Objective To prepare liposomes co-loaded with shikonin and ligustilide (Lip@Shi/Lig), optimize its preparation process, and to investigate its characterization and transdermal performance in vitro. Methods The CCK-8 assay and Synergy Finder analysis tool were used to determine the optimal combination ratio of shikonin and ligustilide based on cell viability. Lip@Shi/Lig was prepared using the thin-film dispersion method, and the optimal formulation of Lip@Shi/Lig was screened through single-factor investigation using encapsulation efficiency as the evaluation criterion. The quality evaluation of Lip@Shi/Lig was conducted using characterization methods, including appearance and morphology observation, particle size, polydispersity index (PDI), ζ potential determination, X-ray diffraction (XRD) analysis, and Fourier transform infrared spectroscopy (FT-IR) analysis. Finally, the transdermal permeability and dermal retention performance of Lip@Shi/Lig were investigated using the Franz diffusion cell method. Results The optimal combined ratio of shikonin and ligustilide was 1∶1. The optimal conditions were determined as follows: egg yolk lecithin concentration of 10 mg/mL, phospholipid-cholesterol ratio of 4∶1, total drug-phospholipid ratio of 1∶15, and ultrasonication time of 5 min. The resulting Lip@Shi/Lig exhibited regular, spherical vesicle morphology, with encapsulation efficiencies of shikonin and ligustilide at (98.16 ±0.67)% and (97.20 ±0.76)%, respectively, particle size of (88.62 ±0.26) nm, PDI of 0.246 ±0.013, and ζ potential of (-36.57 ±1.65) mV. XRD and FT-IR results indicated that shikonin and ligustilide were successfully encapsulated in the liposomes. The cumulative penetration of shikonin and ligustilide in Lip@Shi/Lig within 30 h were (82.97 ±0.72) μg/cm² and (81.57 ±3.59) μg/cm², respectively, with dermal retention rates of (6.12 ±0.18) μg/cm² and (8.08 ±0.04) μg/cm², respectively, both of which were significantly higher than those of the single drug and the single-drug-loaded liposomes. Conclusion Lip@Shi/Lig was successfully prepared with favorable transdermal properties and stability. It significantly enhanced the transdermal penetration and dermal retention of both shikonin and ligustilide, providing a solid experimental foundation for further in vivo studies and potential clinical applications.

shikonin  /  ligustilide  /  co-loaded liposomes  /  drug combination  /  transdermal absorption  /  cell viability  /  film dispersion method