L α phosphatidylcholine from egg yolk

L-α-phosphatidylcholine from egg yolk (EPC), cholesterol (Ch), didodecyldimethylammonium bromide (DDAB), and cholesteryl hemisuccinate (Chems) were provided by Sigma–Aldrich (Barcelone, Spain). Stearylamine (SA) was purchased from Fluka (Munich, Germany). Timolol maleate (TM), acetazolamide (Acz), and dodecylsulfate were purchased from Acofarma (Barcelone, Spain). (2-hydroxy)propyl-β-cyclodextrin (HPβCD, Eur. Pharm. 5th ed., D.S. 6.3) was supplied by Roquette (Lestrem, France). Acetonitrile (ACN), trichloromethane, methanol, ethanol, and 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (Hepes) were obtained from Panreac Química (Barcelone, Spain). Polycarbonate membranes (800 and 200 nm in pore size) were acquired from Millipore (Dublin, County Cork, Ireland). All other chemicals used were of analytical degree.

l-α-phosphatidylcholine from egg yolk, 18-β-glycyrrhetinic acid (GA), and all the solvents were purchased from Sigma-Aldrich (Milan, Italy). Sodium hyaluronate (molecular weight = 800–1200 kDa) was sourced from Farmalabor (Canosa di Puglia, Italy). Spanish Broom dressing was provided by Prof. Giuseppe Chidichimo, from University of Calabria (Arcavacata di Rende, CS, Italy). Flax and hemp dressings were obtained from Linificio e Canapificio S.r.l. (Villa D’Almè, Bergamo, Italy). All other chemicals were purchased from Carlo Erba (Milan, Italy). Mouse 3T3 fibroblast cells were from the American Type Culture Collection (ATCC; Rockville, MD, USA). All reagents for cell culture were obtained from Sigma-Aldrich (St. Louis, MO, USA), if not otherwise specified, and were ultrapure grade. Roswell Memorial Park Institute (RPMI-1640) medium, fetal bovine serum (FBS), and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Gibco-Life Technologies (Carlsbad, CA, USA). All plastic supports were purchased from Falcon, BectonDickinson (Franklin Lakes, NJ, USA). Phosphate buffer solution at pH 7.4 (PBS) was prepared with the following composition: 2.38 g/L Na₂HPO₄ × 12 H₂O, 0.19 g/L KH₂PO₄, 8 g/L NaCl. For GA determination, a phosphate buffer with 9.15 g/L Na₂HPO₄ × 12 H₂O, adjusted at pH 7.0 with H₃PO₄, was also prepared.

Sunitinib malate, hydrogen peroxide (H₂O₂), l-ascorbic acid (AA), hydrochloric acid (37% w/w), disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium hydrogen carbonate, pepsin from porcine gastric mucosa, esterase from porcine liver, α-amylase from porcine pancreas, pancreatin from porcine pancreas, sodium cholate, bile extract porcine and l-α-phosphatidylcholine from egg yolk were purchased by Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO).
All solvents were reagent-grade or HPLC-grade and provided by Carlo Erba Reagents (Milan, Italy).
Dialysis tubes MWCO: 3500 Da and 12 000–14 000 Da were provided by Spectrum Laboratories Inc (Rancho Dominguez, CA).
IR spectra were recorded as films or KBr pellets on a Jasco FT-IR 4200 (Easton, MD). Absorption spectra were recorded with a Jasco V-530 UV/Vis spectrometer (Easton, MD).

Ringer buffer saline (pH = 7.4) was prepared using the following reagents: NaCl (Sigma) 100 mM, KCl (Sigma) 2 mM, CaCl₂ (Sigma) 1.08 mM, HEPES (Sigma) 10 mM. ATP, adenosine, and MBCD (Methyl-β-cyclodextrin) were purchased from Sigma as powders and dissolved in Ringer buffer at the beginning of experiments. Cholesterol (Sigma) and L-α-Phosphatidylcholine from egg yolk (Sigma) were dissolved in ethanol and used for preparation of lipid films.

The quality control compounds atenolol, carbamazepine, coumarin, norfloxacin, and ranitidine hydrochloride (Sigma-Aldrich) of known intestinal permeability were used to validate the analysis set. Stock solutions of the reference drugs were prepared in DMSO (Sigma-Aldrich) at 10 mm and then diluted to reach the concentration of 500 μM in PBS of pH = 7.4, so that the concentration of DMSO does not exceed 5% of the total volume. The acceptor 96-well microplate (MultiScreen®, catalog no. MASSACCEPTOR from Millipore) was filled with 180 μl of pH = 7.4 PBS solution containing 5% of DMSO. The donor 96-well plate (MultiScreen® IP Sterile Plate PVDF membrane, pore size of 0.45 µm, catalog no. MAIPN4510 from Millipore) was coated with 5 μl fresh solution of L-α-phosphatidylcholine from egg yolk (Sigma-Aldrich) in dodecane (20 mg ml⁻¹) and left at 70°C for 5 min.
Then, it was filled with drug solution (180 ul per well). The obtained “sandwich” was left under constant slight shaking (50 rpm) overnight at 30°C.

All solvents used, including water, were Optima LCMS grade (Thermo Fisher Scientific, Scoresby, VIC, Australia). Butylated hydroxytoluene (BHT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium chloride and sodium acetate were analytical grade and purchased from Ajax Chemicals (Auburn, NSW, Australia). Industrial-grade compressed oxygen was purchased from BOC (Cringila, NSW, Australia). Synthetic glycerophospholipid standards PC 18:1(9Z)/18:1(9Z) (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine); PC 16:0/18:1(9Z) (1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine); PC 18:1(9Z)/16:0 (1-(9Z-octadecenoyl)-2-hexadecanoyl-sn-glycero-3-phosphocholine); PC 18:0/18:1(9Z) (1-octadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine); and PC 18:1(9Z)/18:0 (1-(9Z-octadecenoyl)-2-octadecanoyl-sn-glycero-3-phosphocholine) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). L-α-Phosphatidylcholine from egg yolk was purchased from Sigma-Aldrich (St. Louis, MO, USA) and all biological tissues used for extractions and tissue sections were purchased from Kieraville Butchery (Kieraville, NSW, Australia). Where available, biological replicates were obtained and these details are provided as Supporting Information (see footnote to Table S1).