Lecithin

Liposomes, which act as ideal candidates for membrane mimics, were formed as thin lipid membranes were gradually hydrated, and the accumulated crystal bilayers became a swelled liquid. Briefly, 30 mg of lecithin (99%, Avanti Polar Lipids, USA) was dissolved in 6 mL of a chloroform/methanol (2:1, v/v) mixture. The dissolved lecithin was topped with a thin lipid layer using a rotary evaporation apparatus (Heidolph, Germany). To remove any residual organic solvents, the layer was dried at 37 °C for 2 h in a high-vacuum oven. The lipid layer was then transferred to vials, sealed, bundled, and frozen. For lipid membrane hydration, 6 mL of preheated HBS-N (GE, Boston, MA, USA) was added to the liposomes to form multilamellar vesicles (LMVs) above the phase transition temperature. The liposome vesicles were immediately shaken and stirred violently, vortexed for 1 min and sonicated for 40 min above the phase transition temperatures. Finally, small unilamellar vesicles (SUVs) were prepared by extrusion. A syringe was used to extract 2 mL of preheated HBS-N and inject this volume into another syringe to wash the assembled extrusion apparatus (Avanti Polar Lipids, USA). The LMV suspension was filtered 11 times through a 50-nm polycarbonate filter (Avanti Polar Lipids, USA) between the extrusion apparatus, and the SUVs were then obtained.

Ester-terminated poly-lactic-co-glycolic acid, or PLGA (50:50, 0.82 dl/g IV, DURECT Corporation, Cupertino, CA, USA) was dissolved at 1 mg/ml in dimethylformamide (DMF). Hp91 was also dissolved in DMF with the PLGA at concentrations of 1 to 5 mg/ml. Lecithin (molecular weight (MW) 330 Da, Alfa Aesar, Ward Hill, MA, USA) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(carboxy(polyethylene glycol)2000) (ammonium salt) (DSPE-PEG-Carboxy, MW 2,849.54 Da, Avanti Polar Lipids, Alabaster, AL, USA) were dissolved together in 2 ml of 4% ethanol per mg PLGA to be used at a ratio of 9% of total PLGA weight for Lecithin and 52% of total PLGA weight for DSPE-PEG-Carboxy. All stock solutions were made using sterile solvents or endotoxin-free water. The aqueous lipid mixture was heated to 68°C while stirring for 3 min. The PLGA-peptide solution was added dropwise to the heated lipid solution while stirring. The solution was then vortexed at 3,000 RPM for three minutes. An additional 1 ml of water per mg of PLGA used was added dropwise to the NP solution while stirring. The NP solution was stirred without cap for 2 h to allow solvent evaporation. The particles were then washed three times using Amicon Ultra centrifugal filter devices by EMD Millipore (Billerica, MA, USA) with 100 Kd cutoff. Particles were suspended in 10% sucrose and flash frozen for later use.