment assays were firstly used to evaluate the in vitro biocompatibility. For cell attachment and morphology, two kinds of typical skin cell lines, human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs), were cultured on different films. SEM images showed HUVECs and HDFs spread well on three different films with pseudopodia, indicating that three different films could well support the adhesion of HUVECs and HDFs (Figure 5a, Figure S7 in Supporting Information). Cell proliferation assays showed that two kinds of cells in all groups increased significantly with culture time after 5 days (Figure 5b,c). In addition, cells cultured with CTS-CS and 30GO-CTS-CS showed better cell activity than other two groups, implying that CTS-CS and 30GO-CTS-CS possessed improved cytocompatibility with HUVECs and HDFs. To evaluate the in vivo tissue healing ability of 30GO-CTS-CS, a typical skin chronic wound model was applied. The different materials were 15

sliced into rounds (Φ = 10 mm) and covered on the wounds of diabetic mice. The wound area of different groups was recorded at day 0, 3, 6, 9, 12 and 14. As shown in Figure 5e, from day 3 to 9, the relative wound areas of CTS, CTS-CS and 30GO-CTS-CS were significantly lower than that of Control, indicating that CTS, CTS-CS and 30GO-CTS-CS could promote closure of chronic wounds at the early stage of wound healing. Meanwhile, it was observed good synthesis situation of collagen I in CTS, CTS-CS and 30GO-CTS-CS groups after 14 days (Figure S8, Supporting Information). Then, histological analysis of vertical section of skin wound after 14 days was further performed by using H&E staining method (Figure 5f). For the CTS-CS and 30GO-CTS-CS groups, the original skin wound sites formed complete and continuous epithelial tissue. Furthermore, hair follicles and glands were also observed in H&E staining images from CTS-CS and 30GO-CTS-CS groups, while there existed few hair follicles or glands in the Control and CTS groups, showing good in vivo wound healing quality of CTS-CS and 30GO-CTS-CS. Previous studies reported that Si ions were beneficial to cell proliferation of HUVECs and HDFs and could promote healing rate of diabetic wounds [28,31,51]. Therefore, it is reasonable to speculate that the good compatibility and bioactivity of CTS-CS and 30GO-CTS-CS should be closely related to the released Si ions from them (Figure S9, Supporting Information). Overall, hierarchical and porous 30GO-CTS-CS film biomaterials possessed good cytocompatibility and satisfactory effect of skin wound healing, showing their potential application in skin wound healing.

4. CONCLUSION In summary, we have prepared a hierarchical and porous GO-CTS-CS film biomaterial with orderly porous lamellar micron-scale structure and the “brick-and-mortar” layered nanostructure by combining vacuum filtration-assisted assembly and freeze-drying strategies. Benefiting from the layered nanostructure and the addition of GO, the hierarchical GO-CTSCS biomaterial had optimal tensile strength (~10 MPa). Meanwhile, the hierarchical GOCTS-CS biomaterial showed good breathability, water absorption and photothermal 16

performance. Moreover, hierarchical GO-CTS-CS biomaterial exhibited good in vitro photothermal antibacterial and antitumor effects. In vivo studies suggested that the GO-CTSCS biomaterials had satisfactory photothermal antitumor efficiency and the capacity for skin wound healing. Such a high-performance and multifunctional biomaterial may be used as a wound dressing for diverse wounds.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program of China (2018YFC1105201), the National Natural Science Foundation of China (81771989, 51761135103), Innovation Cross Team of Chinese Academy of Sciences (JCTD-2018-13) and Technology Commission of Shanghai Municipality (17540712300).

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Figure 1. Morphologies of hierarchical and porous GO-CTS-CS film biomaterials. (a) Photographs of different films. Fracture section morphologies of different films at (b) low magnification and (c) high magnification. The hierarchical GO-CTS-CS biomaterials (20GOCTS-CS, 30GO-CTS-CS and 40GO-CTS-CS) showed orderly porous lamellar micronscale structure and the “brick-and-mortar” layered nanostructure

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Figure 2. Characterization of hierarchical and porous GO-CTS-CS film biomaterials. (a) Tensile strength and (b) Representative tensile stress-strain curves of different films. (c) Photographs of hierarchical GO-CTS-CS films placed on plume and loaded with wooden arm model and 500-gram weight. (d) Comparison of tensile strength of 30GO-CTS-CS with previous reported wound dressings. (e) Moisture permeation and (f) water absorption of different films. Hierarchical and porous GO-CTS-CS film biomaterials exhibited high tensile strength, good breathability and water absorption. (*p<0.05, **p<0.01, ***p<0.001).

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Figure 3. Photothermal performance and in vitro photothermal antibacterial and antitumor performance. Temperature curves of different films under NIR irradiation at (a) dry state and (b) wet state. (c) The final temperature of GO-CTS-CS with different GO contents under NIR irradiation for 5 min at dry state. (d) Bacterial colonies of E. coli on agar culture plates in different conditions. (e) Colony forming unit ratio of E. coli in different conditions. (f) Bacterial colonies of S. aureus on agar culture plates in different conditions. (g) Colony forming unit ratio of S. aureus in different conditions. (h) Live/dead staining images of tumor cells cultured with different films under different irradiation conditions (red: dead cells, green: live cells). (i) Cell viability of tumor cells cultured with different films under different irradiation conditions. The 30GO-CTS-CS films displayed good in vitro photothermal 26

antibacterial and antitumor effects. (*p<0.05, **p<0.01, ***p<0.001).

Figure 4. In vivo photothermal antitumor efficiency of hierarchical GO-CTS-CS film biomaterials. (a) Infrared thermal images and (b) temperature curves of tumor-induced skin wounds treated with CTS, CTS-CS and 30GO-CTS-CS under NIR irradiation. (c) Changes of tumor volume over time in different groups. (d) Representative photographs of tumor-induced mouse in different groups at day 0 and day 14. (e) Photographs of tumors in different groups after treatment. (f) H&E staining images of tumor-induced skin wounds in different groups after treatment. The 30GO-CTS-CS films exhibited ideal in vivo photothermal antitumor effects.

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Figure 5. In vitro biocompatibility and in vivo skin wound healing efficiency of hierarchical GO-CTS-CS film biomaterials. (a) The morphologies of human umbilical vein endothelial cells (HUVECs) cultured on different films at day 1. Cell proliferation of (b) HUVECs and (c) human dermal fibroblasts (HDFs) cultured with different films, indicating good cytocompatibility of 30GO-CTS-CS. (d) Photographs of skin wounds and (e) relative skin wound area in different groups from day 1 to day 14. (f) H&E staining images of skin wounds in different groups on day 14. 30GO-CTS-CS group formed complete and continuous epithelial tissue with hair follicles and glands, showing satisfactory skin wound healing efficiency. (*p<0.05, **p<0.01, ***p<0.001).

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Scheme 1. Schematic illustration for the design strategy of hierarchical and porous GOCTSCS film biomaterials. (a) Preparation process and (b) multifunctional application of hierarchical GO-CTS-CS film.

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Graphical abstract

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