Preparation of liposomes

Liposomes were prepared according to the method of dried lipid film hydration. Briefly, 96 mg L-α-phosphatidylcholine (EPC) (Avanti Polar Lipid, Alabama, USA) and 24 mg cholesterol (molar ratio of 2:1) (and 24 mg loperamide HCl) (Sigma-Aldrich, Sydney, Australia) were solubilized in 6 ml chloroform:methanol (2:1, v/v) in a 50 ml round bottomed flask and dried by rotary evaporation under reduced pressure (100 mbar; 10 min; 37°C). In addition, 60 μl of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen, Victoria, Australia) was added to tag the liposomes pink so that an even dispersion in the gel could be visually gauged. The resultant thin lipid film was hydrated with the addition of 6 ml of sterile phosphate buffered saline (PBS; pH 6.5) and resuspended in a 37°C water bath. The resultant multilamellar liposomes were then reduced in lamellarity and size to 100 nm via probe sonification (60 amps, 10 mins, 37°C). The size distribution of the liposomal dispersion was determined by dynamic laser light scattering (Zetasizer Nano S™, ATA Scientific). Unencapsulated drug was removed from the liposome suspension using Slide-A-Lyser dialysis cassettes with a 10 kDa MWCO (Thermo Fisher Scientific, Scoresby, Victoria) at 4°C. Encapsulation efficiency (EE%) was determined by disrupting the vesicles with ethanol and evaluating loperamide HCl concentration using HPLC. Loperamide-encapsulated liposomes had a mean particle size of 102 nm and a polydispersity index of 0.203. The size and polydispersity of the control liposome formulation was similar. A low PDI (<0.3) signifies that the mean particle size is an adequate indicator of the size variance in the entire sample. This procedure resulted in high loperamide HCl encapsulation efficiency of >99%, which equated to 3.86 ± 0.068 mg (mean ± SD) of loperamide HCl encapsulated in each milliliter of the liposome suspension. Drug release assays for the loperamide-encapsulated liposomes have previously been conducted with consistent results (Hua, 2014; Iwaszkiewicz and Hua, 2014). Liposomes were stored at 4°C and were used within 14 days. Our laboratory has previously confirmed that the liposomes are stable in size, polydispersity, and loperamide concentration over this time period (Iwaszkiewicz and Hua, 2014). All chemicals and solvents were of at least analytical grade.