Abstract: Flexible liposomes are an excellent drug delivery nanocarrier, however, the leakage of drugs from liposomes has become common technical obstacle in the industry and also hindered its further application seriously. It is very urgent and necessary to avoid or reduce the leakage of drugs from liposomes. In this work, five kinds of essential oils such as Folium Artemisiae Argyi oil (FA), Folium Eucalypti oil (FE), Arabian Jasmine oil (AJ), Syzygium Aromaticum oil (SA) and Fructus Forsythiae oil (FF) were encapsulated in the lipid bilayer of palmatine chloride (PC) loaded flexible nano-liposomes (PFL), then the optimal essential oil and its dosage level were determined by the external leakage curve of PC. The female Japanese white rabbits were used to evaluate the vaginal irritancy potential of liposomes samples. The pharmaceutical properties such as encapsulation efficiency, particle size, zeta potential, deformability and structure of liposomes samples were evaluated. In order to investigate the permeability of liposomes samples to deliver PC across skin and mucous membrane in vitro, the side-by-side diffusion cells were used. The results showed that the leakage of hydrosoluble PC from PFL was reduced at different degrees by the essential oils in the lipid bilayer of PFL, however, the reduction in leakage degree was obviously higher for FA than thoses of FE, AJ, SA and FF (P < 0.05), and the highest reduction in leakage degree was obtained when the FA and lipid mass ratio was 1:6. The encapsulation efficiency, particle size, zeta potential and deformability of PFL were not significantly changed after FA was encapsulated in the lipid bilayer of the PFL (P > 0.05), so did the lamellar structure of PFL. In addition, the transdermal and transmucosal permeability of PC were also enhanced obviously by encapsulating FA in the lipid bilayer of PFL, and there was no vaginal/vulvar irritation observed in the rabbits. In summary, the drug leakage was reduced by encapsulating suitable essential oil (such as FA) in the lipid bilayer of flexible liposomes, and the vaginal mucosa permeability were improved for the drug. These results provide a novel technique in the improvement of flexible nano-liposomes for drug delivery.