Niacinamide

(−)-Epicatechin
of ≥90% and (+)-catechin hydrate of ≥96% purities were
purchased from Sigma-Aldrich (Saint Louis, MO) and recrystallized
from ethanol. Barbituric acid, 5-methylo-1H-benzotriazole,
4-(methyloamino)-pyridine, 5-methylbenzimidazole, acetamide, salicylamide,
niacinamide, isonicotinamide, caffeine, glutarimide, and the solvents
purchased from Sigma-Aldrich were of analytical grade.
The cocrystal
synthesis: Catechins and the BTA coformer in 1:1 and 2:1 molar ratios
were dissolved in ethanol at 30 °C. Colorless crystals were obtained
by slow evaporation of the solvent. The procedure lasted for 2 days
at room temperature. Crystals were collected from the crystallization
vessels.

Poly(vinyl alcohol) (PVA, Mw 13,000–23,000, 87–89% hydrolyzed), sucrose, sulforhodamine B (SRB), poly(methyl methacrylate) (PMMA), fluorescein sodium salt (FSS), and niacinamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethyl Acetate was purchased from Daejung Chemicals and Metals (Daejeon, Korea). Hard-polydimethylsiloxane was purchased from Dow Corning (h-PDMS, MS-10021, Dow Corning, Midland, MI, USA). Porcine skin was purchased from Cronex (Cheongju, Korea). Milli-Q (Millipore, Burlington, MA, USA) water was used for all the experiments.

Niacinamide and polyethylene glycol 400 (PEG 400) were purchased from Sigma Aldrich (St Louis, MO, USA). PLGA polymers, EXPANSORB^® DLG 50-2A (1:1 lactide/glycolide ratio; molecular weight (MW) range: 15–30 kDa) and EXPANSORB^® DLG 50-8A (1:1 lactide/glycolide ratio; MW range: 80–130 kDa) were kindly donated by Merck KGaA (Darmstadt, Germany). Dr. Pen™ Ultima A6 (MN device) was purchased from Dr. Pen Inc. (San Jose, CA, USA). Porcine ear skin was obtained from a local slaughterhouse (Atlanta, GA, USA). Dimethyl sulfoxide (DMSO, Procipient^®) was obtained as a sample from Gaylord Chemical (Slidell, LA, USA). Fluoresoft^® was purchased from Alden Optical (Lancaster, NY, USA). Potassium phosphate dibasic, phosphate-buffered saline 10× solution, HPLC grade acetonitrile, and orthophosphoric acid were purchased from Fisher Scientific (Bridgewater, NJ, USA). HPLC grade methanol was obtained from Medsupply Partners (Atlanta, GA, USA).

Isotopically labelled R15B^414-613 was overexpressed in M9 media (6 g/l Na₂HPO₄, 3 g/l KH₂PO₄, 0.5 g/l NaCl) supplemented with 1.7 g/l yeast nitrogen base without NH₄Cl and amino acids (Sigma-Aldrich, Y1251). 1 g/l ¹⁵NH₄Cl and 4 g/l ¹³C-glucose were supplemented for ¹⁵N and ¹³C labelling respectively. Additionally, filtered-sterile solutions of 2 mM MgSO₄ (final concentration in M9 media), 25 mg FeSO₄.7H₂O, and 1 ml vitamin mix (100 ml vitamin mix stock solution contains riboflavin 100 mg (Sigma-Aldrich, R9504), niacinamide 100 mg (Sigma-Aldrich, N5535), pyridoxine hydrochloride 10 mg (Sigma-Aldrich, P9755), thiamine 100 mg (Sigma-Aldrich, T1270) were added to the media. Cells were grown in M9 media as described for LB media above with the following exception: cells were grown at 37 °C until OD₆₀₀ 0.6 before induction of protein expression with 200 μM IPTG for 18 hours at 22 °C. Isotopically labelled protein was purified as described above for the native protein.

The bicinchoninic acid (BCA) protein assay kit was from Thermo (Waltham, MA, USA). The protease inhibitor cocktail was from Roche (Basel, Switzerland). Tricustin A (TSA), niacinamide (NAM), and sodium butyrate were from Sigma-Aldrich (St. Louis, MO, USA). The acetylated peptide enrichment kit was from Cell Signaling Technology (Danforth, MA, USA). Sep-pak C₁₈ packings were from Waters (MA, USA). The Fast mutagenesis system was from TransGen Biotech (Beijing, China). Nickel-nitrilotriacetic acid (NTA) agarose and QIAquick PCR purification kits were from Qiagen (Dusseldorf, North Rhine-Westphalia, Germany). Restriction endonucleases were from New England BioLabs (Ipswich, MA, USA). Goldview nucleic acid gel stain was from Biomed (Beijing, China).

For sample extraction, 0.5 g of sample was extracted with 1 mL of ultra-pure H₂O for 20 min in an ultrasonic bath (SONOREX, Bandelin, RK 103H, Darmstadt, Germany) at room temperature. The samples were centrifuged (Microcentrifuge Hettich D-78532, Germany) at 11,000× g rpm for 2 min, then the supernatant was filtered through a 0.45 µm cellulose filter.
A Vanquisher H UHPLC (ultra-high performance liquid chromatography) system from Dionex (Thermo Fisher Scientific, Germany) with a DAD detector was used for the analysis of the following vitamins: thiamine (B1), riboflavin (B2), nicotinamide (B3), pyridoxine (B6), cyanocobalamin (B12). The mobile phase was composed of ultra-pure H₂O with 1% acetic acid and MeOH in gradient with a flow of 0.3 mL/min. The chromatographic column used was a Accucore aQ (100 × 2.1 mm, 2.6 mm) from Thermo Fisher kept at 25 °C. The injection volume was 8 µL and the detector was set at 270 nm. Thiamine hydrochloride, riboflavin, niacinamide, pyridoxine hydrochloride, and cyanocobalamin standards were purchased form Sigma-Aldrich, Inc. (Sydney, NSW, Australia). The dilutions were made in ultra-pure water with a 0.1–100 µg/mL linearity range. The quantification limit was 0.1 and the detection limit was 0.03 µg/mL for all analyzed vitamins.