ABSTRACT
Objective To develop a method to determine the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes. Methods In this paper, the thin-film rehydration method was used to prepare doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes. Liposomes and free drugs were separated by dialysis, gel microcolumn centrifugation, and ultra-high speed centrifugation. The content of free drugs and drugs in liposomes was determined by HPLC, and the entrapment efficiency of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes was calculated. Results The optimal formulation of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes was DPPC-DSPE-PEG2000-TAIII-DOX with a molar ratio of 5:1:1:1. The liposomes prepared using thin-film rehydration method had a well-defined spherical shape with a size of (55.4 ± 0.40) nm, a PDI of (0.20 ± 0.02), and a weakly negative zeta potential of (-17.4 ± 0.6) mV. The excipients in the liposomal formulation can be well separated from doxorubicin hydrochloride and timosaponin AIII in the selected chromatographic conditions. The calibrated linear curve of doxorubicin hydrochloride was within 24.9-498.0 μg/mL (r = 0.999 9) and that of timosaponin AIII was within 50.55-1 011.0 μg/mL (r = 0.999 6). Free doxorubicin hydrochloride and timosaponin AIII were well separated from liposome by gel microcolumn centrifugation, and the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AIII was (85.12 ± 1.27)% and (76.51 ± 0.46)% respectively. Conclusion The thin-film dispersion- method can be used for the preparation of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes. The method of gel microcolumn centrifugation is accurate, reproducible, simple, and suitable for determination of the encapsulation efficiency of co-loaded liposomes.