Copper Silicate Hollow Microspheres-Incorporated Scaffolds for

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Copper Silicate Hollow Microspheres-Incorporated Scaffolds for Chemo-Photothermal Therapy of Melanoma and Tissue Healing Qingqing Yu, Yiming Han, Xiaocheng Wang, Chen Qin, Dong Zhai, Zhengfang Yi, Jiang Chang, Yin Xiao, and Chengtie Wu ACS Nano, Just Accepted Manuscript • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Copper Silicate Hollow Microspheres-Incorporated Scaffolds for Chemo-Photothermal Therapy of Melanoma and Tissue Healing Qingqing Yu†,‡,⊥, Yiming Han§,⊥, Xiaocheng Wang†,‡, Chen Qin†,‡, Dong Zhai†, Zhengfang Yi*§, Jiang Chang†, Yin Xiao∥, and Chengtie Wu*† † State Key Laboratory of High Performance Ceramics and Superﬁne Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, P. R. China. ‡ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, P. R. China. § Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P. R. China. ∥ The Institute of Health and Biomedical Innovation, Queensland University of Technology, 80 Musk Avenue, Queensland 4059, Australia. ⊥Q. Y. and Y. H. contributed equally to this work. * Corresponding author: E-mail: [email protected] (C. T. Wu), [email protected] (Z. F. Yi)

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Abstract The treatment of melanoma requires complete removal of tumor cells and simultaneous tissue regeneration of tumor-initiated cutaneous defects. Herein, copper silicate hollow microspheres (CSO HMSs)-incorporated bioactive scaffolds were designed for chemo-photothermal therapy of skin cancers and regeneration of skin tissue. CSO HMSs were synthesized with interior hollow and external nanoneedle microstructure, showing excellent drug loading capacity and photothermal effects. With incorporation of drug-loaded CSO HMSs-into the electrospun scaffolds, the composite scaffolds exhibited excellent photothermal effects and controlled NIR-triggered drug release, leading to distinctly synergistic chemo-photothermal therapy of skin cancer both in vitro and in vivo. Furthermore, such CSO HMSs-incorporated scaffolds could promote proliferation and attachment of normal skin cells and accelerate skin tissue healing in tumor-bearing mice and diabetic mice. Taken together, CSO HMSs-incorporated scaffolds may be used for complete eradication of the remaining tumor cells after surgery and simultaneous tissue healing, which offers an effective strategy for therapy and regeneration of tumor-initiated tissue defects. Keywords: copper silicate, photothermal therapy, chemotherapy, melanoma, tissue healing


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Melanoma is one of the most aggressive skin cancer with an increasing incidence in recent years.1 Surgery, the primary treatment for such a skin disease, suffers from a high risk of relapse and large cutaneous defects.2-4 Since melanoma is highly resistant to chemo/radiotherapy, various alternative treatments have been explored to eradicate tumor cells, including photodynamic therapy (PDT) and photothermal therapy.5,6 Although PDT is an effective adjuvant treatment for non-melanoma skin cancer, the use of PDT for melanoma has not yet been substantially pursued, mainly due to the poor light penetration depth through pigmented lesions, and the antioxidant effect of melanin.1,7 Recently, photothermal therapy has shown great potential for cancer therapy, in which photothermal agents (PTAs) strongly absorbs NIR light and effectively convert photon energy into physical heat to ablate cancer cells, such as gold nanorod,8 gold nanoshells,9 and graphene oxide.6 However, few of them have the dual functions for therapy of skin tumor and regeneration of the tumor-induced skin defects, which can hardly heal by themselves. Furthermore, the immediate repair of damaged tissue after tumor resection is of great importance for long-term healing.10-12 Thus, it is quite necessary to develop a bifunctional biomaterial with cancer therapeutic capacity for treating residual skin tumor cells and simultaneous tissue healing capacity. Cu-based chalcogenides have been widely proved as effective PTAs due to intense NIR absorption, stemming from 3d electron transition of Cu ions from the valence band to the intermediate band, such as CuS,13 Cu9S5,14 CuCo2S4,15 and Cu2−xSe.16 However, there have been few reports using Cu-based chalcogenides as drug vehicles in the area of nanomedicine. Furthermore, previous studies have demonstrated that Cu ions can accelerate wound healing by promoting cell migration,17 angiogenesis,17-20 and collagen deposition.21 In addition, silicates-based biomaterials can promote the re-epithelialization and collagen deposition during wound healing.22,23 Considering the photothermal effect of Cu ions and the tissue regeneration capacity of both Cu and Si ions, it would be interesting to design muitifunctional


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Cu-based silicates, which could be served not only as PTAs and drug vehicles for combined skin cancer treatment, but also as healing agents for skin tissue regeneration after surgery. Until now, nanoparticle injection or transdermal delivery for photothermal therapy still remains notable challenges in clinical applications, such as particle aggregation and potential toxicity in excess of Cu ions,17,24-26 which are not conducive to tissue regeneration. However, such toxicity can be eliminated if Cu ions are slowly released from a localized depot.17 Moreover, it is difficult to inject PTAs intratumorllay for photothermal therapy and chemotherapy after tumor resection in clinical application. A tissue engineering scaffold may provide an easy and efficient strategy to overcome these challenges. Previous studies have reported that fibrous scaffolds can not only locally and controllably release the loaded cargos,27,28 but also provide mechanical support and structural cues for tissue ingrowth,23,29 which play a key role in wound healing. Therefore, it is resonable to incorporate nanoparticles into biocompatible fibrous scaffolds to decrease the aggregation and toxic side effects of the nanoparticles. Herein, we successfully prepared a copper silicate hollow microspheres (CSO HMSs)-incorporated electrospun scaffold, using CSO HMSs as drug-loaded PTAs for melanoma therapy and as the source of therapeutic elements (Cu and Si) for localized wound healing. Firstly, CSO HMSs with high drug loading capacity and excellent photothermal performance were synthesized by a hydrothermal method using SiO2 as a sacrificing template (Scheme 1a). The drug-loaded CSO (Tra-CSO) HMSs were then electrospun into Poly(ε-caprolactone)/Poly(D, L-lactic acid) (PP) matrix to obtain Tra-CSO-PP scaffolds (Scheme 1b), endowing the scaffolds with excellent photothermal effects and NIR-triggered drug release. Such scaffolds could be used for combined chemo-photothermal therapy in a single platform and achieved significantly synergistic antitumor effects both in vitro and in vivo (Scheme 1c). Furthermore, the CSO HMSs-incorporated scaffolds not only promoted the proliferation and adhesion of normal skin cells, but also accelerated wound healing in 4 ACS Paragon Plus Environment

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tumor-bearing mice and diabetic mice. Therefore, the prepared CSO HMSs-containing scaffolds could be used as promising candidates for high-efficiency therapy of melanoma and healing of skin tissues.


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Results and Discussion Characterization of CSO HMSs. CSO HMSs were successfully developed via a facile hydrothermal method using SiO2 spheres as a sacrificing template.30 When the SiO2 spheres were maintained in ammonia solution at high temperature, the silicate ions would produce and react with the Cu ions to generate the copper silicate, which was preferentially deposited on the surface of SiO2 spheres. With the reaction proceeding, the shell of copper silicate was produced and continued growing thicker until the SiO2 spheres were completely consumed, leading to the formation of CSO HMSs. CSO HMSs with interior hollow and external nanoneedle microstructure were observed by transmission electron microscope (TEM) and scanning electron microscope (SEM). As shown in Figure 1a-c, the CSO HMSs had inner hollow cavities of ~800 nm in diameter and spherical shells of ~50 nm in thickness, which were covered with nanoneedles of ~200 nm in length. Such microstructure enabled the CSO HMSs with large specific surface area of 400 m2/g and mesoporous (~2.1 nm) as determined by the N2 adsorption-desorption measurements (Figure S1a,b). The X-ray diffraction (XRD) pattern showed that CSO HMSs could be indexed into chrysocolla (Cu2-xSi2O5(OH)3·xH2O, Figure S2) without any additional impurities and the Si/Cu atomic ratio was 2.17 as determined by X-ray fluorescent spectrometry (XRFS) (Table S1). The selected-area electron diffraction (SAED) pattern of CSO HMSs also conformed the amorphous structure of nanoneedles (Figure S3). Energy-dispersive spectrometry (EDS) element mapping showed that the O, Si, and Cu elements were uniformly distributed (Figure 1d-f). To investigate drug-loading capacity of the CSO HMSs, Trametinib, a MEK inhibitor for the treatment of melanoma,31 was used as a model chemotherapy drug in our study. It was observed that the loading capacity of Trametinib gradually increased with increasing Trametinib concentrations (Figure 1g). The highest drug loading efficiency of CSO HMSs was determined to be 26.9% at the drug concentration of 2 mg/mL (Figure S4). After drug


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loading, the surface area of the CSO HMSs greatly decreased from 400 to 232 m2/g and the average hydrodynamic diameters of CSO HMSs increased from 955 nm to 1100 nm (Figure S1c), indicating that the drug molecular was partly located on the surface of CSO HMSs and partly distributed in the hollow center of HMSs. There were plenty of positive charged sites of Cu2+ ions on the surface of CSO HMSs, which might adsorb the negatively charged amide groups in Trametinib molecules.32 Therefore, the drug molecules could bond to CSO HMSs via electrostatic interaction, thereby greatly decreasing the zeta potential of CSO HMSs from 1.1 mV to -32.9 mV (Figure S1d and Figure 1h). All these results suggested that the CSO HMSs could be effective drug vehicles for chemotherapy. Previous studies have demonstrated that d-d electronic transition of Cu ions makes Cu-based chalcogenides strongly absorb NIR light.13-16 In our study, UV-vis spectra of the CSO HMSs in phosphate buffer solution (PBS) showed that the CSO HMSs exhibited obvious absorbance ~820 nm (Figure S5), which was suitable for photothermal therapy with the 808 nm laser. Thus, the photothermal performance of CSO HMSs was investigated using an 808-nm laser. Obviously, the temperature of CSO aqueous solution (0.02 g/mL) exhibited a laser-power-dependent manner and exceeded 70 °C within 10 min at 0.85 W/cm2, while the temperature of water without CSO HMSs showed no obvious elevation (Figure 1i). Furthermore, the temperature of CSO HMSs displayed no noticeable change after undergoing four cycles of the laser irradiation (0.25 W/cm2, 5 min, Figure S6), suggesting the excellent photothermal stability of CSO HMSs. The photothermal conversion efficiency (η) of CSO HMSs was further calculated to be 48.3 % according to previous methods (Figure S7),15 which was higher than the majority of PTAs such as Cu2-xSe nanocrystals (22 %),16 Cu9S5 nanoparticles (29.7 %),14 Au nanorods (21 %),33 and BP quantum dots (28.4%).34 These results indicated that the CSO HMSs were promising candidates for photothermal therapy of cancer.


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Characterization of scaffolds. Considering the excellent drug-loading capacity and photothermal effects of CSO HMSs, the drug-loaded CSO HMSs were incorporated into the PP fibrous scaffolds by electrospinning for combined cancer therapy. To investigate the effect of CSO contents on photothermal performance of scaffolds, different contents of CSO HMSs (10, 20, and 30 w. t. %) were added into PP matrix to prepare 10CSO-PP, 20CSO-PP and 30CSO-PP scaffolds, respectively. The CSO HMSs were uniformly dispersed in the electrospun solution and could maintained stable even for a week (Figure S8), suggesting that the CSO HMSs were uniformly distributed in polymer matrix during electrospinning within 2 hours. After electrospunning, the morphologies of those scaffolds were observed by SEM. The fibers were randomly distributed to form the continuous structure and the CSO HMSs were uniformly embedded in fibers without obvious aggregation even at the high CSO content of 30 w. t. % (Figure 2a-c,e-g and Figure S9). Subsequently, we systematically investigated the photothermal performance of those scaffolds using the 808-nm laser irradiation. The scaffolds exhibited a CSO-mass-dependent photothermal effect and the temperature of the 30CSO-PP scaffolds reached a plateau of ~50 °C at the power density of 0.65 W/cm2 in air for 5 min (Figure 2i and Figure S10). In contrast, the temperature of PP scaffolds without CSO HMSs hardly changed. Moreover, the temperature of the 30CSO-PP scaffolds increased with rising power density (0.45, 0.65 and 0.85 W/cm2) under wet conditions (0.5 mL water) for 15 min (Figure S11). We also investigated the stability of the the 30CSO-PP scaffolds. The thermostability was explored under laser irradiation (0.65 W/cm2, 15 min), and the morphology was observed by SEM. The results showed that biopolymer matrices maintained their original fiber morphology and dimensions (Figure S12), exhibiting excellent thermostability. To further evaluate the stability of the CSO HMSs in fibers, 30CSO-PP scaffolds were immersed in PBS (pH = 7.4) and treated with ultrasonication (50 KHz, 200 W) for 1 h. The CSO HMSs remained stable and still firmly adhered to the fibers after ultrasonication (Figure S13). 8 ACS Paragon Plus Environment

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To explore the feasibility of 30CSO-PP scaffolds for photothermal therapy, the heat penetration depth from the fibers to the surrounding tumors was investigated by placing the 30CSO-PP scaffolds on the top of pork with different thickness (2, 4, 6 and 8 mm), and irradiated by the 808-nm laser at the power density of 0.65 W/cm2 for 15 min. The temperature of the bottom of pork was recorded. It was found that the pork temperature at the depths of 6 mm increased by 15.6 °C (Figure S14), which was sufficient to kill tumor cells in vivo.35 To endow the scaffolds with photothermal effects and drug release for chemo-photothermal therapy of skin cancer, Trametinib loaded-CSO HMSs (30 w. t. %) were uniformly electrospun into polymer matrix to obtain Tra-CSO-PP scaffolds (Figure 2d,h and Figure S15), which exhibited similar morphology and photothermal effects with 30CSO-PP scaffolds. It has been proved that nanofibers have great potential for ‘on-off’ delivery of drugs, due to their extremely large surface area and porosity, which enhance the sensitivity to external stimuli.28 Furthermore, it is likely that generated heat could accelerate drug molecular motion, and the drug releasing rate was increased with temperature elevating.36 Thus, we investigated NIR-triggered drug release from Tra-CSO-PP scaffolds in PBS (pH = 7.4, Figure 2j). After four ‘on-off’ cycles of laser irradiation, the release percentage of Trametinib (~100 µg in total) reached ~55, 36 and 19%, when laser-induced temperature was 50, 43 and 37 °C, respectively. In contrast, a very small amount of Trametinib was released from Tra-CSO-PP scaffolds in the absence of NIR irradiation. Therefore, the incorporation of drug-loaded CSO HMSs endowed the scaffolds with significant photothermal performance, and the NIR laser could effectively control the drug release from scaffolds. Furthermore, the NIR-triggered release profiles of Cu and Si ions from Tra-CSO-PP scaffolds were also investigated by irradiating the scaffolds in PBS (pH = 7.4) with an 808-nm laser (0.65 W/cm2, 15 min) once per day for 4 days. As shown in Figure S16a,b, there was no obvious acceleration of ion release from Tra-CSO-PP scaffolds under NIR irradiation. In addition, the final temperature of 9 ACS Paragon Plus Environment

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Tra-CSO-PP scaffolds after irradiation (0.65 W/cm2, 15 min) on days 1, 2, 3 and 4 reached ~51.2, 50.7, 49.96, and 50.53 °C, respectively (Figure S16c), suggesting excellent photothermal stability of Tra-CSO-PP scaffolds. In Vivo Biocompatibility Assay. Biocompatibility of the scaffolds was evaluated before antitumor experiments. Human dermal fibroblasts (HDFs), typical skin cell lines, were seeded on PP, 30CSO-PP and Tra-CSO-PP scaffolds for 1, 3 and 5 days. The morphology of scaffolds and HDFs on day 3 was observed by SEM. When treated with the 30CSO-PP and Tra-CSO-PP scaffolds, HDFs with rich filopodia showed enhanced cell adhesion and spreading in contrast to the PP group (Figure 3a-c). Additionally, the fibers of PP, 30CSO-PP and Tra-CSO-PP scaffolds showed no obvious change, suggesting our scaffolds were stable for potential biomedical application. The cell viability was quantitatively examined by cell-counting kit-8 (CCK-8) assay. The results showed that cell viability on day 5 for the 30CSO-PP group was highest among all groups (Figure 3d). The improved cell viability and attachment might result from the sustained release of Si and Cu ions from the 30CSO-PP scaffolds. It is well known that the Cu ions at low yet potentially physiologically relevant concentration can accelerate cell migration and differentiation.17 In addition, the Si ions have been demonstrated to increase the collagen I synthesis and proliferation of skin fibroblasts in vitro.37 Thus, although Trametinib had a little side effect on the proliferation of skin fibroblasts, the composite Tra-CSO-PP scaffolds finally significantly increased the proliferation of skin fibroblasts in vitro as compared to the PP scaffolds. Then, the release profiles of Cu and Si ions were explored by inductively coupled plasma atomic emission spectrometry (ICP-AES). As shown in Figure 3e,f, the concentration of Cu ions released from 30CSO-PP scaffolds was ~13.6, 25.1, and 30.1 µg/mL on days 1, 3, and 5, respectively. The concentration of Si ions was ~19.4, 34.4 and 39.0 µg/mL on days 1, 3, and 5, respectively. In contrast, negligible amount of Cu and Si ions was released from PP scaffolds, further


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comfirming that Cu and Si ions from 30CSO-PP and Tra-CSO-PP scaffolds resulted in the increased proliferation of skin fibroblasts in vitro. The cytotoxicity of 30CSO-PP scaffolds and CSO HMSs was further explored by culturing HDFs with the extracts of 30CSO-PP scaffolds and CSO HMSs. After incubation for 24 h, the cell viability was measured by CCK-8 assay. As shown in Figure S17, the cell viability of HDFs cultured in the extracts of the 30CSO-PP scaffolds on day 3 was comparable to that for the control group. However, the extracts of CSO HMSs showed much toxicity against HDFs, which might be attributed to the fast release of Cu ions in DMEM from CSO HMSs. Then, release profile of Cu ions from 30CSO-PP scaffolds and CSO HMSs in DMEM was investigated by ICP-AES. The results showed that the amount of Cu ions released from 30CSO-PP scaffolds was much lower than that from CSO HMSs (Figure S18). It is likely that the polymer fibers had protective effect on the dissolution of CSO HMSs and thereby decreased the toxic side effects of naked CSO HMSs. In Vitro Anticancer Efficiency. After verifying the safety of the scaffolds, we explored the use of Tra-CSO-PP scaffolds as a therapeutic platform for chemo-photothermal therapy of melanoma skin cancer. To investigate the in vitro single chemotherapy efficacy of the Tra-CSO-PP scaffolds, the skin tumor cells (murine B16F10 melanoma cells) were treated with PP, 30CSO-PP and Tra-CSO-PP scaffolds without laser irradiation and the cell viability was explored by CCK-8 assay. In vitro chemotherapy efficacy of the Tra-CSO-PP scaffolds was studied through the comparison of cell viability of B16F10 for Tra-CSO-PP and 30CSO-PP group. The results (Figure 4a) showed that the cell viability without irradiation (0Laser) for Tra-CSO-PP and 30CSO-PP group was ~90% and ~98%, respectively, suggesting single chemotherapy had insufficient effect on killing tumor cells. To investigate the antitumor effects of chemo-photothermal therapy, the Tra-CSO-PP scaffolds were irradiated (808 nm, 45 °C, 15 min) for 1, 2 and 3 times, the viability of B16F10 cells was studied via CCK-8 assay and confocal laser scanning microscopy (CLSM). It was found that 11 ACS Paragon Plus Environment

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after irradiation for 1, 2 and 3 times, the cell viability for the Tra-CSO-PP+Laser group significantly decreased to ~40.9, 14.0 and 0.1%, while that for the 30CSO-PP+Laser group was ~69.7, 49.6 and 16.7%, respectively. In contrast, no significant difference in cell viability was observed in the PP group with increasing irradiation times. The CLSM images (Figure 4b and Figure S19) also displayed that the amount of living B16F10 cells significantly decreased with increasing irradiation times and Tra-CSO-PP+Laser group showed the maximum antitumor effects. These results indicated that the drug release and hyperthermia effects induced from the Tra-CSO-PP scaffolds led to the highest tumor cell mortality rate among all groups, exhibiting a significantly synergetic effect of chemo- and photothermal therapy of tumor cells in vitro. The synergistic index was further evaluated using the mortality rate of the B16F10 cells (10.0% for Tra-CSO-PP, 30.3% for 30CSO-PP+Laser and 59.1% for Tra-CSO-PP+Laser) and analyzed with the Jin’s formula.38,39 The synergistic index in our study was 1.59, which was larger than 1.15, indicating a synergistic effect of chemotherapy and hyperthermia. It is likely that CSO HMSs-induced hyperthermia not only resulted in cell coagulative necrosis, protein denaturation, mitochondrial dysfunction and a halt in enzyme activity,40,41 but also enhanced drug accumulation in cells and cell sensitiveness to drugs.42 Such synergetic effect was further demonstrated by SEM characterization (Figure S20). The effect of the power density on the viability of B16F10 cells was explored by CCK-8 assay and live/dead staining. As shown in Figure 4c, the cell viability for the Tra-CSO-PP+laser group significantly decreased to ~26.8, 1.2 and 0.7%, while that for the 30CSO-PP+Laser group was ~70.2, 25.3 and 0.8%, when the power density was at 0.45, 0.65 and 0.85 W/cm2 (808 nm, 15 min, one time), respectively. Live/dead staining images (Figure 4d) further confirmed the amount of dead B16F10 increased with the increasing power density and the Tra-CSO-PP+Laser group showed a higher cell killing efficiency than the Tra-CSO-PP and 30CSO-PP+Laser group, indicating a synergistic effect of drug and hyperthermia on anticancer efficiency. All results above indicated the excellent and 12 ACS Paragon Plus Environment

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controllable chemo-photothermal therapeutic efficiency of Tra-CSO-PP scaffolds in vitro. Concerning that single photothermal therapy with the power density of 0.85 W/cm2 could kill tumor cells completely, we thus selected the laser power density of 0.65 W/cm2 for in vivo therapy to investigate the synergistic effect of chemo-photothermal therapy. In Vivo Anticancer Efficiency. Then in vivo antitumor capacity was explored using B16F10 tumor-bearing mice, and the mice were randomized into 7 groups: Control, PP, PP+Laser, 30CSO-PP, 30CSO-PP+Laser, Tra-CSO-PP, Tra-CSO-PP+Laser. The tumors were covered by scaffolds, and followed by 808-nm irradiation (0.65 W/cm2, 15 min, four times). The tumor surface temperatures rapidly increased to ~50 °C, which was sufficient to ablate the tumor cells and had little side effects in wound tissue in vivo,43 while remained below 38 o

C when treated with the PP scaffolds (Figure S21). It was found that the tumors for the

Control, PP, 30CSO-PP, and Tra-CSO-PP groups showed quick growth rates (Figure 5a), suggesting released Trametinib and CSO HMSs themselves were not effective in tumor therapy. However, once exposed to irradiation, both the 30CSO-PP and Tra-CSO-PP scaffolds showed significant inhibition in the tumor growth. Thus, the laser-induced hyperthermia displayed more effective tumor inhibition than the chemotherapy drug. Interestingly, the tumor growth for the Tra-CSO-PP+Laser group was almost completely inhibited, leading to the smallest tumor volume on day 14, while small tumors for the 30CSO-PP+Laser group grew back, resulting in a slightly increased tumor volume again. The tumor tissues on day 14 were weighed (Figure S22), photographed (Figure 5b) and treated with hexatoxylin and eosin (H&E) staining, Ki67 immunohistochemical (IHC) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Figure 5c). According to H&E staining images, the nuclei of tumor cells for the Tra-CSO-PP+Laser group were seriously damaged with abundant karyorrhectic debris. Furthermore, Tra-CSO-PP+Laser induced the maximum inhibition of Ki67 antibody expression of tumor sectioning and showed significantly reduced proliferation of tumor cells 13 ACS Paragon Plus Environment

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after combined antitumor therapy, indicating that antitumor performance was much better than that of stand-one chemotherapy and photothermal therapy for the Tra-CSO-PP and 30CSO-PP+Laser group. Such synergistic effect was further demonstrated by TUNEL assay. Thus, Tra-CSO-PP+Laser scaffolds combining photothermal therapy with chemotherapy distinctly induced apoptosis and inhibited the proliferation of tumor cells, exhibiting a synergistic effect of chemo- and photothermal therapy in vivo. In Vivo Tissue Healing in Tumor-Bearing Mice. To investigate the effect of laser irradiation on the normal tissues adjacent to the tumor, the skin wound of tumor-bearing mice was photographed on days 0, 4, 8, and 14 (Figure 6a). The tumor growths for the Tra-CSO-PP+Laser group was significantly inhibited after four days of treatment, and gradually turned into black scars and fell off. The wound for the Tra-CSO-PP+Laser group even completely healed without tumor recurrence within 14 days. To further evaluate the quality of tissue healing for Tra-CSO-PP scaffolds, the newly regenerated epidermis at wound beds on day 14 was characterized by histologic analysis. As shown in Figure 6b, the epidermis for the 30CSO-PP group was thickest among all groups and was regenerated with the normal architecture. The incomplete epidermis was observed in the Tra-CSO-PP group, while no newly epidermis regenerated in the PP group, suggesting that the quality of wound healing by Tra-CSO-PP scaffolds was obviously higher than PP scaffolds. In addition, greater infiltration of fibroblasts were observed from the Tra-CSO-PP+Laser group as compared to the control group, suggesting the accelerated wound healing in tumor-bearing mice. In Vivo Tissue Healing in Diabetic Mice. In order to eliminate the side effects of the tumor on wound healing, we then used a typical diabetic mouse model to study wound healing efficacy of these scaffolds in vivo. The diabetic mice were randomized into 4 groups: Control, PP, 30CSO-PP, Tra-CSO-PP. A full-thickness circular skin wound was created and covered with scaffolds. Previous studies have reported that Trametinib has negative impact on angiogenesis and cutaneous wound healing.44 Interestingly, both the 30CSO-PP and 14 ACS Paragon Plus Environment

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Tra-CSO-PP groups exhibited a significantly higher wound healing rates than the PP group (Figure 7a,b). To explore the mechanism of accelerated wound healing of Tra-CSO-PP scaffolds, IHC and Masson’s Trichrome staining were performed (Figure 7c). CD31 is considered as a marker of endothelial cells and indicates capillary formation.45 CD31-positive cells for the 30CSO-PP and Tra-CSO-PP groups significantly exceeded that for the PP group (Figure 7d), indicating that capillary formation at wound sites was greatly enhanced by CSO HMSs. The enhanced pro-angiogenesis in vivo could be contributed to the released Cu ions from CSO HMSs, which have been previously demonstrated to stabilize hypoxia-inducible factor-1α and further induce the expression of VEGF.17,19 Moreover, the combination of Cu and Si ions can achieve synergistically stimulatory effects on vascularization.46 In our study, we

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expression

of

angiogenesis-related genes in vivo (Figure 7e) and in vitro (Figure S23). Furthermore, Masson trichrome staining showed most improved re-epithelialization and collagen deposition at the wound beds for the 30CSO-PP group (Figure 7f-h). Collagen, a major component of the extracellular matrix (ECM), plays an important role in ECM re-organization and tissue re-molding.47 The increased Collagen I/III expression for the 30CSO-PP group facilitated ECM re-organization, which in turn induced improved re-epithelialization. It is reported that the Si ions can promote the re-epithelialization and collagen deposition at wound beds via activating the EMT and EndMT signal pathway.22 Furthermore, Si ions have also been demonstrated to increase the proliferation of skin fibroblasts and synthesis of collagen type I in vitro.37 In addition, Cu ions can promote ECM maturation during wound healing by upregulating the expression of cytosolic Atox1, which functions as a Cu chaperone for secretory ECM Cu enzyme LOX.20 Therefore, although Trametinib had a little side effect on the wound healing, the composite Tra-CSO-PP scaffold systems significantly promoted diabetic skin wound healing in vivo by facilitating both revascularization and


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re-epithelialization, which were mainly contributed to the Cu and Si ions released from the scaffolds.


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Conclusions In summary, we have successfully prepared a multifunctional scaffold with the incorporation of CSO HMSs which can be used for both chemo-photothermal therapy of melanoma and skin tissue healing. Firstly, CSO HMSs were synthesized with interior hollow and external nanoneedle microstructure, which enabled them to be effective drug vehicles. Meanwhile, CSO HMSs, a kind of promising PTAs, exhibited excellent photothermal performance. Moreover, the drug-loaded CSO HMSs were electrospun into fibrous scaffolds, endowing the scaffolds with excellent photothermal effects and controlled drug release, leading to significantly synergistic effects on killing skin tumor cells both in vitro and in vivo, as compared to single photothermal treatment or chemotherapy. Furthermore, such CSO HMSs-incorporated scaffolds could promote proliferation and attachment of normal skin cells and accelerate skin tissue healing in both tumor-bearing mice and diabetic mice by stimulating both revascularization and re-epithelialization. Thus, our study suggests that the prepared

CSO

HMSs-containing

scaffolds

offer

a

multifunctional

chemo-photothermal cancer therapy and simutaneous tissue healing.


platform

for

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Methods Materials. Tetraethoxysilane (TEOS), ammonium hydroxide (NH3·H2O, 28%), cupric nitrate trihydrate (Cu(NO3)2·3H2O), tetrahydrofuran (THF), N, N-dimethylformamide (DMF), dimethyloxaloylglycine (DMOG) and Trametinib werepurchased from Sigma-Aldrich (Shanghai, China). Poly(ε-caprolactone) (PCL, Mn = 80,000) and Poly(D, L-lactic acid) (PDLLA, Mn = 1,750,000) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and Jinan Daigang Biomaterial Co. Ltd. (Shandong, China), respectively. All reagents were applied as received with no further purification. Synthesis of CSO HMSs. CSO HMSs were synthesized via a facile hydrothermal treatment involving a chemical-template etching process. Firstly, the SiO2 colloidal spheres were prepared according to the Stöber method,48 and used as self-sacrificing template. The as-prepared SiO2 spheres (0.13 g) were then homogeneously dispersed in distilled water (50 mL) followed by the addition of Cu(NO3)2·3H2O (0.7 mmol) and NH3·H2O (5 mL). After vigorously stirring for 30 min, the resulting suspension was transferred into a Teflon-lined autoclave (50 mL) and maintained at 140 °C for 12 h. The obtained precipitates were collected, washed with distilled water and ethanol, and finally dried at 60 °C. Preparation of Tra-CSO HMSs. To prepare Trametinib-loaded CSO (Tra-CSO) HMSs, 0.05 mg of as-synthesized CSO HMSs was dispersed in 1 mL of Trametinib/DMSO solution at Trametinib concentration of 2 mg/mL. After incubation on a shaker (120 r/min, 37 °C) for 24 h, the mixture was centrifuged, washed twice with distilled water to remove the excess Trametinib molecules and DMSO, and then dried for further use. To determine the drug loading capacity and loading efficiency of CSO HMSs, the Trametinib at various concentrations (0.25, 0.5, 1, 2, 3 and 4 mg/mL) was loaded onto CSO HMSs following the similar procedure. The supernatant was collected by centrifuging. The final equilibrium concentration of Trametinib in the supernatant was detected using UV-vis analysis at 360 nm. 18 ACS Paragon Plus Environment

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The drug loading capacity was calculated using equation (1).

The drug loading efficiency was calculated using equation (2).

Where C0 and C1 (mg/mL) are the initial and final concentration of Trametinib, respectively, and W (mg) is the weight of the CSO HMSs. Preparation of Tra-CSO-PP scaffolds. The Tra-CSO-PP scaffolds were fabricated via a facile electrospinning process. Typically, PCL (0.075 g), PDLLA (0.075 g) and Tra-CSO HMSs (0.045 g) were blended in the mixed solvent of DMF (1.7 mL) and THF (0.3 mL). After continuous stirring at 30 °C for 10 h, the mixed solution was placed in a 5-mL plastic syringe with a 23-gauge needle tip. Electrospinning was conducted under the following conditions: injection rate: 0.03 mL/min; voltage: 10 KV; relative humidity: ~60 %RH. The final fibrous product was collected on a flat aluminum-foil plate placed at 20 cm apart from the needle tip and kept at room temperature for 24 h to remove the residual solvent. Similarly, the PP, 10CSO-PP, 20CSO-PP, and 30CSO-PP scaffolds were prepared with CSO HMSs contents of 0, 10, 20 and 30 w. t. % relative to the polymer mass, respectively. Characterization. The morphologies were observed by transmission electron microscopy (TEM, JEM-2100F, JEOL, Japan). Elemental distribution was observed by scanning electron microscopy (SEM, Magellan400, FEI, USA) equipped with energy-dispersive spectrometry (EDS). The phase composition of CSO HMSs was measured by X-ray diffraction analysis (XRD, Rigaku D/Max-2550V, Geigerflex, Japan). The molar ratio of Si to Cu was measured using X-ray fluorescent spectrometry (XRFS, AXIOS, PANalytical, Netherlands). The specific surface area based on the Brunauer-Emmett-Teller (BET) methods and the pore size distribution of CSO and Tra-CSO HMSs were determined at 77 K by N2 19 ACS Paragon Plus Environment

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adsorption–desorption measurement (Micromeritics ASAP 2010 analyzer, Micromeritics, USA). The surface charges and size distribution were measured by Zeta-potential test (Zeta sizer Nanoseries Nano ZS90, UK). The chemical bond structure was assessed by Fourier transform infrared spectroscopy (FTIR; Nicolet iS50, USA). Photothermal Performance of CSO HMSs. To study the photothermal performance of CSO HMSs, CSO aqueous solution (0.05 mg/mL) was exposed to an 808-nm laser irradiation at various power densities (0.45, 0.65 and 0.85 W/cm2) for 10 min. The temperature changes were recorded by an infrared (IR) thermal camera (PM100D, Thorlabs GmbH, Munich, Germany). To evaluate the photothermal stability, temperature changes of CSO powders (0.05 g) was monitored by an IR-camera under 5 rounds of laser-irradiated heating (808 nm, 0.25 W/cm2, 5 min) and natural cooling cycles. The photothermal conversion efficiency (η) of CSO HMSs was calculated using the following Equation (3),

where h is the heat transfer coefficient, A is the container surface area, Δ Tmax is the maximum temperature change of the CSO HMSs solution (35.5 °C), I is the laser power (728 mW) and Aλ is the absorbance (1.876) at 808 nm. Q0 is the heat input due to light absorption by the solvent. The lumped quantity hA was determined according to Equation (4),

where C and m are the heat capacity (4.2 J/g) of water and mass (0.255 g), respectively. Thus, the η value of CSO HMSs was caculated to be 48.4 %. In order to get the hA, θ is introduced in, which is defined as the ratio ofΔT toΔTmax (5),

Photothermal Performance of Tra-CSO-PP scaffolds. PP, 10CSO-PP, 20CSO-PP, 30CSO-PP and Tra-CSO-PP scaffolds were tested under dry conditions with power densities 20 ACS Paragon Plus Environment

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of 0.65 W/cm2 for 5 min, and wet conditions (0.5 mL PBS) with power densities of 0.85 W/cm2 for 10 min. The thermal images and temperature changes were recorded by an infrared (IR) thermal camera. In Vitro NIR-Triggered Drug and Ion Release. Tra-CSO-PP scaffolds were immersed in PBS buffers (0.5 mL) with various pH values (5.5, 6.8 and 7.4) at 37 oC. At determined time intervals, the medium was collected for UV-Vis analysis and immediately replaced with fresh PBS solution. To study NIR-triggered drug release behavior, the Tra-CSO-PP scaffolds were immersed in PBS (pH = 7.4, 0.5 mL). The NIR laser was turned on to heat the PBS solution to 37, 43 or 50 °C and maintained for 30 min by adjusting power density of laser in real time. The laser was then turned off, allowing the solution to cool down to room temperature (25 °C), and maintained for 1 h. The released Trametinib was collected for UV-vis analysis. The accumulative released Trametinib was calculated using equation (6).

Where Dreleased is the accumulative amounts of released Trametinib and Dtotal is the total amounts of Trametinib incorporated in the scaffolds (~100 µg). The NIR-triggered release profiles of Cu and Si ions from Tra-CSO-PP scaffolds were investigated by irradiating the scaffolds in PBS (pH = 7.4). The Tra-CSO-PP scaffolds were immersed in PBS (pH = 7.4) at 37 °C for 4 days and irradiated by an 808-nm laser (0.65 W/cm2, 15 min) once per day. The concentration of Cu and Si ions in PBS after irradiation was analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). As control, the 30CSO-PP scaffolds without irradiation were maintained at 37 °C. In Vitro Biocompatibility Assay of Tra-CSO-PP Scaffolds. The human dermal fibroblasts (HDFs) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, China) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 µg/mL of streptomycin. The PP, 30CSO-PP, Tra-CSO-PP scaffolds were firstly placed in 21 ACS Paragon Plus Environment

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48-well plates. The HDFs (2.0 × 104 cells/well) were then seeded on scaffolds and incubated for 1, 3 and 5 days in a humidified incubator (5% CO2, 37 °C). As a control, HDFs were cultured in blank wells. At the intervals of 1, 3 and 5 days, the cell viability was measured using CCK-8 assay (Cell counting kit-8, Kumamoto, Japan). In brief, the scaffolds and supernatants were removed. The cells were incubated with CCK-8 solution (200 µL) for another 1.5 h. Thereafter, the supernatant (100 µL) was pipetted into a 96-well plate and the absorbance was measured at 450 nm (ODcontrol, ODsample) by a microplate reader (Epoch, BIO-TEK, USA). Cell viability was calculated using equation (7).

Where ODCCK-8 is the absorbance of CCK-8 solution without cells. To measure the released amount of Si and Cu ions, the PP, 30CSO-PP and Tra-CSO-PP scaffolds were soaked in DMEM for 5 days. The medium was collected on days 1, 3 and 5 for release analysis using inductively coupled plasma atomic emission spectrometry (ICP-AES, Vista AX, Varian, Palo Alto, CA, US). For cell morphology observation, HDFs incubated on scaffolds for 3 days were fixed in 2.5% glutaraldehyde, washed with PBS, dehydrated in a series of ethanol solutions (30, 50, 70, 90, 80, 95, and 100 % (v/v)) and hexamethyldisilazane (HDMS), and finally visualized under SEM. The effect of the CSO HMSs on the angiogenesis-related gene expression of the human umbilical vein endothelial cells (HUVECs) was assessed using real-time quantitative polymerase chain reaction (RT-qPCR). The CSO HMSs were dispersed in complete endothelial cell medium (ECM) with CSO HMSs concentration of 0, 0.1, 0.5, 2 and 10 (ug/mL). The HUVECs were cultured in CSO/ECM dispersion for 3 days in a humidified incubator (5% CO2, 37 °C) and harvested using Trizol reagent (Invitrogen) to extract the RNA. The concentration of RNA was measured by a Nanodrop 2000 reader (Thermo Scientific). cDNA was synthesized using a PrimeScript RT reagent kit (TaKaRa) according to the 22 ACS Paragon Plus Environment

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manufacturer’s instructions. Primers of VEGF, HIF-1a, VEGF receptor 2 (VEGF-2) (KDR), endothelial nitric oxide (eNos) and GAPDH were synthesized according to previous publication.49 The data were normalized against the housekeeping gene GAPDH mRNA expression and were quantified relative to the corresponding gene expression of the control sample (cells cultured in ECM without CSO HMSs), which were standardized to 1. In Vitro Antitumor Efficacy. The skin tumor cells (murine B16F10 melanoma cells) were cultured in DMEM supplemented with 10% FBS, 100 IU/mL of penicillin and 100 µg/mL of streptomycin. The B16F10 cells (5.0 × 104 cells/well) were seeded in 48-well plates and cultured for 24 h in an incubator (37 °C, 5% CO2). The PP, 30CSO-PP or Tra-CSO-PP scaffolds (10 mm in diameter) were added to the culture wells. Subsequently, the NIR laser was turned on and the cells were treated with 45 °C for 15 min. This irradiation treatment was conducted for 1, 2 and 3 times every 12 h. As a control, The B16F10 cells were cultured without scaffolds in the absence of irradiation. The cell viability after irradiation for 1, 2 and 3 times was determined by CCK-8 assay. The synergetic index (QA+B) of the combination of CSO-enabled photothermal therapy and Trametinib treatment was analyzed with the Jin’s formula using equation (8).38,39

Where EA, EB and EA+B are the mortality rate of the B16F10 cells after photothermal therapy only, chemotherapy only, and chemo-photothermal therapy, respectively. In the Jin’s method, QA+B < 0.85 shows antagonism, 0.85≤ QA+B < 1.15 shows additive effects, and QA+B ≥1.15 shows synergism. To further verify the CCK-8 results, live cells were stained with rhodamine phalloidin and 4, 6-diamidino-2-phenylindole (DAPI, Cytoskeleton Inc., Denver, CO, US) for observation of cell cytoskeleton and nuclei under confocal laser scanning microscope (CLSM, TCS SP8, Leica, Germany), respectively. To directly observe the cell morphology after irradiation, the 23 ACS Paragon Plus Environment

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B16F10 cells exposed to irradiation once were used for SEM (SU8220, Hitachi, Tokyo, Japan). The cells were fixed with 2.5% glutaraldehyde, dehydrated by a series of ethanol solutions (30, 50, 70, 90, 80, 95, and 100% (v/v)) and HMDS. To observe the distribution of live and dead cells, cells were also fixed with 4% paraformaldehyde, stained with Ethidium homodimer-1 and Calcein AM for live/dead cell staining images using CLSM. In Vivo Antitumor Efficacy and Tissue Healing of Tra-CSO-PP Scaffolds in Tumor-Bearing Mice. Balb/c-nude mice (6-8 weeks old, male) were purchased from the Kinglake Animal Center (Shanghai, China). All animal experiments were approved by the Institutional Animal Care and Use Committee of East China Normal University. B16F10 cells (1 × 106) were injected into the right flank of mice to establish the subcutaneous melanoma tumor models. When the tumor diameter reached ~8 mm, the mice were randomized into 7 groups (n = 5): Control, PP, PP+Laser, 30CSO-PP, 30CSO-PP+Laser, Tra-CSO-PP, Tra-CSO-PP+Laser. A full thickness excision wound (diameter: 10 mm) was made at the tumor site. The mice were dressed with relevant scaffolds (diameter: 10 mm) according to grouping. For the laser treatment groups, each mouse was treated with the 808-nm irradiation (0.65 W/cm2, 15 min) once a day from day 0 to day 3. The tumor surface temperature and thermal photograph were recorded by an IR-camera. The tumors and skin wounds were photographed on days 0, 4, 8 and 14. The length and width of the tumors were measured every 2 days and relative tumor volumes were calculated using equation (9).

Where Lt and Wt are the length and width of the tumors on day t, L0 and W0 are the length and width of the tumors on day 0. On day 14, the mice were sacrificed and tumor tissues were excised, weighed, and photographed. Histological analysis of tumor tissues was evaluated by hematoxylin and eosin (H&E) staining, fluorescence terminal deoxynucleotidyl transferase-mediated dUTP nick end 24 ACS Paragon Plus Environment

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labeling (TUNEL assay) and Ki67 Immunohistochemical (IHC) staining. The tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin sectioned into 5-µm-thick slices stained with H&E, and finally examined in an optical microscope (Olympus BX53, Japan). TUNEL assay was performed to observe cell apoptosis. The sections were stained with TUNEL fluorescence in situ Apoptosis Detection kit (Yeasen, Shanghai, China) according to the manufacturer’s manual. The cell nuclei were counterstained with DAPI. TUNEL stained slides were visualized under a fluorescence microscope (Olympus BX53, Japan). To further verify the results of TUNEL assay, tumor cell proliferation was evaluated by IHC staining using Ki67 antibody (Abcam, UK) as the marker of cell proliferation. The tissue section samples were dewaxed, boiled in sodium citrate buffers and incubated with Ki67 antibody overnight at 4 °C. Then the samples were visualized by a diaminobenzidine detection kit (Vector Laboratories, USA) at room temperature. Haematoxylin was used to stain the nuclei. The slides were evaluated using an optical microscope (Olympus BX53, Japan). In addition, histological analysis of the wound tissues was evaluated by H&E staining. In Vivo Diabetic Skin Tissue Healing. C57BL/6J mice (7-8 weeks old, male) were purchased from the Kinglake Animal Center (Shanghai, China). The mice were intraperitoneally injected streptozocin (STZ, Sigma Aldrich, St.Louis, MO, 50 mg/kg) for 5 days. The blood glucose level of mice was measured using glucose meters (Accu-Chek Performa) after 2 months post-injected. The mice were considered as diabetic if the non-fasted glycemia level was higher than 20 mM. The diabetic mice were randomized into four groups (n = 8): Control, PP, 30CSO-PP, Tra-CSO-PP. The dorsal hair of mice was shaved and a circular full-thickness skin wound (diameter: 10 mm) was created, covered with a scaffold according to grouping, finally shielded with Tegaderm™ (3M, St. Paul, MN, US). As a control, wounds were only covered with Tegaderm™. The wounds were photographed every two days from day 3 to day 15 and the wound area was measured by Image J. The relative wound area was calculated using equitation (10). 25 ACS Paragon Plus Environment

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Where Ct and C0 are the wound areas on days t and 0, respectively. On day15, the whole wound tissue with a margin of around 5 mm of ambient unwounded skin was excised, fixed with 4% paraformaldehyde and finally sectioned into 5-µm thick slices for Masson’s trichrome staining. Immunofluorescence stain of CD31 was performed for observing the angiogenesis. The sections were deparaffinized with dimethylbenzene, 100%, 95%, 80%, 70% ethanol, then boiled in sodium citrate buffer for antigen retrieval blocked by 5% BSA. CD31 antibody (Abcam, UK) was added to the tissue sections, and then incubated at 4 oC overnight. The sections were added secondary antibodies and incubated at room temperature for 2 h. The DAPI solution (5 mg/ml) was added on the tissue sections for counterstaining cell nucleus and finally photographed by an optical microscope. The whole wound tissue with a margin of around 2 mm of ambient unwounded skin was crushed to obtain the Total RNA using Trizol (Invitrogen, USA) according to the manufacturer’s protocols and cDNA were synthesized from total RNA (1µg) using Prime Script™ RT Master Mix (Takara Bio Inc., Japan). RT-qPCR was analysis using SYBR Green detection reagent (TakaraBio Inc, Japan). In this study, β-actin was used as reference genes. The specificity of primers was confirmed before use. The primer sequences are shown in supplemental Table S2. Statistical Analysis. All quantitative data were expressed as means ± standard deviation (SD). Statistical significance was evaluated using a one-tailed Student’s t-test, followed by the Mann–Whitney U test. Values of p < 0.05 were considered statistically significant.

Associated Content Supporting Information 26 ACS Paragon Plus Environment

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Characterization of CSO HMSs (Figure S1-S5, Table S1). Photographs of electrospun solution for 30CSO-PP scaffolds (Figures S8). Morphologies of scaffolds (Figures S9, S12, S13, S15). The heat penetration depth of 30CSO-PP scaffolds (Figure S14). Photothermal effect of CSO HMSs (Figures S6, S7) and scaffolds (Figures S10, S11). NIR-triggered ion release from Tra-CSO-PP scaffolds (Figures S16). In vitro biocompatibility assay of 30CSO-PP scaffolds and CSO HMSs (Figures S17). The release profile of Cu ions from 30CSO-PP scaffolds and CSO HMSs (Figure S18). Extended information for in vitro (Figures S19, S20) and in vivo (Figures S21, S22) tumor therapy. In vivo angiogenesis-related gene expression (Figures S23). The primer sequences used for the RT-qPCR study (Table S2) (PDF). Author Information Corresponding Author *E-mail: [email protected]. (C. T. Wu) *E-mail: [email protected]. (Z. F. Yi) Author Contributions ⊥ Q. Y. and Y. H. contributed equally to this work. Q. Y. and C. W. designated the idea of the present work. C. W., Z. Y., J. C. and Y. X. supervised the project and commented on the project. Q. Y. and C. Q. synthesized and characterized the CSO HMSs and composite scaffolds, performed in vitro experiments, and analyzed the data. D. Z. performed in vitro experiments of expression of angiogenesis-related genes and analyzed the data. Q. Y., Y. H. and X. W. performed in vivo antitumor experiments and analyzed the data. Q. Y. and Y. H. performed in vivo experiment of diabetic tissue healing and analyzed the data. Q. Y. wrote the manuscript. All the authors contributed to the discussion during the whole project. Acknowledgements This work was partially supported by National Key Research and Development Program of China (2016YFC1100200), Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-SYS027) and Science and Technology Commission of Shanghai Municipality (17441903700, 17540712300).


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Scheme 1. Schematic illustration of the design and application of Tra-CSO-PP scaffolds. (a) Synthesis of cooper silicate hollow microspheres (CSO HMSs) with interior hollow and external nanoneedle microstructure via a facile hydrothermal method using SiO2 as a sacrificing template and the subsequent anti-cancer drug (Trametinib) loading (Tra-CSO HMSs). (b) Fabrication of Tra-CSO HMSs-incorporated Poly(ε-caprolactone)/Poly(D, L-lactic acid) (PP) fibrous scaffolds (Tra-CSO-PP) by electrospinning. (c) Chemo-photothermal therapy of melanoma skin cancer and healing of skin tissues.


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Figure 1. Characterization of CSO HMSs. (a, b) TEM images of CSO HMSs. (c) SEM image and (d, e, f) corresponding EDX element mappings (Si, O, and Cu, respectively) of CSO HMSs. (g) Loading weight of anti-cancer drug (Trametinib) in CSO HMSs. (h) Zeta potential of CSO HMSs after soaking in Trametinib/DMSO solution with different Trametinib concentrations (0, 0.25, 0.5, 1, 2 mg/mL). (i) Photothermal heating curves of CSO aqueous solution (0.02 g/mL) under 808-nm laser irradiation at various power densities for 10 min. The CSO HMSs with interior hollow and external nanoneedle structure possessed high drug-loading capacity and excellent photothermal effects.


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Figure 2. Morphology and NIR-triggered drug release behavior of Tra-CSO-PP scaffolds. SEM images of scaffolds: (a, e) PP (0 w. t. % CSO), (b, f) 10CSO-PP (10 w. t. % CSO), (c, g) 30CSO-PP (30 w. t. % CSO) and (d, h) Tra-CSO-PP (30 w. t. % Tra-CSO). (i) Infrared (IR) thermal images of various scaffolds (A: PP, B: 10CSO-PP, C: 20CSO-PP, D: 30CSO-PP, E: Tra-CSO-PP) in air under 808-nm laser irradiation (0.45 W/cm2) for 5 min. (j) NIR-triggered drug release profile from Tra-CSO-PP scaffolds. Tra-CSO-PP scaffolds were repeatedly irradiated by an NIR laser to maintain different temperature at 37, 43 or 50 °C for 30 min (Red areas) at a time interval of 1 h without irradiation. The incorporation of Tra-CSO endowed the scaffolds with significant photothermal effects and controlled NIR-triggered drug release.


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Figure 3. In vitro biocompatibility assay. SEM images of human dermal fibroblasts (HDFs) on (a) PP, (b) 30CSO-PP, (c) Tra-CSO-PP scaffolds after 3 days. (d) The cell proliferation of HDFs seeded on PP, 30CSO-PP, Tra-CSO-PP scaffolds for 1, 3 and 5 days. (*p < 0.05, **P < 0.01, ***P < 0.001). The release profiles of (e) Cu and (f) Si ions in dulbecco's modified eagle medium (DMEM) after soaking PP, 30CSO-PP and Tra-CSO-PP scaffolds for 5 days. HDFs with rich filopodia showed enhanced cell adhesion and spreading, when treated with 30CSO-PP and Tra-CSO-PP scaffolds.


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Figure 4. In vitro anticancer efficiency. (a) Relative cell viability of skin tumor cells (murine B16F10 melanoma cells) treated with different scaffolds under 808-nm irradiation (~45 °C, 15 min) for 0, 1, 2 and 3 times and corresponding (b) Confocal laser scanning microscopy (CLSM, red: cytoskeleton; blue: cell nuclei) images. (c) Relative cell viability of B16F10 cells treated with different scaffolds under 808-nm irradiation (15 min, one time) at the power density of 0.45, 0.65, and 0.85 W/cm2 and corresponding (d) Live/dead staining images (green: live cells; red: dead cells). (*P < 0.05, **P < 0.01, ***p < 0.001). The Tra-CSO-PP+Laser group showed a higher cell killing efficiency than the Tra-CSO-PP and 30CSO-PP+Laser group, indicating a synergistic effect of drug and hyperthermia on anticancer efficiency.


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Figure 5. In vivo anticancer efficiency. (a) Tumor growth curves in the period of 14 day. (b) Photographs of the excised tumors on day 14. (c) Hexatoxylin and eosin (H&E) staining, Ki67 immunohistochemical (IHC) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and H&E staining images, TUNEL apoptosis assay (green: apoptotic cells; blue: nucleus) of tumor tissues. Tra-CSO-PP+Laser group held maximum tumor inhibition among all groups, suggesting a synergistic effect of chemo- and photothermal therapy of skin cancer.


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Figure 6. In vivo tissue healing in tumor-bearing mice. (a) Representative photographs of tumors and skin wounds on days 0, 4, 8 and 14. (b) H&E staining images of skin wounds at the tumor site on day 14 for the control, PP, PP + Laser, 30CSO-PP, 30CSO-PP+Laser, Tra-CSO-PP and Tra-CSO-PP + laser groups. The edge of the epidermis is indicated with green dotted lines. The green arrows indicate the epidermis thickness. The epidermis layer for the 30CSO-PP group was thickest among all groups and were regenerated with the normal architecture. The incomplete epidermis was observed in the Tra-CSO-PP group, while no newly epidermis regenerated in the PP group, suggesting that the quality of wound healing by Tra-CSO-PP scaffolds was obviously higher than PP scaffolds.


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Figure 7. In vivo diabetic wound healing study. (a) Representative skin wound photographs on days 0, 5, 9, 11 and 15. (b) Wound closure rates of the control, 30CSO-PP and Tra-CSO-PP groups. (c) Immunofluorescence staining (green: CD31, blue: nuclei) and Masson’s Trichrome staining images of wound beds treated with different scaffolds as noted on day 15. Quantitive analysis of (d) CD31-positive blood vessels, (e) gene expression of VEGF, (f) re-epithelialization, (g) expression of collagen III and (h) collagen I on day 15. (*P < 0.05, **P < 0.01, ***P < 0.001). The 30CSO-PP and Tra-CSO-PP groups exhibited significantly enhanced in vivo wound healing effect as compared to the PP and control groups. In particular, the 30CSO-PP scaffolds up-regulated expression of CD31, VEGF, and collagen I/III, indicating expedited revascularization and re-epithelialization by the stimulatory effect of CSO HMSs.


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Copper silicate hollow microspheres (CSO HMSs)-incorporated scaffolds were successfully prepared by electrospinning for chemo-photothermal therapy of melanoma and tissue healing. The CSO HMSs endowed the compisite scaffolds with excellent photothermal performance, controlled NIR-triggered drug release, and enhanced revascularization and re-epithelialization. The multifunctional smart scaffolds offer an effective strategy for cancer therapy and healing of tumor-initiated tissue defects. 39x22mm (300 x 300 DPI)

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