Ethanol

The following compounds were used for the preparation of betulin, and the synthesis of its derivatives: 4-dimethylaminopyridine (DMAP), acetic anhydride (Ac₂O), carbon tetrachloride (CCl₄), tetrahydrofuran (THF), dichloromethane (DCM), palladium on carbon (10% Pd/C), pyridinium chlorochromate (PCC), 3-chloroperbenzoic acid (mCPBA), lithium aluminum hydride (LiAlH₄), tert-butyl(chloro)diphenylsilane (TBDPSCl), purchased from Sigma Aldrich (Steinheim am Albuch, Germany), KOH, hexane, methanol, ethyl acetate, acetone, MgSO₄, ethanol, purchased from Chempur (Piekary Śląskie, Poland). All reagents were of analytical grade.
For the determination of the antioxidant activity and to assess the skin permeation of the compounds obtained: acetonitrile for HPLC was from J.T. Baker (Berlin, Germany). 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TE), were purchased from Sigma Aldrich (St. Louis, MO, USA); Folin–Ciocalteu reagent and gallic acid (GA), were from Merck (Darmstadt, Germany), potassium persulfate and chloride, sodium chloride, methanol, ethanol, acetone and phosphate buffered saline (PBS, pH = 7.4) were purchased from Chempur (Piekary Śląskie, Poland). All reagents were of analytical grade.

Total RNA was extracted from adipocytes using TriReagent (Sigma-Aldrich). Briefly, cells were harvested, centrifuged, and 1 mL of TriReagent was added to the cell pellet. Cells were incubated for 8 min on ice; next, 200 μL of chloroform (Sigma-Aldrich) was added, and samples were mixed vigorously and centrifuged for 20 min at 4 °C. The aqueous phase was transferred to a new Eppendorf tube and RNA was precipitated with isopropanol (Sigma-Aldrich) and centrifuged at the maximum speed for 15 min at 4 °C. The RNA pellet was washed with 70% of ethanol (Chempur, Karlsruhe, Germany), dried, and resolved in molecular biology-grade water. cDNA was obtained using the High Capacity Reverse Transcription Kit (ThermoFisher, Waltham, MA, USA). Gene expression was analyzed in Real-Time PCR with the use of Fast SYBR Green Master Mix (ThermoFisher). Primers were designed manually and the efficiency of primers was checked using the standard curve. For all sets of primers, the efficiency was close to R = 99%. The specificity of Real-Time PCR was checked with the use of melting curve analysis. The sequences and efficiency data of primers were published previously [21 (link),22 (link)]. Normalization was carried out to the housekeeping gene (β-actin) and calculated according to the ΔΔCt algorithm.

Three types of polymers were used for fabrication of nanofibers and films: Poly(caprolactone) (PCL) (Sigma-Aldrich (Saint Louis, MO, USA), Mw = 80,000 g/mol, Tg = −60 °C), poly(l-lactide) (PLLA) (PL49, Corbion (Amsterdam, The Netherlands,), inherent viscosity = 4.9 dL/g, Tg = 58 °C), and poly(l-lactide-co-caprolactone) (PLCL) 70:30 (Resomer^® LC703S, Evonik (Weiterstadt, Germany), inherent viscosity = 1.3–1.8 dL/g, Tg = 32–42 °C). Solvents—acetic acid (AA) (purity degree 99.5%), formic acid (FA) (purity degree ≥ 98%), and hexafluoroisopropanol (HFIP) (purity degree 98.5%) were purchased from Poch (Gliwice, Poland), Sigma-Aldrich (Saint Louis, MO, USA), and Iris Biotech GmbH (Marktredwitz, Germany), respectively. Ethylenediamine (purity degree 99.5%), isopropanol (purity degree 99.8%), ninhydrin (purity degree 99%), and ethanol (purity degree 99.8%) were purchased from Chempur (Piekary Śląskie, Poland). For PCL and PLLA, glass transition temperatures were determined experimentally. Glass transition temperature of PLCL was assumed as stated by the producer.