Cholesterol

Liposomes consisted of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (Lipoid, Steinhausen, Switzerland), 1,2-didodecanoyl-sn-glycero-3-phosphocholine (Lipoid), and cholesterol (Sigma-Aldrich). Liposomes were prepared using the thin-film hydration method from a lipid mixture of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, 1,2-didodecanoyl-sn-glycero-3-phosphocholine and cholesterol at 1:1:1.33 molar ratio, respectively, as described (20 (link)). Particle diameter and surface charge (ζ-potential) were measured by dynamic light scattering using Malvern Zetasizer (Malvern Instruments, Malvern, United Kingdom). Fluorescent liposomes (PSOG488-liposomes) were generated adding lipid-conjugated fluorescent dye Oregon Green 488 (OG488) 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (ThermoFisher Scientific, Waltham, MA, USA) and had a lipid concentration of 30 mmol/L, a diameter size of 836 ± 217 nm (mean±SD), and a ζ-potential of −38.90 ± 2.52 mV. PS-liposomes encapsulating human insulin chains A (PSA-liposomes) and B (PSB-liposomes), were prepared as described (21 (link)) with the following physicochemical features: PSA-liposomes were 30 mmol/L of lipid concentration, 783 ± 104 nm of diameter and −38.15 ± 1.20 mV of ζ-potential; PSB-liposomes had 30 mmol/L of lipid concentration, 1040 ± 71 nm of diameter and −38.05 ± 0.07 mV of ζ-potential.

Potassium antimony (III) tartrate hydrate, here referred to as SbIII, and Diestearoylphosphatidylcholine were obtained from Sigma (Sigma-Aldrich, USA). A commercially available sterile solution of ascorbic acid, at 100 mg/mL, was obtained from Hypofarma, Brazil. Cholesterol was obtained from Lipoid GmBh, Germany.

F96 was synthesized following a previous reported method.^{9 (link)} Hydrogenated soybean phosphatidylcholine (HSPC) and 1,2-distearoyl-sn-glycero-3-phosphoglycerol, sodium salt (DSPG), and cholesterol were obtained from Lipoid (Ludwigshafen, Germany). Phosphate buffered saline tablets were from Sigma Chemical Co. (St Louis, MO, USA). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) was purchases from Thermo Fisher Scientific (Indianapolis, IN, USA). Dextran sulfate sodium (DSS) with an average molecular weight of 36 000–50 000 was purchased from MP Biomedicals, LLC (Solon, OH, USA). All other chemicals were of analytical grade unless otherwise stated in the text.