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Restorative Biodegradable Two-layered Hybrid Microneedles for Melanoma Photothermal/Chemo Co-therapy and Wound Healing


 Tumor killing and wound healing are two complementary and influential processes during the treatment of melanoma. Herein, a two-layered microneedle platform was developed with bifunctional effect of chemo-photothermal synergistic melanoma therapy and skin regeneration. The platform composed of embeddable curcumin nanodrugs/new Indocyanine Green/hyaluronic acid (Cur NDs/IR820/HA) microneedles and sodium alginate/gelatin/hyaluronic acid (SA/Ge/HA) supporting back layer was prepared through a two-step casting process. With uniform incorporation of Cur NDs and IR820, the microneedles exhibited nice photothermal performance under external near-infrared (NIR) light stimulation and excellent tumor co-therapy ability. Once the embeddable microneedles insert into skin, they rapidly dissolve and activate drug release successfully for tumor treatment. Moreover, the SA/Ge/HA supporting back layer left behind to cover the wound and promote the proliferation of endothelial and fibroblasts cells for enhanced skin regeneration. The two-layered microneedles platform can simultaneously eliminate the tumor and accelerate wounding healing, which may be potentially employed as a competitive strategy for the treatment of melanoma.


Background
Skin tissue is a crucial defense that protects the human body from outside environmental disturbance. 1 Nowadays, skin tumors is diagnosed more than one million cases annually, with an increasing incidence.
Melanoma, a highly malignant tumor of melanocyte origin, is one of the most aggressive skin tumors and is characterized by local invasiveness, early metastasis, repeated recurrence, and high mortality. 2,3 Clinically, surgical resection has been the most widely applied treatment for melanoma but suffered from the high risk of recurrence. 4 Other alternative therapeutic strategies have been investigated to eradicate the melanoma cells, including the chemo/radiotherapy and photodynamic therapy (PDT). Unfortunately, melanoma is resistant to the traditional radio/chemotherapy which could bring about endless pain to patients. 5 Moreover, a major limitation of PDT is its poor light penetration depth through pigmented lesions. 6 In addition, for complete clearance of residual tumor tissues and preventing cancer relapse, the surgical treatment for melanoma often causes a large cutaneous defect. 7 The loss of skin might cause infection and impede the healing process. 8 Therefore, promoting skin repair is of vital value for the treatment of melanoma. However, there are few new biomaterials and strategies to meet the dual requirements of melanoma treatment and wound healing. Thus, it is quite urgently to develop a strategy with the integrated therapeutic effects of melanoma cure and simultaneously for accelerating skin repair.
For address this issue, microneedle as a transdermal drug delivery system is a good choose for melanoma therapy and skin regeneration, which we designed a two-layered microneedle platform with a dissolvable microneedle part and a biodegradable backing cover. Microneedle is famous for its use in biomacromolecules delivery for diabetes therapy, [9][10][11] due to its high safety, painless invasion, and simple administration. [12][13][14] In particular, dissolving MNs can reliably pierce into skin, rapidly dissolve, and then release the encapsulated drug into skin within minutes. 15,16 Due to its excellent mechanical property, water-solubility, biocompatibility, and biodegradability of hyaluronic acid (HA), we choose HA to prepare dissolvable MNs. In the MN part, IR820 and Curcumin nanoparticles were loaded for photothermal and chemo-co-therapy for melanoma treatment. In recent years, the integration of photothermal therapy (PTT) and chemotherapy has emerged as an effective and popular treatment technique for a variety of cancers, which could improve the e ciency of cancer therapeutics and decrease the side effects. 17,18 PTT is based on photothermal agents (PTAs), which can convert near-infrared (NIR) light energy into heat to kill the tumor cells. 19 New Indocyanine Green (IR820), a derivative of Indocyanine green (ICG, approved by the Food and Drug Administration (FDA)), 20 is capable of higher photo-thermal conversion rate and better stability under exposure of NIR light than ICG. 21,22 However, IR820 cannot be retained locally and easily spread all over the body because of water-solubility. With the help of microneedles for localized delivery, IR820 can be centralized to maximize the therapeutic effects and reduce the usage dose. 23 Curcumin (Cur) possesses diverse pharmacological effects, including antitumor, 24 anti-in ammatory, 25 antiangiogenic 26 properties and osteogenic induction. 27 However, the low bioavailability and poor aqueous solubility have restricted its clinical use. 28 To overcome these disadvantages, a facile and green method has recently been developed to synthesize carrier-free curcumin nanodrugs (Cur NDs), which exhibited good stability in physiological environments and better therapeutic efficacy for cancer than free Cur. 29,30 Surgical resection is the most common modality for treating melanoma in clinic. The large skin defect from surgery is di cult to heal and could cause chronic wounds. Unfortunately, few studies addressed the issues of tumor-induced skin repair. 31 Although some previous biomaterials such as injectable hydrogels, 32 electrospun membrane, 33 and nanofibers 34 have been reported to promote skin-tumorinduced skin repair, the fabrication processes were complicated and restricted their use. However, MNs are easily prepared, conveniently used, and can access the target site in a painless and non-invasive way. 35 Simultaneously, the supporting back layer of MNs can be designed with tissue induced activity to promote the proliferation of skin cells, thus accelerating the skin repair. Therefore, the dissolvable microneedle part and biodegradable backing cover composed the dual function system with efficient anti-tumor and enhancing tissue regeneration ability.
Because melanoma is a super cial skin cancer, which rapidly spread into the surrounding skin, local therapy by MNs along with PTT is a more suitable method to melanoma than the systemic therapy system The local therapy strategy can enhance delivery e ciency to the target tumor site, avoid excessive circulation and lesson side effects to normal tissues. 36,37 In this study, we developed a bifunctional two-layered microneedles (MNs) system that consists of dissolving microneedle for chemo-photothermal therapy of melanoma, and a biodegradable supporting back layer for skin regeneration. As displayed in Figure 1(A), the chemotherapeutic Cur NDs and photothermal agent of IR820 were dispersed into the HA solution to endow the microneedle with anticancer efficiency. Furthermore, the supporting back layer was prepared by adding the sodium alginate/gelatin/hyaluronic acid (SA/Ge/HA) solution onto the microneedle. Subsequently, calcium ions was used to cross-link sodium alginate and generate network structure. 38 When the two-layered microneedles inserted into skin and irradiated by the NIR light, the Cur NDs/IR820/HA microneedles dissolve and release Cur NDs and IR820 to achieve the chemo-photothermal effect, and the remaining SA/Ge/HA back layer could stimulate regeneration of skin tissue (Figure 1(B)). The synthesis, physicochemical structure and properties, antitumor treatment and skin repair e cacy of two-layered MNs were investigated with in vitro and in vivo experiments.

Preparation and Characterization of Microneedles
Herein, a two-layered MN system was developed via a two-step casting process. As displayed in Figure  2(A), the MNs were differently colored because of the addition of different composition. After the incorporation of Cur NDs and IR820, the color of Cur NDs /HA MNs and IR820/HA MNs microneedles turned white to orange and dark green, respectively. While the Cur NDs/IR820/HA MNs displayed the color of black green for the mixture of Cur NDs and IR820. Furthermore, Figure 2

Mechanical Strength Tests
To clarify whether the HA MNs are mechanically strong enough to penetrate through the stratum corneum, the morphology changes of MNs tips against static forces were observed. As displayed in Figure 3(B), the sharp tips of MNs present more and more bending after putting with 10 g (~2.45 × 10 −4 N/needle) to 500 g (~1.225 × 10 −2 N/needle) weights on MN patch 41 . The microneedles did not break even though they were been pressed by 500 g weight. The mechanical strength experiments illustrate that the mechanical strength of HA MNs is competent for transdermal drug delivery.

In Vitro Skin Insertion Test
To verify whether the HA MNs could successfully penetrate into the skin, the MNs were applied to nude mice skin. The bright eld photograph of mice skin displays the uniform microchannels created due to the insertion of HA MNs (Figure 3(C)). The rows of blue dots by trypan blue staining in Figure 3 In Vitro Cell Compatibility and Anti-cancer Cell Experiment Cell compatibility of microneedle tips and backing layer materials were tested on B16F10 and NIH3T3 cells. From the CCK-8 test results ( Figure 5(A)), both the two kinds of cells exhibited desirable viability incubated with different concentrations of HA. Interestingly, high concentration of materials led to enhanced cell viability, while cell viability in lower concentration of leachates exhibited no differences between control group, indicating that HA was biocompatible and acted as nutrition for cells. 42 Accordingly, as Figure 5(B) demonstrated, the results veri ed that the back layer SA/Ge/HA scaffold was also non-toxic and safe.
The in vitro anti-cancer e cacy of Cur NDs/IR820/HA MN was evaluated via LIVE/DEAD staining and CCK-8 assay using B16F10 cells. In the LIVE/DEAD images ( Figure 5(C)), the green and red uorescence indicated the alive and dead cells, respectively. Compared with control and HA MNs groups, the sustained release of Cur NDs in Cur NDs/HA MNs led to minor cell apoptosis, while the IR820 MNs + laser group triggered photothermal therapy which can generate hyperthermia and contribute to the death of majority of tumor cells. The localized hyperthermia and increased cell membrane permeability assisted in inducing more tumor cells apoptosis in the Cur NDs/IR820/HA MN + laser group 43 . The quantitative cell survival rate results demonstrate that the cell viabilities were 103.88%, 86.36%, 24.73%, and 2.67% in the groups of HA MNs, Cur NDs/HA MNs, IR820/HA MNs + laser, and Cur NDs/IR820/HA MNs + laser respectively, compared with control group (Figure 5(D)). Besides, to evaluate the cell killing effect of MNs, we further cultured the tumor cells for 48 h after the 808 nm laser irradiation. According to the CCK-8 results ( Figure 5(E)), HA MNs groups showed less cell cytotoxicity, indicating that the material itself was cell compatible. In addition, with the participation of Cur NDs in Cur NDs/HA MNs group, the cell viability of B16F10 decreased to 89.7%, suggesting the single chemotherapy e cacy was insu cient. Tumor cells in IR820/HA MNs + laser group were remarkedly inhibited (23.4%), owing to the hyperthermia triggered by IR820. In contrast, the Cur NDs/IR820/HA MNs + laser group displayed the maximum antitumor e cacy (12.3%). The results suggested that the hyperthermia effects could accelerate the localized release of Cur NDs and also increase the cell membrane permeability and uidity, 44 exhibiting the successful chemo-cophotothermal anti-cancer effect via curcumin and IR820.

In Vivo Anti-cancer Treatment
The in vivo anti-cancer capacity was carried out on B16F10 tumor-bearing C57BL/6 mice, and the time scheme of treatment was shown in Figure 6(A). The mice were randomized into 6 groups: Control, HA MNs, Cur NDs/HA MNs, Cur NDs/IR820/HA MNs, IR820 MNs + laser, and Cur NDs/IR820/HA MNs + laser. The mice in different groups were inserted with the corresponding MNs. The in vivo NIR images of MNs under NIR laser irradiation (808 nm, 0.75 W/cm 2 , 5 min) in G2, G5 and G6 were recorded, which demonstrated a changing color of the tumor site (Figure 6 (B)). As shown in Figure 6 (C), G2 had no obvious temperature rise, while G5 and G6 heated up quickly in the rst 30 s and kept arising slowly and steadily increased to ~50℃, which was su cient to induce the ablation of tumor tissues.
As demonstrated in Figure 6 (D) and (F), the tumor volumes and mice body weights in G1 and G2 increased at an alarming rate. G3 and G4 groups showed slight tumor inhibitory effect, indicating that single Cur NDs was not su cient for tumor therapy. In contrast, G5 and G6 exhibited a desirable tumor killing e ciency with the participation of photothermal therapy, and G6 presented a better tumor inhibition effect, which attribute to the Cur NDs worked synergistically with IR820 for enhanced thermalco-chemotherapy of melanoma. From day 0 to 10, the body weight of the mice in G1, G2, G3, and G4 increased rapidly compared with the other two groups, which might result from the overgrowing tumors without e cient treatment. At 10 days after treatment, the body weight of G1-4 declined drastically. Besides, an emaciated gure and diminished appetite were observed, suggesting the progression to the cachectic stage. In contrast, the weight of mice in G5 and G6 were more stable than the other four groups, which proved that thermal-co-chemotherapy is e cient for tumor elimination. At 14 days, the mice were subjected to humanitarian death and the tumor tissues were removed in an integrated form. From the digital picture of separated tumors (Figure 6 (E)), the G6 demonstrated excellent tumor growth inhibition effect. Similarly, the mean tumor weights (Figure 6 (G)) in G6 was the smallest compared with other groups, indicating that Cur NDs/IR820/HA MNs with laser could e ciently inhibit the tumor growth.
The histological analysis of tumor was carried out via Ki67 staining. The expression of Ki67 in tumor cells signi cantly reduced in the Cur NDs/IR820/HA MNs + laser group, indicating that thermal-cochemotherapy inhibited the proliferation of tumor cell (Figure 7 (A)). The key organs of mice (heart, liver, spleen, lung and kidney) were harvested for H&E staining. From the results (Figure 7 (B)), no signi cant toxicity and damages were found in these main organs. From the lung sections, an obvious decrease of lung metastasis was seen in G5 and G6, indicating that photothermal therapy was effective for inhibiting tumor metastasis progression. Taken together, our results suggested that the Cur NDs/IR820/HA MNs with laser irradiation could inhibit tumor proliferation and thereby exert chemo-photothermal therapy effect with no signi cant in vivo toxicity.

In Vivo Animal Skin Repair
In order to evaluate the skin repair e ciency of our materials, we adopted full-thickness skin defect of SD rats as animal experimental model and the rats were randomly divided into three groups (n = 8): Control group, HA MNs group, and Cur NDs/IR820/HA MNs group. At day 7, all the three groups showed a decrease of the wound area. Fortunately, better wound closure was observed in Cur NDs/IR820/HA MNs group (Figure 8 (A)). The Cur NDs/IR820/HA MNs group presented a lower relative wound area compared to other groups at day 14 (Figure 8 (B)). To further analyze the skin repair effect histologically, the H&E staining was performed. At day 7, in ammation response occurred in all three groups, but in ammatory cells including lymphocytes and neutrophils in control and HA MNs groups were more than that in Cur NDs/IR820/HA group (Figure 8 (E)). Besides, a thicker epidermal layer was observed in Cur NDs/IR820/HA group. As shown in Figure 8(C), at day 14, except the repair of epithelia tissue, the cutaneous appendages such as hair follicles also took shape. Especially, the Cur NDs/IR820/HA MNs group showed complete regeneration of skin tissue with skin appendages and increased cell density, indicating the speeding up of skin repair. In the Masson staining (Figure 8 (D)), the collagen formation was analyzed to further evaluate the skin repair process. A higher amount of and more tight organized collagen bers in Cur NDs/IR820/HA group suggested a higher collagen deposition level, which was bene cial for skin recovery. Moreover, the collagen level in the Cur NDs/IR820/HA MNs group was signi cantly higher relative to the HA MNs group. Natural polymers, including alginate, gelatin and hyaluronic acid, are widely used in the eld of tissue engineering scaffolds, and in particular, for skin regeneration applications. 45 The SA/Ge/HA supporting back layer was bene cial for new skin tissues formation. 46 Additionally, the nice repair results in Cur NDs/IR820/HA group may also attribute to antiin ammation effects of Cur NDs released from Cur NDs/IR820/HA MNs. 47

Conclusion
Considering the dilemma of clinical management for melanoma, a microneedle-based noninvasive photothermal/chemotherapy platform was proposed for enhanced tumor treatment and followed skin tissue repair. This hybrid system demonstrated desirable prospects including 1) The microneedle-based system is relatively simple and convenient, and avoids the operation of trauma, 2) Photothermal/chemotherapy e ciently controls tumor progression, 3) The curcumin nanodrugs not only induce melanoma cells apoptosis, but also reduce in ammation and promote skin regeneration process, 4) The SA/Ge/HA back layer is biocompatible and biodegradable for promotion wound healing. This twolayered microneedle system is not only designed for skin tumor management through a noninvasive path, but also capable of treating wound defect.

Preparation and Characterization of Microneedles
The Cur NDs/IR820/HA MN patches were prepared through a two-step casting process. Firstly, the Cur NDs were synthesized via a reprecipitation approach. 30 In brief, Cur dissolved in ethanol solution was dropwise added into distilled water under vigorous stirring and form Cur nanoparticles. The Cur NDs were concentrated by ultrafiltration and then obtained by lyophilization. Afterwards, 10 ml of HA aqueous solution (30 mg/ml) was mixed with 8 ml of Cur NDs (600 μg/ml) and 2 ml of IR820 solution (2 mg/ml). After forming a uniform solution, 1 ml of the HA mixture solution was added into the polydimethylsiloxane (PDMS) microneedle mold, and the filled template was dried at 55 °C overnight. Subsequently, an additional 1 ml of SA/Ge/HA solution (weight ratio = 2:1:1) were casted onto the mold to form the backing layer. Afterwards, 1.0% calcium gluconate aqueous solution was sprayed to crosslink the backing layer. Finally, the Cur NDs/IR820/HA MN patches were peeled from the PDMS template. The Cur NDs/HA MNs and IR820/HA MNs were fabricated as mentioned above without the incorporation of IR820 or Cur NDs, respectively. The HA MNs were fabricated without IR820 and Cur NDs.
The bright-field microscopic photographs of MNs were captured by a stereomicroscope (OLYMPUS, SZX16, Tokyo, Japan). The morphology of the microneedle structures, the Cur NDs and the lyophilized SA/Ge/HA backing layer were characterized by scanning electron microscopy (SEM; JSM-5900LV, JEOL, Tokyo, Japan). The hydrodynamic size of the Cur NDs was determined by a DLS instrument (Malvern Zetasizer Nano ZS). The average pore size of lyophilized SA/Ge/HA backing layer was estimated with the Smile-View software.

Dissolution Characteristics of Microneedle Arrays
For in vitro dissolution analysis of MNs, the Cur NDs/HA MNs were applied onto porcine skin and fixed with a gum tape. The MNs were taken off from the skin at indicated time intervals (15, 30, and 45 min) and imaged by brightfield stereomicroscopy to visualize the dissolution of microneedles.

Mechanical Strength Tests
To evaluate the mechanical strength of HA MNs, the MNs were tested with a serial of weights. Brie y, the MN patches were positioned vertically (microneedle tips facing up), and weights of 10, 50, 100, 250, and 500 g were put on the tips of MN patches. The deformation of the microneedles was visualized with stereomicroscopy.

In Vitro Skin Insertion Test
The in vitro skin insertion ability of HA MNs was assessed by inserting the MNs into the separated back skin of nude BALB/c mice with a force of 10 N and remained for 5 min. After removal, the treated skin was observed using stereomicroscopy and trypan blue staining method. In brief, the inserted mice skin was stained with trypan blue dye for 5 min, and the residual dye was washed off with phosphate-buffered saline (PBS). The skin insertion capacity was observed by stereomicroscopy. In addition, the collected skin samples were evaluated by H&E staining.

In Vitro Photothermal Performance of MNs
To evaluate the photothermal performance of MNs, the MNs were exposed to 808 nm NIR laser irradiation (MW-GX-808/5000 mW, Leishi, Changchun China) at an output power of 0.75 W/cm 2 for 5 min. The realtime thermal images and temperature changes of the MNs were recorded by a Fluke Ti32 Infrared thermal camera (Infrared Cameras, Fluke, Avery, WA, USA) every 30 s.

In Vitro Cell Compatibility and Anti-Cancer Cell Experiments
To study the cell compatibility of microneedle tip and backing layer material, two kinds of cells (B16F10 and NIH3T3 cells) were used. Firstly, the cytotoxicity of microneedle tip material (HA) was veri ed by B16F10 and NIH3T3 cells. B16F10 or NIH3T3 cells (5000/well) were seeded into the 96-well plates and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in an incubator (5% CO 2 , 37℃) for 24 h. The cell culture medium was then replaced by HA MNs solution (dissolved in DMEM medium, without the back layer) with different concentrations. After another 24 h of incubation, the CCK-8 kit was utilized to detect the cell viability according to the manufacturer's protocol. In brief, working solution was prepared with DMEM medium (CCK-8 kit concentration 10%), then 100 ml of working solution was added to each well. After incubation in the dark (37℃) for 1-4 hours, the absorbance values were measured at 450 nm via Beckman DU7400 spectrophotometer (Beckman coulter, Miami, FL, USA). To investigate the cell compatibility of the backing layer material (SA/Ge/HA), the NIH3T3 cells were employed and the experimental procedure was carried out as described above.
The in vitro anticancer activity of MNs was evaluated using LIVE/DEAD staining. B16F10 cells were seeded into a 6-well plate (2×10 5 cells/well), and different groups of microneedles were placed on the chambers of Transwell. Then the microneedles were irradiated with an NIR laser for 5 min (808 nm, 0.75 W/cm 2 ). After 2 h, the cells were stained with LIVE/DEAD Cell Imaging Kit and observed by fluorescence microscope (DM2000, Leica, Germany).
In addition, CCK-8 assay was also employed to determine the cell viability of MNs treatment. The dissolved solutions of HA MNs, Cur NDs/ HA MNs, IR820/HA MNs and Cur NDs/IR820/HA MNs were added into the culture wells, respectively. Subsequently, the cells were irradiated with an NIR laser light (808nm, 0.75 W/cm 2 , 5 min), and then cultured for 48 h. The cell survival rate of B16F10 was tested via the CCK-8 kit.

In Vivo Anti-cancer Study
The subcutaneous melanoma tumor model was established by injecting 100 μl of 1×10 6 B16F10 cells into the right back of C57BL/6 mice (6-8 weeks old, female, ~20 g). When the tumor size grow to approximately 50-70 mm 3 , the mice were randomized into 6 groups (n = 5) as follows: G1, Control; G2, HA MNs; G3, Cur NDs/HA MNs; G4, Cur NDs/IR820/HA MNs; G5, IR820 MNs + laser; G6, Cur NDs/IR820/HA MNs + laser. After anesthetization via iso urane, the mice were treated with microneedles according to the grouping. The G5 and G6 groups were exposed to the NIR laser (808 nm, 0.75 W/cm 2 , 5 min). The tumor surface temperature and thermal images were captured timely via the NIR thermal imaging camera. The body weight and size of tumor were measured every 2 days. The tumor volume was calculated as follows: tumor volume (V) = (tumor length) × (tumor width) 2 /2. At day 14 after treatment, the mice were sacri ced and the tumor tissues was harvested, weighed and photographed. After xation with 4% paraformaldehyde, the tumor samples were dehydrated with graded ethanol series, embedded into paraffin, sectioned into serially 5 μm thick slices, and then stained with Ki67. Additionally, the key organs of mice including heart, liver, spleen, lung, and kidney were all collected for histological analysis.
In Vivo Animal Skin Repair Experiment Skin repair experiment was carried out on SD rats (8 week-old, male, 200-220 g). After anesthetization, the dorsal hair of SD rats was shaved and a 1.5 cm × 1.5 cm skin defect was constructed on the back. The rats were randomized into 3 groups (n = 8): Control group, HA MNs group, and Cur NDs/IR820/HA MNs group. The microneedles were inserted into the defect area, after which the defects of all groups were protected with Tegaderm (3M, St. Paul, MN, USA). At day 0, 7 and 14, the wound healing was photographed and the wound areas were measured by ImageJ software. Subsequently, four rats were sacri ced in each group at day 7 and day 14, respectively, and the whole wound sites (including the wound and the surrounding normal skin tissues) were excised and stained with H&E and Masson staining. At 7 and 14 days, the number of inflammatory cells in H&E stained micrographs was calculated through and Image J software.

Statistical Analysis
All quantitative data were presented as means ± standard deviation (SD). SPSS 11.0 software (Chicago, IL, USA) were utilized for statistical analysis. Statistical differences of groups were analyzed by one-way ANOVA, and a P values < 0.05 was considered statistically signi cant.