Multi-lamellar self-assembly of cationic liposomes by epidermal growth factors

Abstract

Liposomes are biodegradable, non-toxic, and are used for intercellular delivery of proteins. However, the proteins are prone to degradation in organic solvents or dramatic temperature changes during the liposome preparation processes. In addition, controlling the size distribution of the liposomes incorporating the proteins is difficult, and the loading efficiency is only around 10 to 20 %, which is undesirable since proteins tend to be expensive. To solve these problems, I developed multi-lamellar formations using self-assembly by using the interactions between lipid membranes of the liposomes and polyelectrolytes in solution. Multi-lamellar complexes have been researched which protect intercalants from outer environment and exhibit dense alignment, inducing high loading efficiency of the intercalants. Because of the various charges of the lipids and the isoelectric points of the proteins, electrostatic attractions could be induced by the charge difference between the liposomes and the proteins. Here I demonstrated the formation of multi-lamellar complexes with membrane conformation of the cationic liposomes mediated by the anionic proteins in a layer-by-layer manner. DLS showed that the EGF/DOTAP complexes had narrower size distributions compared to the DOTAP liposomes. The dispersion stability of the EGF/DOTAP complex was retained for 100 days. The loading efficiency measured by BCA assay and ELISA were shown to be more than 95%. Molecular dynamics simulation was used to calculate the possibility of compact alignment of the EGFs on the DOTAP liposome membrane. FRET showed that the distance was 4 to 5 nm. To investigate the structure of the EGF/DOTAP complexes, the thicknesses of the DOTAP membranes and aqueous layer including the EGFs were calculated by cryo-TEM images. In addition, the mechanism of intercalation-aided formation of EGF/DOTAP complexes was investigated using Langmuir trough, which is used for analyzing the lipid surface changes by interactions between the lipid membrane and the proteins. Finally, the biological stability with structural and chemical changes of the EGF extracted from the complexes was shown to be more stable compared to the EGF in solution.