Asymmetric liposomes prepared with a simple cyclodextrin-lipid complex method to improve performance of vesicles beyond conventional liposomes

Developing liposomes delivery system via membrane asymmetry can offer more advantages to liposomal research. This research aims to produce asymmetric liposomes via a simpler cyclodextrin-lipid complexation and exchange method compared to that first produced by E. London and co-workers. Our procedures include formulating the cyclodextrin, CD, complex with the lipid (e.g. DOTAP) i.e donner. Large unilamellar vesicles (LUV, acceptor) formulated via thin film hydration method, where the required lipids, 4mM, were dissolved in chloroform, a thin film was formed. HEPES buffer hydrated thin film. Finally, equal volumes of the cyclodextrin-lipid complex and acceptor LUV dispersion were mixed to allow lipid exchange. Then asymmetric liposomes were separated by centrifugation. Fluorescence quenching and zetapotential, ZP, methods confirmed liposomes' asymmetry. Liposomes were analysed using size and ZP analysis, UV/VIS spectrophotometry to calculate entrapment efficiency (EE), and pH gradient with Bromocresol green, BCG, used to qualitatively confirm EE. Optimised formulation containing POPC in outer leaflet and POPC (15%), DOTAP (45%), and cholesterol (40%) was used to entrap salmon sperm DNA. The EE was determined using nanodrop lite, where 6µl of DNA (10mg/ml) was added to 2mM of asymmetric liposomes at a ratio of 19.4:1 liposome to DNA. Results of ZP established the asymmetry of liposomes as when neutral lipid POPC has been used as an acceptor LUV (inner) and DOTAP was formulated as a CD-lipid complex donor (outer layer), ZP of liposomes increased from -7.1 to 33.9mV and the vice versa when the CD-lipid complex formed with POPC to be outer layer led to reducing the ZP from 23.5 to 16.2mV. Optimised formula exhibited good asymmetric membrane integrity and EE via using BCG as a visual indicator and good encapsulation of DNA, with EE of  92.5%, size of 198.48nm and ZP of 26.4mV.