Materials
Dopamine (DA), D-methionine, riboflavin and nitrotetrazolium blue chloride (NBT) were purchased from Macklin (Shanghai, China). Polydimethylsiloxane (PDMS) mold was purchased from Taizhou Microchip Pharmaceutical Technology (Taizhou, China). Hyaluronic acid sodium salt (100 kDa) and super active hyaluronic acid (HA) (5 kDa) were purchased from Hefei BOSF Biotechnology Co., Ltd. 30% H2O2 was obtained from Sinopharm Chemical Regent Co., Ltd. Cell Counting Kit-8 (CCK8) was purchased from MeilunBio®. 2, 7-dichlorofluorescein diacetate (DCFH-DA), MitoTracker® Green FM, Annexin V-EGFP/PI Apoptosis Detection Kit, Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus), Hieff® qPCR SYBR Green Master Mix (No Rox) and Calcein-AM/PI Double Stain Kit were purchased from Yeasen biotech Co., Ltd. MMP assay kit with JC-1, Hydrogen Peroxide Assay Kit, Trizol and DAPI were purchased from Beyotime (Shanghai, China). RhB, DPPH, and ABTS were purchased from J&K Scientific Co., Ltd. Anti-HMGB1 antibody was obtained from Abcam (catalog no. Ab79823). p-STAT1 (Ser707) antibody was pruchased from Abmart (catalog no. PC3427S). p-STAT3 (Tyr705) antibody was obtained from Cell Signaling Technology (catalog no. 9145T). MelanA antibody was obtained from Affinity (catalog no. AF0204).
Characterizations
Transmission electron microscopy (TEM) images were captured by a HT7700 transmission electron microscope (JEM-F200, Japan). Scanning electron microscope (SEM) images were captured by HITACHI UHR FE-SEM SU8000 Series (SU8010, Japan). The dynamic light scattering (DLS) size distribution was measured by Zetasizer Nano ZS ZEN3600 (Malven, England). UV/Vis absorption spectra were measured on a Cary 60 UV/Vis spectrophotometer (Agilent Technologies, USA). The immunofluorescence images were captured by Leica Stellaris 5 (Heidelberg, Germany). The morphology images of microneedles were captured by stereomicroscope (T1-HD206, China). The Quantitative Real-time PCR Analysis were conducted by iQ5 PCR assay systems (Bio-Rad Laboratory, USA). The mechanical strength of microneedles was conducted by Electronic Universal Material Testing Machine (Instron 5944, USA). The permeation ability of microneedles was tested by multiphoton microscope (FVMPE-RS, Japan). All the fluorescent pictures were captured by fluorescence microscope (Leica Stellaris 5, Germany).
Synthesis of PDA and PDA-JAKi nanoparticles
180 mg DA was dissolved in 90 mL H2O. When it was heated to 50℃, the 760 µL NaOH (1 M) was added immediately and stirred for 5 h. After cooling into room temperature, the PDA nanoparticles were obtained by centrifugation at 18,000 rpm for 15 min. The deposit re-suspended in pure water and centrifugated at 4000 rpm to remove large particles. To obtain PDA-JAKi, various concentrations (0.5, 1, 1.5, 2 mg) of JAKi were feed into 2 mg PDA solution, after stirring for 4 h, the final product was centrifugated and the supernatant was collected for UV/Vis absorbance analysis. The loading efficiency of JAKi was determined using the formula: loading efficiency = 100% × (total JAKi – unloaded JAKi) / total JAKi. The loading capacity of JAKi was determined using the formula: loading capacity = 100% × loaded JAKi / total PDA-JAKi.
Catalytic ability against H2O2
50 and 100 µg/mL PDA and PDA-JAKi were co-incubated with 1 M H2O2 for 12 h at room temperature. After centrifugation, A hydrogen peroxide assay kit was utilized to quantify the content of H2O2 in the supernatant. The specific operation was carried out according to the instructions.
Free radical scavenging assays
For DPPH (1,1-diphenyl-2-picrylhydrazyl radical) depletion assay, 100 mM DPPH was co-incubation with different concentrations (0, 12.5, 25, 50, 100 µg/mL) PDA-JAKi for 20 min in the dark. Then, the reaction mixture solution was centrifuged and the supernatant was collected for further detection by UV/Vis at 515 nm. In addition, the 10 µg/mL PDA-JAKi was chosen to obtain the absorbance curve of DPPH over time.
For •OH depletion assay, the ability of •OH depletion was monitored by ABTS (2,2’-azino-bis(3-ethylbenzothiazoline 6-sulfonate)). 920 µL of H2O, 10 µL of H2O2 (200 µM), 20 µL of FeSO4·7H2O (18 mM), and 10 µg of PDA-JAKi were mixed together. After sonication for 5 min, the supernatant was collected by centrifugation and then incubated with 50 µL ABTS (10 µM) for different times (0, 1, 10, 30, 90, 120 min). Finally, the UV/Vis absorbance of the reaction solution at 750 nm was recorded.
For O2−• depletion assay, 12.5 mM methionine, 75 µM nitrogen blue tetrazole, 20 µM riboflavin and different concentrations (0, 12.5, 25, 50, 100 µg/mL) of PDA-JAKi were resuspended in 1 mL of PBS. The reaction solution was collected for UV/Vis analysis at 560 nm after UV irradiation for 15 min.
Preparation of microneedle patches
180 mg/mL HA hydrogel solution (sodium hyaluronate: super active hyaluronic acid = 1:5, w/w) was added to the PDSM mold. Then, PDSM mold was placed into horizontal centrifuge and centrifuged three times at 4000 rpm for 5 min. The whole product was dried at 37℃ over night. Finally, the HA MNs were carefully removed from molds for the following experiments. PDA MN, JAKi MN and PDA-JAKi MN were prepared by almost the same process as HA MN, with the PDA, JAKi and PDA-JAKi already doped in HA hydrogel solution respectively. For rhodamine B (RhB) -loaded HA MN preparation, RhB was added into the HA hydrogel solution for next steps.
Mechanical strength of MN detection
The various MN patches were placed on the steel plate and moved vertically direction with detector probe at a speed of 0.1 mm s–1. When it touched the tip, recorded the displacement and force immediately. Meanwhile, when the tip of MN patch started to break, the destructive force of the MN patch was recorded.
Permeability ability of HA MN detection
The PDA-JAKi MN patches were inserted into the skin of mice. After 10 min of application, 10 mg/mL methylene blue (MB) aqueous solution was applied to stain the microholes. After another 10 min application, the MB aqueous solution was removed and the images were recorded with stereomicroscope. To further understand the penetration, the mice skin was collected and fixed with 4% paraformaldehyde. The hematoxylin and eosin (H&E) staining was performed by Wuhan Service Biotechnology Co., Ltd. The RhB-loaded HA MN patches were prepared in advance. They were applied to the freshly excised skin of mice. The RhB-loaded HA MN were removed and the penetration depth was observed using a multiphoton microscope (ex = 540 nm, em = 625 nm) after 10 min of application.
Drugs release
The JAKi MN were placed in PBS solution (pH 7.4). As the MN dissolved, the JAKi was released. The supernatant was then collected for cumulative drug release accroding to the absorbance of JAKi at 285 nm.
For PDA-JAKi release detection, the 100 µg/mL PDA-JAKi was dissolved in 5 mL PBS solutions (pH 6.0 and pH 7.4) and stood in the 37℃ incubator. At the indicated time, 1.5 mL of the supernatant was collected and centrifuged, and the 1.5 mL fresh PBS solution was added. The supernatant was measured by UV/Vis at 285 nm.
Cell experiments
For cytotoxicity assay, HaCaT and PIG1 cells were seeded into 96-well plates (5 × 103 cells per well) and incubated overnight. Subsequently, the initial DMEM was disposed of and replaced with fresh DMEM solution that included varying concentrations of JAKi and PDA-JAKi individually. After further incubation for 24 h, cell viabilities were assessed by CCK-8 assay. Similarly, various doses of H2O2 ranging from 0 to 1000 µM were added into fresh DMEM, cell viabilities were assessed by CCK-8 assay after 24 h incubation. The DMEM was replaced with fresh DMEM containing 1 mM H2O2 and various concentrations of JAKi and PDA-JAKi. After co-incubation for 24 h, the cytotoxicity was conducted by CCK-8 assay. Control group represents no treatment. Untreated group represents cells treated with H2O2 or INF-γ only.
To evaluate cellular apoptosis using flow cytometry, PIG1 cells were seeded into 6 wells with a density of 1 × 105 and cultured for 12 h. Subsequently, the cells were exposed to DMEM supplemented with H2O2, H2O2 + PDA, and H2O2 + PDA-JAKi (1 mM H2O2, 50 µg/mL PDA, 50 µg/mL PDA-JAKi) for a duration of 24 h. Then, the cells were gathered and subjected to staining using the Annexin V-FITC/PI Apoptosis Detection Kit. The stained cells were promptly examined using a flow cytometer (Beckman Coulter, USA).
For live and dead cell observation, the HaCaT cells were seeded into a 6-well plate overnight and further exposed to different treatments for 24 h: H2O2, H2O2 + PDA, H2O2 + JAKi, H2O2 + PDA-JAKi (1 mM H2O2, 50 µg/mL PDA, 50 µg/mL PDA-JAKi, 5.5 µg/mL JAKi, at queal dose of PDA-JAKi). And then, Calcin-AM solution (2 mM, 1:1000) and PI solution (1.5 mM, 1:1000) were added to stain the cells for 10 min. After that, the cells were observed using a fluorescence microscope. The quantitative analysis of the CA/PI was performed using the ImageJ software.
To detect the MMP change, JC-1 Assay Kit was employed to detect the alterations. PIG1 cells were placed in a 6-well plate with a density of 1 × 105 cells per well and incubated for 12 h. Following various treatments (Control, H2O2, H2O2 + PDA, H2O2 + PDA-JAKi; 1 mM H2O2, 50 µg/mL PDA, 50 µg/mL PDA-JAKi) for 24 h, the cells were conducted by JC-1 Assay Kit. And the fluorescence images were acquired using a fluorescence microscope.
To observe the mitochondrial integrity, HaCaT cells was seeded into a 6-well plate at a density of 1 × 105 cells per well. After overnight incubation, the cells were treated with H2O2, H2O2 + PDA, and H2O2 + PDA-JAKi (2 mM H2O2, 50 µg/mL PDA, 50 µg/mL PDA-JAKi) for 30 min, respectively. MitoTracker Green probe was utilized to stain mitochondria in living cells. The nucleus was stained with DAPI. And the fluorescence images were acquired using a fluorescence microscope.
To investigate antioxidant stress activity in vitro, HaCaT cells were co-incubated with 1 mM H2O2 and PDA/JAKi/JAKi-PDA, respectivly. After 24 h co-incubation, cells were collected for further CAT, SOD and MDA activity detection accroding to the manufacturer’s instructions.
For in vitro ROS detection, the HaCaT cells were seeded into a 6-well plate (1 × 105 cells per well) and cultured overnight. Afterwards, the origin DMEM was removed, and H2O2, H2O2 + PDA and H2O2 + PDA-JAKi (1 mM H2O2, 50 µg/mL PDA, 50 µg/mL PDA-JAKi) were introduced to the fresh DMEM for 4 h. Then, PBS solution with DCFH-DA probe was stained for 30 min. Cells were washed with PBS three times and observed by a fluorescent microscope or immediately detected by a flow cytometry.
For PDA-JAKi uptake, HaCaT cells were plated in dishes and incubated overnight. 100 µg/mL PDA was added and incubated with the cells for 24 h. The cells were stained with the DAPI for 10 min. Finally, the cells were observed by confocal microscopy (Leica Stellaris 5, Germany).
Transmission electron microscopy
100 µg/mL PDA-JAKi was incubated with HaCaT cells. At the indicated time, cells were harvested for observation of melanosomes by TEM.
Western blotting
To confirm inhibition of the JAK-STAT pathway induced by PDA-JAKi, HaCaT cells were pretreated with IFN-γ (20 ng/mL) for 24 h. The original DMEM was then replaced with fresh DMEM containing PDA/JAKi/JAKi-PDA for a further 24 h incubation. After different treatments, the cells were harvested in an ice bath using RIPA lysis buffer, followed by immunoblotting of cell lysates using primary antibodies against p-STAT1, p-STAT3 and GAPDH at 4 °C overnight. A chemiluminescence imaging system (Bio-Rad, USA) was employed to determine the expression levels of proteins. The semi-quantitative analysis of the protein bands was calculated with the ImageJ software.
Immunofluorescence staining in vitro
PIG1 cells were seeded into confocal dishes (1 × 105 cells per dish) and incubated for 12 h. Afterwards, the cells were treated with H2O2, H2O2 + PDA and H2O2 + PDA-JAKi (1 mM H2O2, 50 µg/mL PDA, 50 µg/mL PDA-JAKi) for 24 h. PIG cells were stained with anti-HMGB1 antibody for immunofluorescence imaging.
RNA isolation and quantitative real-time PCR (qPCR) analysis
Total RNA was isolated from HaCaT cells using Trizol reagent. It was reverse transcribed to cDNA using Hifair® 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus). The qPCR analysis was carried out using the Hieff® qPCR SYBR Green Master Mix (No Rox). All procedures were performed according to the instructions. Relative mRNA expression was normalised to the GADPH gene. The primers used in this study were listed as follows (Table 1).
ELISA assay
On one hand, HaCaT cells were co-treated with 50 µg/mL PDA, 5.5 µg/mL JAKi, and 50 µg/mL PDA-JAKi for 12 h with 1 mM H2O2. On the other hand, HaCaT cells were pre-treated with IFN-γ (20 ng/mL) for 24 h and then treated with PDA/JAKi/PDA-JAKi for 24 h. Supernatants were collected after different treatments, the chemokine levels were measured using CXCL9, CXCL10 and CXC16 ELISA kits in accordance with instructions.
Animal experiments
Female C57BL/6 mice aged 4–6 weeks were used in this experiment. All mice were purchased from Beijing Weitong Lihua Animal Experiment Co., LTD. All animal rearing and experiments were carried out in accordance with the regulations of the Animal Care and Trial Committee of Wenzhou Medical University. The vitiligo mice model was established according to previously reported literature [18]. In brief, a vitiligo mouse model was established by applying 5% hydrogen peroxide to the dors al skin of mice twice a day for six weeks. The vitiligo mice were randomly divided into 6 groups including: (1) Control group; (2) Untreated group; (3) HA MN group; (4) PDA MN group; (5) JAKi MN group; (6) PDA-JAKi ointment group; (7) PDA-JAKi MN group. The therapeutic dose of PDA and PDA-JAKi was 25 mg/kg and the therapeutic dose of JAKi was 2.75 mg/kg. The control group indicated that the mice were not treated in any way and the untreated group indicated that the mice were treated with H2O2 only. The ointment was purchased from Mannings. The PDA-JAKi ointment was prepared by simply mixing the ointment with the PDA-JAKi. The treatments were administered twice a day and two pieces at a time for the microneedles-mediated groups. For the PDA-JAKi ointment group, the ointment was applied thinly to the corresponding area of the back and gently massaged until absorbed. At the end of the treatments, the mice were sacrificed, and the treated skin was collected for ELISA assay, CD8+ T cells immunofluorescence staining, H&E staining, Masson-Fontana staining, ROS immunofluorescence staining and melanA immunofluorescence staining. Subsequently, the major organs were then harvested to confirm the compatibility of the therapeutic agents. All the samples were further performed by Wuhan Service Biotechnology Co., Ltd.
Statistical analysis
All the results above represented mean ± standard deviation. Statistically differences were determined by Student’s t-test or One-way ANOVA. Post hoc test using Graphpad Prism software 8.2.1. P values < 0.05 was considered as a significant difference between data (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significance).
