www.acsnano.org
Electrospun Micropatterned Nanocomposites Incorporated with Cu2S Nanoflowers for Skin Tumor Therapy and Wound Healing Xiaocheng Wang,†,‡ Fang Lv,§ Tian Li,†,‡ Yiming Han,§ Zhengfang Yi,§ Mingyao Liu,§ Jiang Chang,† and Chengtie Wu*,† Downloaded via UNIV OF THE SUNSHINE COAST on June 25, 2018 at 19:13:29 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, People’s Republic of China ‡ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, People’s Republic of 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, People’s Republic of China S Supporting Information *
ABSTRACT: Surgical excision of skin cancers can hardly remove the tumor tissues completely and simultaneously result in cutaneous defects. To avoid tumor recurrence and heal the tumor-induced wounds, we designed a tissue engineering membrane possessing bifunctions of tumor therapy and skin tissue regeneration. The micropatterned nanocomposite membrane was successfully fabricated by incorporating Cu2S nanoflowers into biopolymer fibers via a modified electrospinning method. With uniformly embedded Cu2S nanoparticles, the membranes exhibited excellent and controllable photothermal performance under near-infrared irradiation, which resulted in high mortality (>90%) of skin tumor cells and effectively inhibited tumor growth in mice. Moreover, the membranes supported the adhesion, proliferation, and migration of skin cells as well as significantly stimulated angiogenesis and healed full-thickness skin defects in vivo. This proof-ofconcept study offers a facile and reliable strategy for localized skin tumor therapy and tissue regeneration using bifunctional tissue engineering biomaterials, showing great promise for tumor-induced wound healing applications. KEYWORDS: electrospinning, biomaterials, skin cancer therapy, wound healing, tissue engineering
S
1100 nm) energy into heat, has received increasing attention due to deep penetration (several centimeters) of NIR light in biological tissues.11−13 Recently, semiconductor cuprous sulfide nanoparticles (e.g., Cu2−xS,14−16 Cu7S417 and Cu9S518,19) have been employed as promising PTAs due to their intrinsic NIR region absorption and efficient heat generation ability. Numerous reports have demonstrated their excellent photothermal efficacy to ablate cancer cells both in vitro14,17 and in vivo.15,16,18,19 The direct application of cuprous sulfide nanoparticles in the tumor sites is capable of killing the tumor cells under NIR irradiation.18,19 However, nanoparticles applied in the form of powders are easily detached from the wound sites after surgical excision of skin tumors. In addition, the direct contact of nanoparticles with wound beds easily induces inflammation reaction of skin tissues and thereby
kin tissue plays a crucial role as a sensor of the periphery and the protective barrier against the external environment.1 Nowadays, skin cancer is the most common human malignancy with over a million cases detected annually.2,3 Surgical excision remains the most widely used and preferred modality for the treatment of skin cancers.4,5 However, the secondary intention healing for the cutaneous defects after surgery is often underestimated and neglected.6 Moreover, the remaining asymptomatic tumor tissues require complete clearance to avoid recurrence.7 Unfortunately, there are few alternative therapeutic strategies to fulfill the dual requirements of tumor therapy capacity and simultaneous skin tissue regeneration after surgical removal of skin tumors. Therefore, it is urgently needed to construct a biomaterial with bifunctional properties for treating skin tumors and simultaneously for stimulating wound healing. Photothermal therapy (PTT) has been widely explored as a noninvasive or minimally invasive treatment for a variety of cancers in recent years.8−11 In particular, the photothermal agent (PTA), which converts the near-infrared (NIR, λ = 700− © 2017 American Chemical Society
Received: August 17, 2017 Accepted: October 23, 2017 Published: October 23, 2017 11337
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
Cite This: ACS Nano 2017, 11, 11337-11349
Article
ACS Nano
Figure 1. Morphology characterization and element distribution of CS-PLA/PCL membranes. Representative SEM images of (A, B) 0CSPLA/PCL, (C, D) 10CS- PLA/PCL, (E, F) 20CS- PLA/PCL, (G, H) 30CS-PLA/PCL, (I, J) 40CS-PLA/PCL, and (K, L) 50CS-PLA/PCL membranes at different magnification. Representative high-resolution (M, O) SEM and (N, P) TEM images of (M, N) 0CS- and (O, P) 30CSPLA/PCL membranes. (Q) HAADF-SEM images and (R, S) EDS elemental mappings (Cu in red and S in blue) of 30CS-PLA/PCL membranes. (T) EDS spectrum of 30CS-PLA/PCL membranes. All of these CS-PLA/PCL membranes exhibited a patterned porous structure (300 μm), and the diameter of the Cu2S-incorporated fibers showed a decreasing trend with the increase in the nanoparticle contents.
property for general tumor therapy. However, there is no report about the potential tissue regeneration application by using Cu2S nanoparticles. Taking advantage of the photothermal effect of Cu2S and the angiogenesis of Cu ions, it would be interesting and reliable to develop a composite membrane by integrating Cu2S nanoparticles into the electrospun fibers to achieve the bifunctions of tumor therapy via photothermal performance of Cu2S and skin tissue regeneration via angiogenesis effect of Cu ions simultaneously in one single platform. In the present study, we have successfully developed a bifunctional electrospun membrane via a facile patterning coelectrospinning method. Our preliminary studies have shown that the poly(D,L-lactic acid)/poly(ε-caprolactone) (PDLLA/ PCL) membranes with patterned microstructures not only distinctively promoted adhesion, proliferation, and angiogenesis of endothelium cells but also accelerated in vivo wound healing as compared to randomly deposited fibrous membranes.21,33 To endow the electrospun fiber membranes with anticancer efficiency, Cu2S nanoparticles were uniformly encapsulated within the patterned fibers during the electrospinning process, and their effects on the fiber structure and photothermal property were systematically investigated. The significant photothermal heating ability for Cu2S incorporated PDLLA/ PCL (CS-PLA/PCL) membranes allows us to further apply them to ablate the skin tumor cells (B16F10 cells and A375 cells) in vitro and inhibit the B16F10 tumor growth in vivo. For
results in the release of dissolved ions from nanoparticles in an uncontrolled manner, which limits their application for tissue regeneration. Therefore, it would be optimal to integrate the PTAs with other tissue repairing strategies to develop a multifunctional biomaterial for treating tumor-induced cutaneous defects. Electrospun membranes have been extensively used for tissue engineering primarily because their microstructures can be easily regulated by a simple electrospinning process.20−23 With the extracellular matrix (ECM)-like architecture, inherently high surface to volume ratio and biocompatible components, the membranes provide both mechanical support and biological stimulation for cell adhesion, migration and proliferation.20,24 For good electrospinnability and biological properties, the blend solutions of several polymers are more commonly used to prepare electrospun polymeric fibers than single-component biopolymer solutions.25−27 In addition, various therapeutic agents can be easily incorporated into the fibers during the electrospinning process to enhance the healing efficiency and provide bioactive characteristics.22,28 For example, copper ions have been demonstrated to stimulate the proliferation and angiogenesis of endothelial cells,29−31 and the electrospun membranes integrated with copper-doped bioactive glasses have been reported to facilitate vascularization and wound tissue ingrowth in rats by Zhao et al.32 Although Cu2S-based nanoparticles have been widely explored as PTAs for tumor therapy, the previous mainly focused on the photothermal 11338
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano
Figure 2. Photothermal performance of CS-PLA/PCL membranes. Real-time infrared thermal images and corresponding photothermal heating curves of 30CS-PLA/PCL and 0CS-PLA/PCL membranes in the (A, C) dry and (B, D) wet environments under continuous irradiation of 808 nm laser at the power density of 0.50 W·cm−2 for 5 or 10 min. (E) Final temperature change of CS-PLA/PCL membranes with different Cu2S contents and laser power densities after irradiation for 10 min in the wet environment. 30CS-PLA/PCL membranes could be rapidly and effectively heated up under 808 nm laser irradiation, showing a strong photothermal conversion efficacy.
wound healing evaluation, the human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) were used as model cell types for in vitro regenerative studies including cell attachment, proliferation, and migration. Furthermore, the in vivo wound healing efficacy was investigated to heal full-thickness skin defects using a diabetic C57BL/6 mouse model.
indicating the absence of Cu (II) and their oxides.37,38 The XPS peaks of S could be assigned to the binding energy of S 2p3/2 (161.5 eV) and S 2p1/2 (162.8 eV).14,38 The atomic ratio of Cu/S was 1.92, very close to stoichiometry of Cu2S, as determined by an inductively coupled plasma optical emission spectrometer (ICP-OES). Subsequently, a series of patterned CS-PLA/PCL fiber membranes was successfully prepared by embedding different amounts of Cu2S nanoparticles via a patterned electrospinning process (Figure S2). The CS-PLA/PCL membranes with the Cu2S contents of 0, 10, 20, 30, 40, and 50 wt % relative to the polymer mass were named as 0CS-PLA/PCL, 10CS-PLA/PCL, 20CS-PLA/PCL, 30CS-PLA/PCL, 40CS-PLA/PCL, and 50CS-PLA/PCL membranes, respectively. All of these CSPLA/PCL membranes exhibited a micropatterned structure with altered fiber density in different regions, and the fibers deposited in the circular holes (∼300 μm) constructed a loose fibrous structure (Figure 1A−L and Figure S3). Although the Cu2S nanoparticles were not distributed homogeneously within individual fibers (Figure 1M,O and Figure S4), they were dispersed throughout the entire electrospun membranes. With the increase of Cu2S content in the precursor dispersion, the particle amount in the fibers proportionally increased. Interestingly, the diameter of the fibers showed a decreasing trend from approximately 1200 to 200 nm. The particles could not be fully embedded inside of the electrospun fibers, which led to the fibers with very rough surface (Figure 1N,P and Figure S5). The uniform distribution of Cu2S nanoparticles within the fibers was also visualized by high-angle annular dark-
RESULTS Morphologic and Compositional Characterization. In this study, Cu2S incorporated nanocomposite PLA/PCL (CSPLA/PCL) fiber membranes were prepared by a patterning electrospinning method. First, Cu2S nanoparticles were synthesized through a facile hydrothermal route as previously reported.34 The scanning electron microscope (SEM, Figure S1A,B) revealed the obtained particles with a diameter of 200− 600 nm and sponge-like architecture consisting of plenty of ultrathin nanosheets. The X-ray diffraction (XRD, Figure S1C) pattern of the particles matched well with the crystal phase of digenite (Cu2S, JCPDS no. 02-1294). The composition of Cu2S nanoparticles was further confirmed by X-ray photoelectron spectroscopy (XPS, Figure S1D−F). The experimental binding energies for Cu 2p3/2 and Cu 2p1/2 were 932.4 and 952.1 eV, respectively, which were well consistent with the values (932.1−932.9 eV for Cu 2p3/2 and 952.4−952.8 eV for Cu 2p1/2) for monovalent copper sulfide, Cu2S, from the NIST XPS database35 and more recent reports.14,15,34,36−38 The two peaks were clearly separated by 19.7 eV, and there were no shakeup satellite peaks in the region from 936 to 946 eV, 11339
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano
Figure 3. In vitro anticancer efficiency of CS-PLA/PCL membranes. (A, B) Confocal LSM (red: cytoskeleton; blue: cell nuclei) images of skin tumor cells (A: murine B16F10 melanoma cells, B: human A375 melanoma cells) on membranes and (C) live/dead staining images (green: live cells; red: dead cells) of B16F10 melanoma cells on glass slices treated by 0CS- or 30CS-PLA/PCL membranes without laser irradiation and 0CS- or 30CS-PLA/PCL membranes with laser irradiation (808 nm, 0.50 W·cm−2, 15 min). Relative cell viability of melanoma cells (B16F10 and A375 in D; B16F10 in E−G) treated under different conditions: (D) 0CS- or 30CS-PLA/PCL membranes without NIR treatment and 0CS- or 30CS-PLA/PCL membranes with NIR treatment (808 nm, 0.50 W·cm−2, 15 min); (E) 30CS-PLA/PCL membranes under different temperature (808 nm, 15 min); (F) 30CS-PLA/PCL membranes with different irradiation times (808 nm, 0.50 W·cm−2, 15 min); (G) 30CS-PLA/PCL membranes with different irradiation duration (808 nm, 0.50 W·cm−2) (*p < 0.05, **p < 0.01, ***p < 0.001). 30CS-PLA/PCL membranes effectively killed skin tumor cells, exhibiting significant in vitro anticancer effect under NIR irradiation.
field scanning electron microscopy (HADDF-SEM, Figure 1Q) and energy-dispersive spectroscopy (EDS) elemental mapping (Figure 1R,S). The characteristic peaks of Cu and S in the EDS spectrum of 30CS-PLA/PCL membranes further confirmed the existence of Cu2S in the obtained nanocomposite membranes (Figure 1T). Photothermal Performance. To investigate the photothermal performance of CS-PLA/PCL membranes, temperature changes and thermal images of the membranes upon NIR irradiation (0.50 W·cm−2) under dry (air) and wet (500 μL of phosphatic buffer solution (PBS)) conditions were recorded. As depicted in Figure 2A−D, the surface temperature of 30CSPLA/PCL membranes rapidly increased and reached a plateau of approximately 61 °C within 5 min in a dry environment and 52 °C within 10 min in a wet environment. In contrast, the 0CS-PLA/PCL membranes showed no obvious heating effect under the same condition (32 and 30 °C in the dry and wet environments, respectively). Unfortunately, 40CS- and 50CSPLA/PCL membranes could not maintain their shapes when exposed to NIR irradiation (0.50 W·cm−2 for 5 min under dry conditions), which were seriously damaged under the photothermal temperature of approximately 65 °C (Figures S6E,F and S8). By contrast, the 0CS-, 10CS-, 20CS-, and 30CS-PLA/
PCL membranes showed no obvious change in shape under the same irradiation conditions since the temperature was below 61 °C (Figure S6A−D). The internal structural deformation was further visualized by SEM (Figure S7), and the electrospun fibers of 30CS- and 40CS-PLA/PCL membranes partly melted under the temperature above 60 °C. Comparatively, 30CSPLA/PCL membranes preserved better macroporous patterned structures than 40CS-PLA/PCL membranes. When the CSPLA/PCL membranes were immersed in PBS, they could maintain their structural integrity and exhibited controllable photothermal heating ability with the temperature ranging from room temperature to 62 °C by altering laser power densities and Cu2S contents (Figure 2E and Figures S9 and S10). The above results indicated the excellent and controllable photothermal performance of CS-PLA/PCL membranes. Prior to the evaluation of anticancer efficacy, we tested the photothermal stability of 30CS-PLA/PCL membranes by soaking them in cell culture medium for 3 days. As shown in Figure S11, the sample temperature after 1, 2, and 3 days reached approximately 55.2, 55.4, and 54.1 °C, respectively, slightly lower than the temperature of 56.5 °C on day 0 under continuous NIR irradiation for 10 min at 0.60 W·cm−2. To investigate the photothermal effect with the increase of the 11340
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano depths of tissues, we placed the 30CS-PLA/PCL membranes on one side of pork with different thickness (1, 3, 5, 7, 9, and 11 mm), which was then exposed to NIR irradiation (0.40 W/cm2, 10 min), and the temperature of the other side was recorded. It was found that the pork temperature at different depths (1, 3, 5, 7, 9, and 11 mm) reached approximately 54.7, 50.6, 47.2, 43.1, 40.4, 36.8, and 33.5 °C, respectively (Figure S12). In Vitro Anticancer Efficiency. To evaluate the in vitro anticancer efficiency of CS-PLA/PCL membranes, two types of skin tumor cells (B16F10 cells and A375 cells) were seeded on the membranes and then irradiated with an 808 nm laser (0.50 W·cm−2, 15 min). As shown in Figure 3A,B, few tumor cells were observed in the laser-irradiated 30CS-PLA/PCL membranes, and the cellular morphology was seriously damaged (Figure S13). However, tumor cells spread well and closely adhered on the surface of 0CS-PLA/PCL membranes irrespective of irradiation and 30CS-PLA/PCL membranes without NIR irradiation. To further investigate the cell killing efficacy of CS-PLA/PCL membranes on surrounding tumor cells, B16F10 cells were first cultured on glass slices and then incubated with 0CS- or 30CS-PLA/PCL membranes with NIR irradiation. Live/dead images (Figure 3C) of tumor cells showed red-fluorescence when adjacent to the 30CS-PLA/PCL membranes after irradiation, indicating that cellular membrane integrity was destroyed. In contrast, the cells in the other three groups exhibited green fluorescence, indicating they were almost alive. CCK-8 assay (Figure 3D) showed that the cell viability of B16F10 cells and A375 cells of 30CS-PLA/PCL + laser group (51 °C, 15 min, one time) decreased to 26.4% and 23.1% respectively, significantly lower than that of other control groups (0CS-PLA/PCL, 30CS-PLA/PCL, and 0CS-PLA/PCL + laser group). Moreover, the cell viability of B16F10 cells adjacent to the laser-irradiated 30CS-PLA/PCL membranes further decreased with the increase of photothermal temperature (14.7% for 57 °C), irradiation times (8.7% for three times), and duration (6.3% for 20 min), as clearly displayed in Figure 3E−G. All of the results above indicated the excellent and controllable photothermal therapeutic efficiency of 30CSPLA/PCL membranes in vitro. In Vivo Tumor Growth Inhibition Efficiency. Encouraged by the high in vitro anticancer effect on tumor cells, we further sought to confirm the tumor therapeutic efficiency of 30CS-PLA/PCL membranes in vivo. 0CS- and 30CS-PLA/PCL membranes were utilized to cover the wound area in the tumor sites and followed by exposure to the 808 nm laser at a power density of 0.40 W·cm−2 for 15 min (Figure S14). The temperature still remained approximately 41 °C in the laser group (without membranes applied) and 0CS-PLA/PCL + laser groups, while the temperature rapidly increased and exceeded 50 °C in the 30CS-PLA/PCL + laser group (Figure 4A, B). Tumor volumes obviously decreased under such high temperature after 4 days of treatment, while tumor growth rates in other five groups continuously increased in an uncontrollable manner (Figure 4D). Most importantly, the tumors in the 30CS-PLA/PCL + laser group gradually disappeared without recurrence, and the original wound even healed within 14 days, as shown in the comparison of the tumor photos before (day 0) and after various treatments (day 14) in Figure 4C and Figure S15. The tumor weight and photographs from six groups after various treatments for 14 days were shown in Figure 4E,F. The mean tumor weight in the 30CS-PLA/PCL + laser group was the lightest among all groups, further confirming that 30CS-
Figure 4. In vivo photothermal therapy. (A) NIR thermal images and (B) photothermal heating curves of B16F10 tumor-bearing mice treated with 30CS-PLA/PCL membranes, 0CS-PLA/PCL membranes, and without membranes when exposed to the 808 nm laser irradiation (0.40 W·cm−2, 15 min). (C) Representative photographs of mice after various different treatments indicated taken on days 0 and 14. (D) Growth curves of skin tumors in different groups of mice after various treatments indicated. (E) Weight and (F) photographs of the tumors after various treatments on day 14. Hyperthermia induced by laser-irradiated 30CS-PCL membranes efficiently inhibited tumor growth in vivo.
PLA/PCL membranes could effectively inhibit tumor growth under the NIR irradiation. The in vivo microscopic therapeutic efficacy was further assayed by the typical Ki67 immunohistochemical staining, hexatoxylin and eosin (H&E) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Figure 5). Ki67 expression of tumor cells in the 30CSPLA/PCL + laser group was severely decreased, indicating significantly reduced proliferation of tumor cells after photothermal therapy. H&E images revealed that most cells were dead from necrosis, showing eosinophilic cytoplasm and nuclear damage with abundant karyorrhectic debris. The TUNEL assay indicated that the tumor cells from 30CSPLA/PCL + laser group were largely apoptotic after irradiation, while these in the other five groups were well alive. Quantitative analysis (Figure 5B−D) further indicated the significant difference among groups. All of the results suggested that 30CS-PLA/PCL membrane-induced hyperthermia could significantly suppress the tumor growth by inhibiting the cell proliferation and inducing mixed necrosis/apoptosis of tumor cells in vivo. In Vivo Wound Healing in Tumor-Bearing Mice. As shown in Figure S15, the tumor volume of the 30CS-PLA/PCL + laser group was reduced after 4 days of treatment, gradually disappeared with black scars left at the original sites after 8 days and the wound even completely healed without tumor recurrence within 14 days. By contrast, the wounds hardly closed with increasing tumor volumes in the other five groups 11341
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano
Figure 5. In vivo microscopic anticancer efficacy. (A) Immunohistochemical analysis, H&E stained images, and TUNEL apoptosis assay (green: apoptotic cells; blue: nucleus) of tumor tissues after photothermal therapy. Quantification of (B) the expression of Ki67, (C) H&E, and (D) TUNEL assay (***P < 0.001). 30CS-PLA/PCL membranes distinctly inhibited the in vivo tumor cell proliferation and induced mixed cell necrosis/apoptosis under the NIR irradiation.
Figure 6. In vivo wound healing in tumor-bearing mice. (A) Photographs of angiogenesis state in the tumor-induced wound beds for the control, 0CS-PLA/PCL, 30CS-PLA/PCL, and 30CS-PLA/PCL + laser groups as compared to normal skins and normal diabetic wounds without membranes applied on day 14. (B) H&E staining of sectioned wounds for the control, 0CS-PLA/PCL, 30CS-PLA/PCL, and 30CSPLA/PCL + laser groups 14 days after treatments. The skin tissues in the 30CS-PLA/PCL + laser group were regenerated with the normal architecture and regular capillary networks in remarkable contrast to the incomplete epidermis and tumor-specific vasculature as observed in the other groups.
(i.e., control, laser, 0CS-PLA/PCL, 30CS-PLA/PCL and 0CSPLA/PCL + laser groups). The outgrowth of newly regenerated epidermis in the skin wounds after photothermal therapy was characterized by histologic analysis (Figure 6). The skin tissues in the 30CS-PLA/PCL + laser group were regenerated with the normal architecture and regular capillary
networks, which greatly differed from the incomplete epidermis and tumor-specific vasculature at the wound beds as observed in the other groups 14 days after treatments. In Vitro Regenerative Activity. For the application of tumor-induced skin wound healing, apart from photothermal therapy, it is of equal importance that the implanted 11342
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano
Figure 7. Enhanced in vitro regenerative activity of skin cells by CS-PLA/PCL membranes. (A) SEM images of HDFs after seeding on the 0CS- or 30CS-PLA/PCL membranes for 24 h. (B) The cell proliferation of HDFs cultured with 0CS- or 30CS-PLA/PCL membranes for 5 days. (C) In vitro scratch assay of HUVECs cultured with membranes. (D) Quantification of the relative wound area. 30CS-PLA/PCL membranes supported attachment and proliferation of HDFs and significantly promoted the migration of HUVECs as compared to other groups (*p < 0.05, **P < 0.01, ***P < 0.001).
Figure 8. Enhanced in vivo diabetic wound healing by CS-PLA/PCL membranes. (A) Representative skin wound photographs on days 0, 4, 8, and 12 and (B) wound closure rates of the control, 0CS-CS-PLA/PCL, and 30CS-PLA/PCL groups. (C) Representative photographs of angiogenesis state in the wound beds (up) and immunofluorescence staining (down; green: CD31, blue: nuclei) at the wound healing region 12 days after treatment. (D) Quantification of CD31-positive blood vessels at the wound healing region from control, 0CS-PLA/PCL, and 30CS-PLA/PCL groups on day 12. 30CS-PLA/PCL membranes showed the best wound healing efficiency among three groups (*p < 0.05, **P < 0.01, ***P < 0.001).
membranes possess skin regenerative activity. Therefore, two typical skin cell lines, human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs), were cultured to investigate the in vitro bioactivity of CS-PLA/PCL membranes. The morphologies of HDFs on the 0CS- and 30CS-PLA/PCL membranes were characterized by SEM and confocal laser scanning microscopy (CLSM). It was found that
the cells spread well and were closely attached to the membranes after 24 h of incubation (Figure 7A and Figure S16). CCK-8 assay (Figure 7B) showed that the proliferation rates of HDFs incubated with 0CS- and 30CS-PLA/PCL membranes were significantly higher than that of the control group after 5 days of culture. When the 30CS-PLA/PCL membranes were immersed in the cell culture medium, Cu ions 11343
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano
PCL membranes for skin tissue regeneration. Our results have demonstrated the excellent and controllable photothermal effect of CS-PLA/PCL fiber membranes after integration with Cu2S nanoparticles. The skin tumor cells could be effectively killed by NIR-irradiated CS-PLA/PCL membranes both in vitro and in vivo, exhibiting significant photothermal anticancer efficiency. Moreover, the membranes supported the adhesion, proliferation, and migration of normal skin cells as well as significantly stimulated angiogenesis and effectively healed fullthickness skin defects in mice. The present work indicates that the electrospun CS-PLA/PCL fiber membrane may be a suitable candidate for effectively healing tumor-induced skin wounds, which meets the dual requirements of tumor therapy and wound healing. One significant advance of the study is that we successfully prepared micropatterned fibrous membranes by incorporating Cu2S nanoparticles into biopolymer matrix. For good electrospinnability and stability, the blend of PLA/PCL solution was used as the substrate considering the good mechanical strength and stability of PCL and the accurately controllable assembly of PLA fibers during electrospinning, as demonstrated in our previous studies.27 Compared with randomly deposited fibers, the macroporous structure of patterned membranes is beneficial to supply oxygen and nutrient to cells and also provides topographic cues for promoting cell migration and ingrowth.21,33 When Cu2S nanoparticles were added to the PLA/PCL solution, the particles might form localized condensates of charges in the electrostatic field and disturb the jet formation and flow continuity during electrospinning. To determine the highest Cu2S contents that could be doped, different amounts of Cu2S particles were added into PLA/PCL solution. Because the biopolymer could be served as stabilizer molecules to wrap around the Cu2S and prevent nanoparticle aggregation, the Cu2S particles were well dispersed in the polymer solution via a simple ultrasonic treatment, which led to the homogeneous distribution of Cu2S embedded in the polymer fibers. However, the addition of Cu2S increased the charge density in ejected jets, and thus higher electric-field intensity was imposed to the jets to balance the self-repulsion of the excess charges, resulting in smaller diameter of the achieved electrospun fibers.39,40 As expected, the proportional increase in Cu2S contents led to a gradual decrease in the fiber diameter, and the continuously spinning process was even impeded when the Cu2S contents exceeded 50 wt %. Fortunately, the micropatterned structure of the prepared membranes could be well maintained by carefully modulating electrospinning parameters even when the particle content reached 50 wt %. The fibers in the patterned membranes exhibited altered fiber density in different regions. The fibers deposited in the insulating regions of the steel template for patterning electrospinning had a much lower fiber density and usually constructed a loose fibrous structure, which is expected to facilitate the transport of oxygen and nutrient to cells for supporting cell migration and ingrowth.24,33 More importantly, the uniformly encapsulated Cu2S nanoparticles in the CS-PLA/ PCL membranes could not only play an important role in ablating tumors under NIR irradiation but also be beneficial for healing tumor-induced wounds. Second, the prepared nanocomposite membranes displayed excellent photothermal property for killing skin tumor cells in vitro and inhibiting tumor growth in vivo. As we know, the photothermal conversion efficacy of typical PTAs (such as goldbased nanostructures) is attributed to localized surface plasmon
could be rapidly released from the 30CS-PLA/PCL membranes with an ionic concentration of 0.497 μg·mL−1 in the first 24 h and followed by a much slower release thereafter at a cumulative concentration of 0.780 μg·mL−1 on day 4 (Figure S17). An in vitro scratch assay was conducted to explore the effect of 30CS-PLA/PCL membranes on skin cell migration. All HUVECs shared a similar wound width after being scratched by a pipet tip (Figure 7C). The cells moved toward the wound area, and the scratch was almost disappeared in the 30CS-PLA/ PCL group after 6 h. The relative wound area (Figure 7D) significantly decreased to 5.1% for the 30CS-PLA/PCL group, as compared to control group (28.1%) and 0CS-PLA/PCL group (18.4%), indicating that 30CS-PLA/PCL membranes could accelerate the migration of HUVECs. To investigate the photothermal effect on normal skin cells, HUVECs were treated with 0CS- or 30CS-PLA/PCL membranes and exposed to the NIR irradiation (0.50 W· cm−2, 15 min). The morphologies of HUVECs on laserirradiated 30CS-PLA/PCL membranes were seriously damaged, while cells spread well and were closely attached to the 30CS-PLA/PCL membranes and 0CS-PLA/PCL membranes irrespective of irradiation (Figure S18A−D). CCK-8 assay (Figure S18E) indicated that the cell viability of HUVECs adjacent to the laser-irradiated 30CS-PLA/PCL membranes significantly decreased to 40.8%, suggesting that normal skin cells could be killed by the NIR-induced heat. In Vivo Diabetic Wound Healing Efficacy. To exclude the negative influence of tumor growth on wound healing, the in vivo wound healing efficacy was further investigated in a typical STZ-diabetic C57BL/6 mouse model instead of tumorbearing models. The healing process of a full thickness incisional wound was tracked over 12 days. As presented in Figure 8A,B, the 30CS-PLA/PCL group showed a significantly higher wound healing rate than the control and 0CS-PLA/PCL groups. Although there was no significant difference on days 2, 4, and 6 post-injury (at early stage), the wound area of the 30CS-PLA/PCL group was markedly reduced as compared to other groups after 8 days (at late stage). The remaining wound area on day 12 for the control, 0CS-PLA/PCL, and 30CS-PLA/ PCL groups was 28.9%, 25.5%, and 13.6%, respectively. Furthermore, the revascularization in the wound beds was investigated by optical microscope observation and immunohistochemical analysis of CD31 expression. As shown in Figure 8C, there were significantly more blood vascular networks and CD31 positive vessels (green) observed in the 30CS-PLA/PCL group than the control and 0CS-PLA/PCL groups. The CD31 expression level in the 30CS-PLA/PCL group (138.6%) was significantly higher than that in the 0CS-PLA/PCL (74.8%) and control (100%) groups (Figure 8D), indicating that capillary formation in the wounds was enhanced by the 30CS-PLA/PCL membranes. Taken together, our findings suggested that the 30CS-PLA/PCL membranes could effectively promote skin wound healing in vivo.
DISCUSSION In the current study, we first proposed the concept to combine the photothermal therapy with tissue regeneration for treating the skin tumors and simultaneously for stimulating skin wound healing. To this end, we developed a bifunctional nanocomposite membrane by integrating Cu2S nanoflowers into biopolymer fibers for tumor therapeutic efficiency while improving the bioactivity of the patterned electrospun PLA/ 11344
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano resonance (LSPR) originated from free electrons.41,42 However, the LSPR of semiconductor cuprous sulfides derives from the collective oscillations of holes because there are many copper vacancies in the copper-deficient stoichiometries (Cu2−xS).38,43,44 The optical absorption can also be enhanced by constructing photonic-crystal microstructures and/or lasercavity mirrors, especially when the size of particles or cavities is close to the wavelength of light.13,45 In our case, Cu2S particles presented sponge-like architecture consisting of plenty of ultrathin nanosheets with a diameter of 200−600 nm, which could serve as excellent laser-cavity mirrors of the 808 nm laser (Figure S1B). When the Cu2S microsponges were exposed to the laser, photons were either absorbed by the nanosheets or reflected in these open cavities until most of them were absorbed. The significantly enhanced photon absorption resulted in higher oscillation frequency of holes and improvement of photothermal conversion efficiency. Therefore, with Cu2S nanoparticles physically encapsulated in the biopolymer fibers, the 30CS-PLA/PCL membranes exhibited significant photothermal heating ability under the NIR irradiation in comparison to 0CS-PLA/PCL membranes without Cu2S particles. In contrast to the negligible shape change of 30CSPLA/PCL membranes after irradiation (61 °C), the 40CS- and 50-PLA/PCL membranes could not maintain their structural integrity, and they were seriously damaged under the photothermal temperature of approximately 65 °C. It is known that pure PCL melts at ∼60 °C, while PLA possesses a higher melting point than 150 °C. Therefore, the copolymer membrane might keep its shape due to the support of PLA under the temperature slightly above 60 °C. Our results indicated that the CS-PLA/PCL membranes could maintain their macroporous structures under wet conditions and exhibited controllable photothermal heating ability with the temperature ranged from room temperature to 62 °C by altering laser power densities and Cu2S contents in the membranes. The excellent and controllable photothermal effect of CS-PLA/PCL membranes awards them the attractive potential application in tumor ablation therapy. To investigate the in vitro anticancer efficiency of CS-PLA/ PCL membranes, two types of skin tumor cells (B16F10 and A375 cells) were used, and it was found that almost all cells in 30CS-PLA/PCL membranes or adjacent to the membranes were killed under the 808 nm irradiation at a power density of 0.50 W·cm−2. The cell mortality of B16F10 cells was ∼93.7% under the hyperthermia (51 °C for 20 min) induced by laserirradiated 30CS-PLA/PCL membranes, indicating the high photothermal therapeutic efficiency of 30CS-PLA/PCL membranes in vitro. Photothermal therapy is known to apply hyperthermia between 40 and 45 °C to deactivate tumor cells, and higher temperature (>45 °C) is sufficient to result in irreversible cellular damage.11 Assuming the temperature of in vivo human body is 36 °C, after being covered by 30CS-PLA/ PCL membranes, the tumor sites could be easily heated above 45 °C with a short irradiation period of the 808 nm laser at a low and safe power density of 0.40 W·cm−2 (the conservative limit of 808 nm laser intensity set is ∼0.33 W·cm−2). Considering the propagation of light energy and heat transfer in tissues, the tumor surface temperature in our study was elevated above 50 °C after irradiation for 15 min. Our results showed that the tumor growth was effectively inhibited by the high temperature induced by 30CS-PLA/PCL membranes under the 808 nm laser irradiation. To date, necrosis and apoptosis are the commonly reported mechanisms of cell death
triggered by PTT.46 Necrosis is characterized by disintegrated cell membranes and subsequent release of the intracellular contents into the extracellular milieu. By comparison, apoptosis is programmed cell death with undamaged membrane integrity. More specifically, low-energy irradiation (50 °C) can result in necrosis.9 By prolonging the irradiation time, primary apoptosis ultimately gives rise to secondary necrosis.10,47 Besides, the tumor cell proliferation can be inhibited by the two effects above.46 To further provide insight into how the tumor cells response to photothermal treatment, we analyzed the in vivo cell death by histological staining. The proliferation of cancer cells was distinctly inhibited by the laser-irradiated 30CS-PLA/ PCL membranes. In addition, both necrosis and apoptosis of tumor cells were observed in the 30CS-PLA/PCL + laser group, which is possibly due to the relatively low-energy irradiation in the hyperthermic peripheral zone and deeper tumor tissues. Therefore, the photothermal effect of CS-PLA/ PCL membranes suppressed the skin tumor growth by inhibiting cell proliferation and triggering mixed necrosis/ apoptosis of cancer cells. Third, it was interestingly found that the 30CS-PLA/PCL membranes implanted in tumor-induced wounds not only inhibited tumor recurrence but also facilitated the skin tissue regeneration in the tumor-bearing mice. This encouraging result suggested that the tumor cells could be eradicated by photothermal effect at the early stage of treatment and the tissue regeneration ability of 30CS-PLA/PCL membranes was not affected by short-time NIR irradiation and played an important part in the following wound healing process at late stage. Even though normal skin cells adjacent to 30CS-PLA/ PCL membranes could also be killed under NIR irradiation, the skin cells originated from surrounding healthy tissues would migrate to the wound beds and contribute to wound healing after photothermal therapy at the following stage. To exclude the influence of tumor growth on wound healing, we specially investigated the regenerative effect of CS-PLA/PCL membranes themselves on normal skin cells in vitro and chronic skin wounds in vivo. Successful wound healing is known as a complex and dynamic biological process, which requires a coordinated cellular response involving keratinocytes, fibroblasts, and vascular endothelial cells.48 Fibroblasts play an important role in the synthesis of proteins and collagen, whereas endothelial cells are primarily responsible for angiogenesis. Our in vitro study showed that both 0CS- and 30CSPLA/PCL membranes supported the adhesion, spreading, and proliferation of human dermal fibroblasts. More importantly, the 30CS-PLA/PCL membranes significantly accelerated the migration of endothelial cells as compared to 0CS-PLA/PCL membranes without Cu2S particles, indicating the enhanced in vitro regenerative activity by the incorporation of Cu2S particles. Cu ions have been previously reported to stimulate proliferation and migration of endothelial cells in a dose dependent manner29 and to enhance microvessel ingrowth, collagen synthesis, and deposition in vivo32,49 It is noted that Cu-ion concentration previously used for angiogenesis stimulation of endothelial cells generally ranged from 1 to 500 μM (i.e., 0.064 to 32 μg·mL−1).29−31 Our preliminary studies showed the released Cu ion from Cu-containing mesoporous bioactive glass at a concentration of 14.2 μg· mL−1 could stimulate hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) expression in human bone marrow stromal cells (hBMSCs).28 In this 11345
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano study, the concentration of Cu ions ranged from 0.497 μg·mL−1 to 0.780 μg·mL−1 during the in vitro cell culture, which was much lower than that reported in previous literatures. In addition, our results showed that the small amount of released Cu ions from the 30CS-PLA/PCL membranes could be safe for dermal fibroblasts and played an important role in the significantly increased migration of endothelial cells. When the 30CS-PLA/PCL membranes were applied to full-thickness skin wounds, Cu ions would be released from the gradually degraded polymer membranes and played a chemoattractant role in accelerating the wound closure rate and capillary formation, as observed in the present study. These results indicated the enhanced regenerative efficacy of Cu2S-loaded membranes, which makes them more suitable and safer for healing the cutaneous wounds resulting from surgery excision of tumor tissues.
mixed, stirred continuously for 8 h, and fed in a hypodermic syringe (10 mL) before electrospinning. The electrospinning parameters were as follows: electric field strength: 8−12 kV; air gap distance: 15 cm; inner diameter of spinneret: 0.7 mm; flow rate of solution: 0.015 mL/ min; relative humidity: 50−60%, and the electrospinning process was conducted at room temperature. In this process, a custom-made stainless steel mesh (pore diameter: 300 μm, Figure S2C) was used as the collector, which was placed horizontally on a conductive plate (Figure S2A) by applying certain pressure on both ends of the mesh. As a control, unloaded PLA/PCL membranes were prepared by dissolving PDLLA (0.25 g) and PCL (0.25 g) in the mixture of DMF (3.9 mL) and THF (1.1 mL) and named as 0CS-PLA/PCL membranes. After electrospun fibers were directly deposited on the surface of the mesh (Figure S2B), the patterned membranes were gently peeled from the collector, physically fixed on aluminum foils, and then sectioned into circular shape (diameter: 10 mm) before use (Figure S2D). All of the membranes were placed under vacuum for 24 h at 25 °C to remove the residual solvent. Characterization. The overall microscopic images of CS-PLA/ PCL membranes were obtained using an optical microscope (S6D, Leica, Germany). The morphology and elemental mapping of the membranes were examined by a transmission electron microscope (TEM, Tecnai G2 F20, FEI Electron Optics, Netherlands) and a scanning electron microscope (SEM, S-4800, Hitachi, Japan) equipped with an energy-dispersive spectrometer (EDS). The crystal structure and phases of the samples were characterized by X-ray diffraction (XRD, D8 ADVANCE, BRUKER AXS GMBH, Germany). X-ray photoelectron spectroscopy (XPS) measurements were performed on a special spectrometer (ESCAlab250, Thermo Fisher Scientific, MA, US). The light-induced temperature changes and thermal images of membranes were recorded by an infrared thermal imaging system (PM100D, Thorlabs GmbH, Germany). The concentrations of Cu and S were measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES, Vista AX, Varian, Palo Alto, US). Photothermal Performance of CS-PLA/PCL Membranes. The photothermal performance of the CS-PLA/PCL membranes was tested in a 48-well culture plate under dry (air) and wet (500 μL of PBS) conditions upon exposure to an 808 nm laser (diameter: ∼12 mm, 0.50 W·cm−2) for 5 or 10 min. The temperature changes and thermal images were recorded by an infrared thermal imaging system in real time. The light-induced temperature changes of CS-PCL/PLA membranes with different Cu2S contents were acquired by irradiation with various laser power densities (0.20, 0.40, 0.60, and 0.80 W·cm−2) for 10 min under wet conditions. The controllable photothermal temperature elevation induced by laser-irradiated 30CS-PLA/PCL membranes was recorded by real-time adjustment of the laser power densities under the wet conditions. We also investigated the photothermal effect of 30CS-PLA/PCL membranes on one side of pork tissue with different thickness (1, 3, 5, 7, 9, and 11 mm) to mimic the heat transfer from the fibers to the tumors. The side covered by fibers was irradiated under the 808 nm laser at the power density of 0.40 W·cm−2 for 10 min, and the temperature of the other side was recorded by the infrared thermal camera to mimic the in vivo intratumoral temperature. In Vitro Photothermal Therapy. Two skin tumor cell lines, B16F10 cells (murine melanoma cells) and A375 cells (human melanoma cells), were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, HyClone) in a humidified incubator (5% CO2, 37 °C). CS-PCL/PLA membranes with circular shape (diameter: 10 mm) were sterilized by ultraviolet light overnight. The cells were seeded in 48-well plates for 24 h (5.0 × 104 per well, 500 μL medium), and the 0CS- or 30CSPLA/PCL membranes were then gently transferred into the plates. Cells were adjacent to the top side of all membranes, which was away from the steel templates during electrospinning. Tumor cells cultured with 0CS- or 30CS-PCL/PLA membranes were exposed to the 808 nm laser at a power density of 0.50 W·cm−2 for 15 min. As a control, cells were grown with membranes or without membranes under the same conditions without irradiation. After 24 h, the cell viability was quantified using a CCK-8 assay (Cell counting kit-8, Kumamoto,
CONCLUSION In summary, a bifunctional Cu2S-incorporated electrospun membrane was developed for treating tumor-induced cutaneous defects by using the Cu2S nanoflowers as a localized PTA for tumor therapy and as a source of Cu ions for wound healing. The obtained nanocomposite membranes could be easily heated up under NIR irradiation, which thereby effectively killed skin tumor cells in vitro and inhibited the tumor growth in vivo. Moreover, the patterned membranes supported the cell adhesion, proliferation, and migration of normal skin cells in vitro as well as effectively stimulated angiogenesis and healed skin defects in vivo. All of these results suggested that the Cu2S-incorporated electrospun membrane is a promising candidate for healing tumor-induced skin wounds. Our work here presents a facile concept and effective strategy to incorporate tumor therapeutic agents into tissue engineering biomaterials for localized tumor therapy and soft tissue regeneration in one single platform. MATERIALS AND METHODS Materials. Poly(D,L-lactic acid) (PDLLA, Mn = 1,750,000) was provided by Jinan Daigang Biomaterial Co. Ltd. (Shandong, China). Poly(ε-caprolactone) (PCL, Mn = 80,000) was purchased from SigmaAldrich (Shanghai, China). N,N-dimethylformamide (DMF), tetrahydrofuran (THF), Cu(NO3)2·3H2O, glutathione (GSH), and cetyltrimethylammonium bromide (CTAB) of analytical grade were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All reagents were used as received. Preparation of Micropatterned CS-PLA/PCL Membranes. Cu2S nanoparticles were synthesized through a facile hydrothermal route as previously reported.34 Typically, Cu(NO3)2·3H2O (0.2 g), GSH (0.307 g), and CTAB (1.0 g) were dissolved in deionized water (30 mL) to form a homogeneous solution after stirring for 30 min at 40 °C. The obtained solution was then transferred into a Teflon-lined stainless steel autoclave and heated to 120 °C for 24 h. The black products were washed with deionized water and ethanol, separated by centrifugation, ground ,and finally dried at 60 °C under vacuum. Micropatterned composite CS-PLA/PCL membranes was prepared according to a previously reported method with slight modification.21 In a typical preparation of xCS-PLA/PCL (x = 10, 20, 30, 40, and 50) membranes, the mixed solvent was obtained by stirring DMF (3.9 mL) and THF (1.1 mL) for 1 h. The blend of PDLLA (0.25 g) and PCL (0.25 g) was dissolved in the above solvent (4 mL) and stirred for 1.5 h at 30 °C to obtain a homogeneous and viscous PLA/PCL solution. Meanwhile, a desirable amount of Cu2S nanoparticles (0, 10, 20, 30, 40, or 50 wt % relative to the polymer mass) was dispersed in the mixed solvent (1 mL) and ultrasonicated for 1.5 h to obtain CS suspension. The PLA/PCL solution and CS suspension were then 11346
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano
consecutive days (from day 0 to day 3). The tumor surface temperature and thermal images were monitored and recorded by an infrared thermal imaging system. The tumor sizes were measured by a caliper every 2 days and calculated as follows: tumor volume (V) = (tumor length) × (tumor width)2/2. The mice were sacrificed after 2 weeks, and the tumors were harvested, weighed, and photographed. The tumors were then fixed in 4% (v/v) paraformaldehyde solution, embedded in paraffin, sectioned into 5 μm thickness, and stained with hematoxylin and eosin (H&E) for histological analysis. Immunohistochemical staining was performed using Ki67 antibodies as the markers of cell proliferation. The tumor slides were subjected to epitope retrieval in sodium citrate solution, washed in phosphate buffer solution-Tween (PBST), and incubated with specific antibodies at 4 °C overnight. Following staining, sections were visualized by CLSM (Leica, Solms, Germany). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to evaluate the apoptosis of tumor cells. Typically, the tumor slices were incubated with deparaffin and protease K, stained with TUNEL solutions and DAPI, and finally observed using CLSM. Quantitative analysis was accomplished using Image-Pro Plus 6.0. To investigate the wound healing efficacy of CS-PLA/PCL membranes in tumor-bearing mice, the tissues composed of the wound beds and surrounding healthy skins (2 × 2 cm) were excised for cutaneous biopsies. The vascular infiltration state was captured by a digital camera (Canon, Tokyo, Japan). The skin samples were fixed with 4% paraformaldehyde solution, dehydrated in graded ethanol series, embedded in paraffin, sectioned into 5 μm thickness, and evaluated using H&E staining. In Vitro Skin Cell Adhesion, Proliferation, and Migration Assay. Human dermal fibroblasts (HDFs, 2−4 passages) were cultured in DMEM supplemented with 10% FBS (HyClone) in a humidified incubator (5% CO2, 37 °C). The cells were seeded in 24well plates (1.0 × 104 per well, 1 mL medium) and incubated for 24 h. A transwell loaded with a 0CS- or 30CS-PLA/PCL membrane (diameter: 10 mm) was gently transferred into each well, and the cells were incubated for 1, 3, and 5 days. Cell proliferation was quantified using CCK-8 assay according to the manufacturer’s instruction. For cell morphology and adhesion observation, HDFs were seeded in a 48-well culture plate (2 × 104 per well, 500 μL medium) containing 0CS- or 30CS-PLA/PCL membranes (diameter: 10 mm). After 24 h, the cellular samples were fixed with 2.5% (v/v) glutaraldehyde for 20 min, washed in PBS for 3 times, dehydrated in graded ethanol series (30, 50, 60, 70, 80, 90, 95 and 100%) and hexamethyldisilazane (HDMS), and finally observed by SEM. HDF cells were also fixed with 4% paraformaldehyde, and the cell cytoskeleton and nuclei were stained with rhodamine phalloidin and DAPI (Cytoskeleton Inc., USA) for observation under a fluorescence confocal microscopy (TCS SP8, Leica, Germany). For cell migration assay, an in vitro scratch assay was conducted as previously reported.50,51 Human umbilical vein endothelial cells (HUVECs, 2−4 passages) were seeded into 24-well plates (1.0 × 105 per well, 1 mL medium) and cultured in endothelial cell medium (ECM) for 24 h to achieve a confluent monolayer. A vertical scratch was then created using a p200 pipet tip. Then, each well was washed with 1 mL medium to remove the cell debris and smooth the scratch edge. A transwell loaded with a 0CS- or 30CS-PLA/PCL membrane (diameter: 10 mm) was gently transferred into each well before adding 1 mL of fresh ECM. Cells cultured without membranes were used as control. After 6 h, cells were fixed with 4% paraformaldehyde, stained with crystal violet hydrate solution, photographed using a microscope (DMI3000, Leica, Germany), and quantitated as follows: Relative wound area (%) = A/A0 × 100%. Here, the initial wound area (A0) and wound area after 6 h incubation (A) were determined using ImageJ software. To investigate the photothermal effect on normal skin cells, HUVECs were cultured in 48-well plates (5 × 104 per well, 500 μL medium) for 24 h, and then 0CS- or 30CS-PLA/PCL membranes were gently transferred into the plates. Cells cultured with 0CS- or 30CS-PCL/PLA membranes were exposed to the 808 nm laser at a
Japan). Briefly, the supernatant and membranes were removed and followed by incubation with 200 μL of CCK-8 solution (10% in the culture medium) for 1.5 h. Thereafter, 100 μL of the solution was pipetted into a fresh 96-well plate, and the absorbance at 450 nm was measured using a microplate reader (Epoch, BIO-TEK, USA). To evaluate the effect of different temperature on the cell viability, B16F10 cells were cultured with 30CS-PCL/PLA membranes as above and then exposed to an 808 nm laser. The temperature induced by laser-irradiated 30CS-PLA/PCL membranes was real-time monitored by an infrared thermal imaging system. By carefully controlling the laser power densities, the cells were treated under the desired temperature (37, 41, 45, 49, 53, or 57 °C) for 15 min. The tumor cell viability was quantified by the CCK-8 assay as described above. To evaluate the effect of irradiation times on the cell viability, the B16F10 cells were cultured with 30CS-PCL/PLA membranes and irradiated at the power density of 0.50 W·cm−2 for 15 min for the first time. After 12 h, the viability of the four samples was quantified by the CCK-8 assay, and the remaining eight samples were again irradiated (0.50 W· cm−2, 15 min) for the second time. After another 12 h, four samples were subjected to the CCK-8 assay, and the remaining four samples were irradiated (0.50 W·cm−2, 15 min) for the third time. The cell viability was quantified by the CCK-8 assay 12 h later. To evaluate the effect of irradiation duration on cell viability, the samples were exposed to the 808 nm laser at 0.50 W·cm−2 for 5, 10, 15, or 20 min, and the cell viability was also quantified by the CCK-8 assay as described above. To observe the morphology and adhesion of tumor cells on 0CS- or 30CS-PLA/PCL membranes after photothermal therapy, B16F10 cells or A375 cells (5.0 × 104 cells per well, 500 μL of medium) were cultured in the 48-well plates containing 0CS- or 30CS-PLA/PCL membranes and irradiated (0.50 W·cm−2, 15 min, one time) after incubation for 24 h. Cells in the 0CS- or 30CS-PLA/PCL membranes were not irradiated as control. After 12 h, the B16F10 cells were postfixed with 2.5% (v/v) glutaraldehyde for 20 min, followed by 30, 50, 70, 90, 80, 95, and 100% (v/v) ethanol and dried in hexamethyldisilazane (HMDS) for 5 min. The morphology of B16F10 cells in the CS-PLA/PCL membranes was observed under SEM (SU8220, Hitachi, Tokyo, Japan). B16F10 cells or A375 cells were also fixed with 4% paraformaldehyde and stained with rhodamine phalloidin and DAPI (Cytoskeleton Inc., Denver, CO, US) to observe the cell cytoskeleton and nuclei under a confocal laser scanning microscope (CLSM, TCS SP8, Leica, Germany). To qualitatively evaluate the photothermal effect of CS-PLA/PCL membranes on the surrounding tumor cells, 5.0 × 104 B16F10 cells were cultured in 24-well culture plates containing glass slides for 24 h. 0CS- or 30CS-PLA/PCL membranes were gently placed onto the glass slides and irradiated for once (0.60 W·cm−2, 15 min). The membranes were then removed, and cells on the glass slides were stained with Ethidium homodimer-1 and Calcein AM and observed by CLSM. To investigate the photothermal stability of CS-PLA/PCL membranes under in vitro culture, the photothermal curves of 30CSPLA/PCL membranes were recorded under the NIR irradiation (0.60 W·cm−2, 10 min) after soaking the membranes in DMEM (one membrane in 1 mL) for 0, 1, 2, and 3 days at 37 °C. In Vivo Photothermal Therapy and Wound Healing. All animal experiments in this study were performed according to the protocols approved by the Animal Investigation Committee of the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University. The tumor model was established by subcutaneous injection with B16F10 cells (2 × 106 cells in 150 mL PBS) in the hind part of each Balb/c mouse (female, 5−7 weeks old). After tumor inoculation for 7 days (tumor volume ≈ 70 mm3), the mice were randomly divided into six groups (n = 5) as follows: (i) control group, (ii) laser group, (iii) 0CS-PLA/PCL group, (iv) 30CSPLA/PCL group, (v) 0CS-PLA/PCL + laser group, and (vi) 30CSPLA/PCL + laser group. A full thickness wound (diameter: 10 mm) was created at the tumor site, and the wound area of CS-PLA/PCL groups was covered by the 0CS- or 30CS-PLA/PCL membrane (Figure S14). For the laser treatment groups, each mouse was exposed to the 808 nm laser (0.40 W·cm−2, 15 min, one time) for four 11347
DOI: 10.1021/acsnano.7b05858 ACS Nano 2017, 11, 11337−11349
Article
ACS Nano power density of 0.50 W·cm−2 for 15 min. As a control, cells were grown with membranes or without membranes under the same conditions without irradiation. After 24 h, the cell viability was quantified using a CCK-8 assay. For analysis of the Cu-ion release from the composite fibers, a 0CSor 30CS-PLA/PCL membrane was immersed in 1 mL cell medium and kept at 37 °C for 4 days. The supernatants were collected every day, and the concentration of Cu ions was measured by ICP-AES. In Vivo Diabetic Wound Healing. Diabetic mice (female, C57BL/6) were induced by intraperitoneal injection of streptozocin (STZ, Sigma-Aldrich, 60 mg·kg−1) in 0.1 M citrate buffer as described elsewhere.52 The diabetic mice were randomized into a control group (n = 8), 0CS-PLA/PCL group (n = 8), and 30CS-PLA/PCL group (n = 8). The dorsal hair of diabetic mice were shaved 1 day prior to wounding. A standardized full thickness wound (diameter: 10 mm) was created and covered with a 0CS- or 30CS-PLA/PCL membrane before shielded with Tegaderm (3M, St. Paul, MN, US). Digital photographs of diabetic wounds were taken every 2 days, and the wound area was measured using ImageJ software. The wound size was calculated as follows: Relative wound area = At/A0 × 100%, where A0 was the initial wound area on day 0 and At was the wound area on day t (t = 0, 2, 4, 6, 8, 10, and 12). To evaluate newly formed blood vessel in the wound beds, mice were sacrificed after 12 days, and the skin tissues (2 × 2 cm) in the wound sites were excised for cutaneous biopsies. The vascular infiltration state was captured by a digital camera (Canon, Tokyo, Japan). Subsequently, the skin samples were fixed with 4% paraformaldehyde solution, dehydrated in graded ethanol series, embedded in paraffin, and then sectioned into 5 μmthick slices. Immunohistochemical analysis was performed to examine the angiogenesis in the wound beds. The skin tissue sections were rehydrated and boiled in sodium citrate buffer for about 20 min and then incubated with anti-CD31 antibody (Abcam, Cambridge, UK) overnight at 4 °C. DAPI was used to stain the nuclei. The distribution of CD31 protein was observed by CSLM. Statistical Analysis. All data used were obtained at least in quadruplicate. The results were expressed as the means ± standard deviations (SD) and analyzed using a one-tailed Student’s t test to evaluate the significance of differences between two groups. A probability value (p-value) of
