# Polycaprolactone Blended Cosmetic Microneedle for Transdermal Hispidin Delivery System
## 2 Materials and Methods
### Characterization  
Mechanical properties and microstructure of MNs: The material characterization of PLA/PCL was performed using various material analyses. Tensile strength was measured using a Universal Testing Machine (LR10K Plus, LLOYD, Steyning way, BR, UK). For tensile tests, PLA/PCL blends were poured into the sampling molds and cooled for 48 h. To determine the best properties, fabricated samples were stretched at room temperature to determine the maximum strain until fracture. After extensive tests, we obtained the stress-strain curve of each sample. Scanning electron microscopy (SEM, APREO, FEI, Hillsboro, OR, USA) was used to confirm the suitability of the coating process. We also measured the thickness of the materials by analyzing the cross-sectional area of coated MNs. Hispidin delivery test: We estimated the absorbance using Ultraviolet-Visible-Near Infrared (UV-Vis-NIR) spectrophotometry (Lambda 750, Perkin Elmer, Waltham, MA, USA). The characteristic peak of hispidin was measured by estimating a standard curve. We estimated the absorbance of each concentration (0.12, 0.1, 0.08, 0.06, 0.04, and 0.02 wt.%) of hispidin and constructed a standard curve of hispidin. Agarose gel was used to measure the permeability of hispidin into the skin. Agarose gel is a homogeneous and semi-clear substance which mimics porcine skin for skin transitional substances [21]. The coated MNs were applied to the agarose gel (3 g) for 5 min with hand pressure. After MNs were removed, agarose gel was dissolved into DMSO (111 mL). The amount of hispidin in DMSO was measured by UV-Vis, and the permeability of hispidin was estimated using a standard curve by comparing with a coated amount of pristine hispidin. Cell Culture and Viability: B16F10 melanoma cells and Raw 264.7 murine macrophage cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (GIBCO Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured in a 5% CO\({}_{2}\) incubator maintained at 37 \({}^{\circ}\)C. An MTT assay was used to ascertain the cytotoxicity of the three mixed components. B16F10 melanoma cells were dispensed into a 96-well plate at a cell number of 1 \(\times\) 10\({}^{4}\) per well; the samples were added by concentration and incubated in a CO\({}_{2}\) incubator at 37 \({}^{\circ}\)C for 24 h. RAW264.7 macrophage cells were seeded into 96-well plates at a concentration of 5 \(\times\) 10\({}^{4}\) cells per well, with different concentrations (0 \(\mu\)g/mL to 50 \(\mu\)g/mL) of hispidin, PLA, and PCL (10 to 10,000 ppm) and treated for 24 h. Subsequently, a solution of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide (MTT; Quanti-Max TM, Biomax, Seoul, Korea) was added to each well and then incubated in an incubator (37 \({}^{\circ}\)C, 5% CO\({}_{2}\)) for 4 h. A UV MAX kinetic microplate reader (Synergy MT microplate reader, BioTek, Winooski, USA) was used to measure the absorbance at 450 nm. The experiment was conducted three times under the same conditions. NO Production and Melanin Content Assay: The RAW264.7 cells were treated with hispidin, PLA, and PCL, as well as stimulation with LPS (lipopolysaccharide, Sigma Aldrich, St. Louis, MO, USA). NO generation was determined using Griess reagent (nitric oxide plus detection kit, Intron Biotechnology, Gyeonggi-do, Korea), with nitrite production measured according to the absorbance at 540 nm. Melanin Content Assay: B16F10 melanoma cells were dispensed into a six-well plate at 2 mL and incubated for 48 h in DMEM solution containing 10% FBS. Then, 50 nM of \(\alpha\)-MSH was added and incubated for 72 h at 37 \({}^{\circ}\)C. One day earlier, the samples were incubated for 24 h at 37 \({}^{\circ}\)C at each concentration.