Encapsulation Efficiency (EE) and Loading Efficiency (LE)

To determine encapsulation efficiency (%EE) and loading efficiency (%LE), firstly, the standard curve of DOX was drawn. The DOX solution was diluted to eight different concentrations with 5, 10, 15, 20, 25, 30, 40, and 50 μg/mL. Using the ultrapure water as blank control. The excitation wavelength of microplate reader was set at 480 nm, and the absorbance values of DOX solutions with different concentrations were measured. The standard curve of absorbance and concentration was drawn. The standard equation of DOX absorbance(y) and concentration(x) was y = 0.0222x, and the correlation coefficient R² was 0.9998, indicating that there was a good linear relationship between DOX concentration and absorbance value in the range of 0–50 μg/mL.

The procedure in detail as follows: After doxorubicin encapsulation was completed, take two copies of 200 μL DOX loaded liposome solution, one of which was adjusted to 500 μL with buffer, then added 1 mL acidic isopropanol for demulsification, and react on the oscillator for 10 min. The blank control was 500 μL HBS buffer, added with 1 mL acidic isopropanol. Then, took 100 μL samples each and added them to the 96-well plate. The wavelength of the microplate reader SH-9000 (Corona Electric, Ibaraki, Japan) was set at 480 nm. Measure the absorbance and calculate the doxorubicin content. Passing the other copy through P-10 column, eluting with HBS buffer solution with pH 7.8, collecting the liposome part, and using buffer solution to increase the volume to 500 μL, after demulsification, measuring the absorbance value with microplate reader, and calculating the DOX content, which represents the DOX content loaded by liposomes.

EE was calculated as the amount of drug loaded in the liposomes divided by the initial amount of the drug. Encapsulation efficiency (%EE) = Amount of drug loaded in liposomes/Amount of drug added × 100%. LE was calculated as the molar ratio of the drug loaded into liposomes to the total of lipids. Loading efficiency (%LE) = Amount of drug in liposomes/Amount of liposomes × 100%. The encapsulation efficiency (%EE) of DOPC-lip@DOX, DOPE-lip@DOX, DOPE-DVar7-lip@DOX were 98%, 90%, and 93%, respectively. The loading efficiency (%LE) of DOPC-lip@DOX, DOPE-lip@DOX, DOPE-DVar7-lip@DOX was 9.8%, 9.0%, and 9.3%, respectively.