Material
The dopamine hydrochloride and the pluronic F-127 were obtained from Sigma (St Louis, USA). Capsaicin (≥ 99.47%) was obtained from lemeitian medicine (Chengdu, China). Fluorescent hydrophobic dyes, including cy5.5 and coumarin 6 were derived from yuanye Bio-technology (Shanghai). CKGGRAKDC was obtained from Nanjing Peptide Biotech Ltd. BODIPY fluorescent dye was sourced from MedChemExpress Co., Ltd. The Calcein-AM/PI viability/cytotoxicity kit, reactive oxygen species detection kit, oil red O staining kit, CCK-8 regent, and H&E staining kit were provided by Beyotime Co. Antibodies, including anti-Cyto C, anti-PPARγ, anti-UCP1, anti-cEBPα, and anti-TRPV1 were provided by Abcam Co., Ltd.
Synthesis and characterization of TmCNP
The mesoporous PDA (mPDA) was synthesized using a versatile nanoemulsion assembly approach. To encapsulate Cap within the mesopores, mPDA and Cap were gently mixed and rotated at a mass ratio of 5:1 and continuously rotated to produce the Cap-loaded mPDA. Then, the unencapsulated Cap was removed by dialysis. An excess of CKGGRAKDC solution was slowly added to the Cap-loaded mPDA system at a mass ratio of 1:10. The solvents were then removed through freeze-drying, yielding a nanosphere powder.
The obtained TmCNP powders were dispersed in ultrapure water (1 mg/ml), and a single drop was placed onto a copper grid, followed by counterstaining with phosphotungstic acid. The shape image of TmCNP was observed using TEM equipment (FEI Talos F200X). The ζ potential and size were assessed using DLS Instruments (Malvern, UK).
In vitro release kinetics of drug from NPs
C6 was utilized as a substitute for Cap in these experiments. The C6 loading efficiency of mPDA and TmPDA was first measured, with values of approximately 31.0% (538.34 ± 21.76 µg/mg) and 39.37% (649.34 ± 19.76 µg/mg), respectively. 10 mg of TmPDA@C6 were dissolved in 2 ml PBS and placed into a dialysis bag (Mw = 3500 Da), which was subsequently immersed in 10 m of PBS. During dialysis, 0.2 ml samples were collected at predetermined time points, and an equal volume of fresh PBS was added. To investigate the laser-triggered drug release behavior of the nanoparticles, C6-loaded NPs were irradiated with an 808 nm laser at a power density of 1 W/cm² for 5 min at predetermined time points (2, 4, 6, and 8 h). The PBS solution was then collected for fluorescence absorption value to quantify the amount of C6 released. The release of C6 from the NPs was determined using fluorescence microplate analysis.
Hemolysis experiment
Red blood cells (RBCs) from nice were diluted with PBS to a final concentration of 4%. A 0.5 ml aliquot of the RBC suspension was incubated with an equal volume (0.5 ml) of TmCNP solutions at various concentrations, ranging from 10 to 500 µg/ml, at 37 °C for 1 h. Positive controls (100% hemolysis) were prepared using 0.1% Triton X-100, while negative controls (0% hemolysis) were prepared with PBS. After incubation, the samples were centrifuged at 3200 rpm for 15 min, and the absorbance of the supernatant was measured at 576 nm. The hemolysis rate = (Asample-Anegative)/(Apositive-Anegative) × 100%.
In vitro biocompatibility
The cytotoxicity of NPs and MN materials was evaluated using the CCK-8 assay. Briefly, 3T3-L1 cells in good growth status were harvested and seeded into 96-well cell culture plates (8 × 104 cells/ml), followed by overnight incubation. The cells were then exposure to NPs (10 µg/ml) or to 48-hour MN material extracts in cell culture medium, followed by incubation for the specified duration. The treated 3T3-L1 cells were further analyzed using CCK-8 regent and Calcein-AM/PI staining dye. The results were quantified via a microplate reader and visually assessed using a fluorescence microscope.
Primary adipocytes study
Abdominal adipose tissue was harvested from 7-day-old mice, minced, and digested in HBSS with 2% BSA and 2 mg/ml collagenase I for 25 min at 37 °C on a shaker (100 rpm). Preadipocytes were then collected by centrifugation at ~ 500 g for 8 min and plated in DMEM/F-12 media supplemented with 10% serum. After cell fusion, adipocyte differentiation medium was introduced, containing 125 nM indomethacin, 5 mM isobutylmethylxanthine, 800 nM insulin, 1 µM rosiglitazone, and 5 µM dexamethasone. On day 2 differentiation, the cells were maintained in media containing 1 nM rosiglitazone and 10 nM insulin, with media changes occurring every other day.
For the cell uptake study, hydrophobic cy5.5 was used as a surrogate for Cap to visualize uptake. Induced adipocytes and 3T3-L1 preadipocytes were treated with TmPDA&cy5.5 (cy5.5: 10 µg/ml) at 37 °C for 6 h. Redundant NPs were removed, and cell nuclei were labeled with DAPI for 8 min. After removing the remaining dye, cellular uptake of the fluorescent nanoparticles was observed and captured under a confocal microscope.
Adipocytes treated with TmCNP for 24 h was collected and lysed for protein analysis (PPARγ and cEBPα). The mRNA levels of intracellularly expressed TRPV1, UCP1, and Cyto C were determined using a reverse transcription kit. Primers applied for Q-PCR assays are listed in Table S1. Lipid droplets were stained with BODIPY green fluorescent dye and oil red O, and visualized via fluorescence microscopy. The mtDNA copy number was determined by Q-PCR, using the nuclear-encoded gene B2M as an internal reference.
For intracellular ROS analysis, the induced adipocytes were treated as indicated and then incubated with fresh media containing 5 µM DCFH-DA (Beyotime Biotechnology) at 37 °C for 25 min. The cells were subsequently washed four times with PBS and captured using confocal microscopy.
Stimulation of adipocytes with palmitic acids
After 6 days of induction, the culture medium of the adipocytes was replaced with F12/DMEM supplemented with 4% serum and 0.5% fatty acid-free BSA. The cells were then treated with 500 µM palmitic acid (containing 10% BSA) for 4 days. Following this treatment, nanoparticles were applied to evaluate the changes in lipid droplets and triglyceride levels within the cells.
Fabrication of TmCNP-loaded cryoAGMN
AGMNs were designed and prepared using a PDMS mold. A 200 µl volume of an optimized hydrogel mixture, consisting of 5% (w/v) AlgMA and 5% (w/v) GelMA containing 0.2 mg of TmCNP, was cast into the mold and centrifuged at 3,500 rpm for 4 min to fill the needle cavities. Next, 50 µl of a 5% cold PNIPAM solution containing 2.5% PLA was evenly applied to the flat cavity of the mold and irradiated under UV light for 30 s to form a separable layer. For the back layer, 50 µl of 10 wt% AlgMA solution was uniformly applied on top of the solidified PNIPAM gel. The fabricated patch was then frozen at − 80 °C for 2 h and carefully detached from the mold using adhesive tape.
Characterization of AGMNs
The cryo-formed AGMNs were imaged using an optical microscope immediately after removal from an ultra-low temperature refrigerator. The morphologies of the needle tips marked with Alexa fluor 405 fluorescence dye, along with the FITC-mixed separating layer and the rhodamine-labeled back layer, were photographed through microscope.
Mechanical performance testing of cryo-AGMN
The mechanical performance of the cryo AGMNs was evaluated using an Instron tensile testing machine to assess their insertion capability. The microneedle was positioned flat on a stage that had been pre-cooled in a -80 °C freezer for two hours, ensuring that the needle tips were oriented upward. The device was programmed to apply a vertical force at a constant speed of 0.5 mm/min. The testing equipment automatically recorded the force exerted on the needle tips at various displacements, generating a displacement-force curve. Data from each test were recorded and subsequently analyzed.
Transdermal delivery of NPs using cryo-AGMNs in vivo
Male C57BL/6J mice were sourced from Vital River Lab Animal Technology Co. Prior to the experiments, abdominal hair of HFD mice (male, 14–16 weeks, 35–40 g) was removed using depilatory cream under anesthesia. Two cy5.5-labeled cryo-AGMNs were applied to the left and right flanks of the mouse abdomen under NIR808 light. Nanoparticle delivery was confirmed via in vivo imaging (IVIS Spectrum, Perkin Elmer) at specific time points (days 0, 1, 3, 7, and 14). We used image analysis software to identify microneedle application sites. Fluorescence intensity within the region of interest (ROI) was measured, and average radiant efficiency (photons/s/cm²/sr/µW) was calculated to quantify signal strength. The ROI values were adjusted by subtracting the fluorescence intensity of untreated skin from the same animal to minimize background signal interference.
Obesity model and treatment
In experiments aimed at treating diet-induced obesity, 6-week-old male C57BL/6J mice were gave either a high-fat diet (60% kcal; D12429) or a normal chow diet (10% kcal; D12450B) for a duration of 8 weeks.
For the obesity reversion test, HFD mice were assigned into six groups; group I (low fat diet, LFD), untreated mice on a LFD; group II (HFD), mice on an HFD; group III (HFD + Cap), mice subcutaneous injected with Cap solution (Cap content: 8 mg/kg) and fed on an HFD; group IV (HFD + TmCNP): mice subcutaneous injected with TmCNP solution and fed on HFD; group V (HFD + MN@TmCNP), mice abdominally punctured with MN@TmCNP and fed on HFD; group VI (HFD + NIR&MN@TmCNP): mice on an HFD treated with MN@TmCNP under NIR irradiation (1 W/cm² at 808 nm) for 5 min. The administration was performed at the beginning of 8 weeks of HFD feeding. Microneedle treatments were administered every four weeks, while infrared irradiation was applied every three days. Groups IV-VI: Cap content: 8 mg/kg, TmCNP: 200 mg/kg.
Body weight of the sample was weighted and recorded weekly. Metabolic measurements, including GTT and ITT, were conducted at designated time points: at 18 and 22 weeks of age for animals in the obesity treatment study. Digital images of the mice were captured at the conclusion of the experiment (22 weeks of age). After an overnight fast, the mice were sacrificed and various tissues were collected for further analysis.
TRPV1 Inhibition following MN treatment
To investigate the role of TRPV1 in mediating the metabolic effects of microneedle treatment, HFD mice were intraperitoneally injected with capsazepine (CPZ, 15 mg/kg; HY-15640, MedChemExpress) 30 min prior to each microneedle application to inhibit TRPV1 activity. Control groups received equivalent volumes of vehicle solution. At the end of the treatment period, liver and serum samples were collected to assess lipid accumulation, and scWAT was harvested for immunofluorescence staining of UCP1 expression.
Thermal photography
Digital images were acquired using thermal infrared imaging camera. After anesthesia, the mice were positioned on a table with their abdomens facing upward, approximately 20 cm from the camera, for a 5-min video recording. The temperature of the designated area in the thermographic images was automatically measured by the camera’s built-in system.
Histological analysis
Mice were euthanized at prescribed time points, and tissues including gel implants, liver, and fat pads, were separated. These tissues were washed in cold PBS, fixed in 10% neutral formalin for 20 h, then embedded in paraffin. Then, sections were cut to a thickness of 5 μm and subsequently stained with hematoxylin and eosin. After mounting with neutral balsam, all samples were imaged and analyzed with ImageJ software.
Immunostaining
For immunohistochemical analysis of adipose tissue, sections were initially deparaffinized, rehydrated. Subsequently, heat-induced antigen retrieval was performed to enhance antigen detectability. After washing three times with PBS and inactivating endogenous peroxidases using a 3% H₂O₂ solution for 10 min, the sections were blocked with 5% BSA. Primary antibodies (anti-cEBPα, anti-PPARγ, and anti-TRPV1) were then incubated overnight at 4 °C. After three times with PBS, the sections were incubated with secondary antibodies at a 1:250 dilution of for 40 min at room temperature, followed by three washes in PBS. For cell nucleus staining, sections were mounted in hematoxylin solution for 3 min. All stained sections were imaged using a light microscope.
Serum lipid measurements
To analyze the serum lipid levels, mice were fasted overnight prior to serum collection. The serum concentrations of TCHO and TG were detected via a commercial test kit (Jiancheng, Nanjing), following the product instructions.
Liver function tests
Serum levels of aspartate amino transferase (AST) and alanine amino transferase (ALT) in mice were measured with commercial Kits (Jiancheng Bioengineering Institute) according to the manufacturer instructions.
Statistics analysis
Results are displayed as mean ± standard deviations (SD). Significant differences between groups were calculated by Student’s unpaired t-test, one-way, or two-way ANOVA (Tukey’s, Skide’s and Dunett’s multiple comparison test). Non-parametric data were analyzed by the Mann–Whitney test. *p < 0.05; **p < 0.01; ***p < 0.001 considered as statistically significant. Statistical analysis was performed with GraphPad Prism 10.0 (GraphPad Software).
