Soype

POPE and 1,2-diacyl-sn-glycero-3-phosphoethanolamine from soybean (SoyPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. TMA-DPH was purchased from Molecular Probes, Inc. (Eugene, OR, USA). HEPES buffer contains 10 mM HEPES, 150 mM NaCl, 1 mM EDTA (pH 7.4). LUVs were extruded with Whatman® nuclepore polycarbonate filters (Sigma-Aldrich, St. Louis, USA) in a mini-extruder (Avanti Polar Lipids, Alabaster, USA). Turbidity was measured in a spectrophotometer (Beckam Instruments, Inc., Fullerton, USA), whereas steady-state fluorescence anisotropy measurements were made using a Horiba Jobin Yvon Spex Fluorolog 3-22/Tau 3 spectrofluorometer (Kyoto, Japan). MLVs containing 0.4 mol% TMA-DPH were obtained by co-dissolving the lipids (POPE:SoyPE 3:1), probe, and glycoside in chloroform, followed by solvent evaporation under nitrogen stream and high vacuum desiccation. Dried lipid films were then hydrated with 10 mM HEPES buffer (pH 7.4) to the desired final concentration (0.5 mM lipid, and 0/50 μM glycoside). Lipid suspensions were subjected to five cycles of freezing and thawing at 25 °C. Anisotropy was measured by exciting samples at 360 nm and collecting emission at 430 nm, using 2.7 nm slit width. Temperature was maintained with an isothermal bath and was monitored with a thermistor, inserted into the sample chamber.

Fmoc-protected amino acid derivatives, coupling reagents, and Rink amide p-methylbenzhydrylamine (MBHA) resin were purchased from Calbiochem-Novabiochem (Laufelfingen, Switzerland). Fmoc-l-propargylglycine (Fmoc-Pra-OH) was purchased from Polypeptide (Strasbourg, France). Doxorubicin hydrochloride and the other chemicals were purchased from Sigma–Aldrich, Fluka (Buchs, Switzerland), or LabScan (Stillorgan, Ireland) and were used as received, unless otherwise stated. All phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL); in particular the soybean phospholipid mixture is composed of Soy PC (3.8 mg) Soy PE (3.0 mg) Soy PI (1.8 mg) Soy PA (0.7 mg) Soy LPC (0.7 mg) with 0.1% butylated hydroxytoluene (BHT). Culture medium DMEM, fetal bovine serum and tissue culture plastic ware were purchased from Microtech (Naples, Italy). Annexin V-FITC Apoptosis Detection Kit was purchased from eBioscience (San Diego, CA, USA). Dihydroethidium (DHE) was purchased from Sigma-Aldrich (Milan, Italy).