Sodium hyaluronic acid (Mn = 1.44 × 103 kDa) was obtained from Freda Biochem Co., Ltd (Shandong, China). Curcumin was purchased from Dalian Meilun Biological Co., Ltd. Sodium alginate, IR820, and gelatin (from porcine skin) were obtained from Sigma-Aldrich (USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and all other cell culture media and reagents were bought from Gibco™ (Grand Island, NY, USA). Live/dead cell imaging kit was acquired from Thermo Fisher Scientific Inc (Waltham, MA, USA). CCK-8 kit was purchased from Houston, Texas, USA. The polydimethylsiloxane (PDMS) microneedle templates were acquired from Guangzhou SLLK Co. Ltd (Guangzhou, China).
SD rats (8 week-old, male, 200 ± 20 g) were purchased from Chengdu Dashuo Animal Experiment Co. Ltd (Chengdu, China). C57BL/6 mice (8 week-old, female) and nude BALB/c mice (4 week-old, female) were obtained from Beijing HFK Bioscience Co. Ltd. (Beijing, China). The animal researches were admitted and supervised by the animal committee of West China Hospital of Stomatology, Sichuan University.
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 . In brief, Cur dissolved in ethanol solution was dropwise added into distilled water under vigorous stirring and then formed Cur NDs. 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 PDMS microneedle mold, and then 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 with the backing layer. Finally, the Cur NDs/IR820/HA MN patches were peeled from the PDMS template after drying process. 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. Briefly, 100 pores were chosen randomly from the SEM images of the lyophilized SA/Ge/HA backing layer scaffold to calculate the average pore size using the “Length Measurement” tool.
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 test
To evaluate the mechanical strength of HA MNs, the MNs were tested with a serial of weights. Briefly, 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. Afterwards, 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/cm2 for 5 min. The real-time 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 test
To study the cell compatibility of microneedle materials, two kinds of cells (B16F10 and NIH3T3 cells) were used. Firstly, the cytotoxicity of microneedle tip material (HA) was verified 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% CO2, 37 ℃) for 24 h, respectively. The cell culture medium was then replaced by HA MNs solution (dissolved in DMEM medium, without the backing 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, 100 µl CCK-8 working solution (10%) was added into each well. After incubation in the dark (37 ℃) for 1–4 h, 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. Moreover, the CCK-8 assay was also used to quantitatively analyze the NIH3T3 cell proliferation with the leaching solution from two-layered HA microneedle material (HA microneedle tip + SA/Ge/HA backing layer) for 1, 2, and 3 days after cell seeding.
In vitro anti-cancer cell experiments
The in vitro anticancer activity of MNs was evaluated using live/dead staining. B16F10 cells were seeded into a 6-well plate (2 × 105 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/cm2). 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 after 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 (808 nm, 0.75 W/cm2, 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 × 106 B16F10 cells into the right back of C57BL/6 mice (6–8 weeks old, female, ~ 20 g). When the tumor size grew to approximately 50–70 mm3, the mice were randomized into 6 groups (n = 5) as follows: Control (G1); HA MNs (G2); Cur NDs/HA MNs (G3); Cur NDs/IR820/HA MNs (G4); IR820 MNs + laser (G5); Cur NDs/IR820/HA MNs + laser (G6). After anesthetization via isoflurane, the mice were treated with microneedles according to the grouping. The G5 and G6 were exposed to the NIR laser (808 nm, 0.75 W/cm2, 5 min). The tumor surface temperature and thermal images were captured timely via the NIR thermal imaging camera. The body weight of mice 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 sacrificed and the tumor tissues was harvested, weighed and photographed. After fixation 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 (3 M, St. Paul, MN, USA). At day 0, 7 and 14, the wound healing was photographed and the wound areas were measured by Image J software. Subsequently, four rats were sacrificed 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 day 7 and 14, the number of inflammatory cells in H&E stained micrographs was calculated through and Image J software.
All quantitative data were presented as means ± standard deviation (SD). SPSS 11.0 software (Chicago, IL, USA) was utilized for statistical analysis. Statistical differences of groups were analyzed by student’s t-test or one-way ANOVA, and the P value < 0.05 was considered to be statistically significant.