The encapsulation mechanism of palmatine in anionic liposomes during flash nanoprecipitation

Liposomes, with their unique bilayer structure and biocompatibility, have attracted widespread attention in the food, pharmaceutical, and cosmetic industry. However, low production rate, spontaneous fusion, and the leakage of the encapsulated drugs (especially small hydrophilic molecules) have limited the application of such delivery vesicle. In this study, flash nano-precipitation (FNP) technology is used to achieve high throughput liposome preparation. By introducing an anionic phospholipid, dihexadecyl phosphate (DHP), the zeta-potential of liposome was largely increased to enhance the colloidal stability. Representative hydrophilic molecules (anionic tartrazine, cationic palmatine, and zwitterionic rhodamine B) were successfully encapsulated by FNP produced anionic liposomes at high encapsulation efficiency (40 % - 80 %), where, counterintuitively, the cationic palmatine showed the least encapsulation. The encapsulation mechanism of palmatine during FNP was then further investigated by quasi-crystal microbalance (QCM-D), Raman spectroscopy, and fluorescence probing. The results demonstrated that palmatine molecules, instead of adsorbing on the anionic lipid membrane surface, inserted into the lipid bilayer and interacted simultaneously with the C-N bond in the phospholipid head group and the C-C bond in the hydrophobic tail. The insertion of palmatine molecules caused the exposure of the hydrophobic region in lipid bilayer, increased the microenvironmental polarity, and enhanced the fluidity of the membrane. This study provided molecular level insight into the encapsulation mechanism of small molecules in liposome vesicles during FNP, which could provide guidelines for future liposome research and production.