# Polycaprolactone Blended Cosmetic Microneedle for Transdermal Hispidin Delivery System
## 2 Materials and Methods
### Characterization
After the incubation was completed, 1 N NaOH and 10% DMSO were added to the cell pellet obtained by centrifugation at 13,000 rpm for 5 min; samples were boiled at 95 \({}^{\circ}\)C for 20 min, completely dissolving the melanin, and 150 \(\upmu\)L samples were transferred to a 96-well plate, followed by microplate reader measurement of the absorbance at 490 nm. The same conditions were repeated three times to obtain an average value and calculate the amount of melanin produced in each well. Isolation of Total RNA and RT-PCR: Total RNA was isolated with an RNeasy Mini kit ( RNeasy Mini kit, Qiagen, Hilden, USA). RNA was reverse-transcribed with an oligo (dT) primer using the Super Script First-Strand Synthesis System (Intron power complementary DNA (cDNA) synthesis kit, Intron Biotechnology, Korea). RT-PCR was performed using Recombinant Taq DNA polymerase, according to the manufacturer's instructions (2\(\times\) PCR master mix solution, Intron, Gyeonggi-do, Korea). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) level was used as a control. The following primers were used: iNOS forward 5\({}^{\prime}\)-GCAGAATGTGACCATACG-3\({}^{\prime}\) and reverse 5\({}^{\prime}\)-ACAACCTGTGGTTAAGGC-3\({}^{\prime}\); COX-2 forward 5\({}^{\prime}\)-CCACTTCAAGGAGCTTGA-3\({}^{\prime}\) and reverse 5\({}^{\prime}\)-AGTCATCTGCTACGGGAGA-3\({}^{\prime}\); GAPDH forward 5\({}^{\prime}\)-ACCACAGTCCA TGCCATCAC-3\({}^{\prime}\) and reverse 5\({}^{\prime}\)-CCACCCTGTTGTGTAGCC-3\({}^{\prime}\). The conditions for PCR were as follows: 30 cycles of 94 \({}^{\circ}\)C denaturation for 30 s, 60 \({}^{\circ}\)C annealing for 30 s, and 72 \({}^{\circ}\)C extension for 30 s. The PCR products were analyzed by electrophoresis on 2% agarose gels stained with Red safe nucleic acid staining solution (Intron). **Western Blotting:** Cell extracts were resolved by SDS/PAGE (BR456-1043, Bio Rad, Korea), transferred to a PVDF membrane (BR162-0177, Bio Rad, Seoul, Korea), and immunoblot with anti-iNOS (#2977S, Cell Signaling, Massachusetts, USA), anti-COX-2 (#12282S, Cell Signaling, USA), or anti-\(\beta\)-Actin (Ab8227, Abcam, MA, USA) antibodies. Reactive bands were detected by chemiluminescenceee using Clarity(tm) Western enhanced chemiluminescence (ECL) Substrate (BR170-5060, Bio Rad, Seoul, Korea). **Strains**: For the _Staphylococcus aureus_ (Korea Collection for Type Cultures; KCTC 1916), _Staphylococcus epidermidis_ (KCTC 1917), and _Pseudomonas aeruginosa_ (KCTC 2153) experiments, strains from the Korea Cell Line Bank were used. After adding them to the TSA (Tryptic Soy Agar) medium, the culture was enriched for 18 to 24 h at 35 to 37 \({}^{\circ}\)C. In addition, for _Escherichia coli_ (KCTC 1039), a strain from the same institution was used. After adding it to TSB (Tryptic Soy Broth) medium, the optical density (OD) value (600 nm) was fixed in a range of 0.760 to 0.802 for 18 to 24 h at 35 to 37 \({}^{\circ}\)C using the method previously described (Table 1). Measurement of Antibacterial Activity Using Paper Disc Method: The antimicrobial efficacy of each substance was measured using the paper disc method according to [PERSON] [22]. The strain in the main culture was determined by increasing the activity under the optimal culture conditions and then incubating TSB (Tryptic Soy Broth) for about 6 h in the optimal medium to fix the OD value of the culture solution at 0.8. Then, 100 \(\upmu\)L of the culture medium of each strain was evenly spread on the optimal medium in TSA (Tryptic Soy Agar); the used concentrations of hispidin were 1, 5, 10, and 20 \(\upmu\)M/disc. A sterilized paper disc (Whatman No. 5 paper, Sigma-Aldrich, St. Louis, MO, USA) was