| dc.description.abstract | The C-terminus of Indolicidin (IL) was stearylated to form amphiphilic ILs, which can self-assemble in an aqueous environment. A helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), has been combined with ILs to promote gene delivery. To elucidate the promotion effects of DOPE, we additionally investigated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), which owns a trimethylated head group, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), which owns saturated hydrocarbon tails. These 3 lipids were individually mixed with ILs in a molar ratio of 0.3:1 and denoted as ILs0.3E, ILs0.3C, and ILs0.3S, respectively. By this, we may evaluate the structure effects of head groups and tail saturation on self-assembly structure, gene delivery, and transfection efficiency. The transmission electron microscopy (TEM) results showed that ILs exhibited dendritic cylinder structures, whereas ILs0.3E and ILs0.3C formed spheres. Interestingly, ILs0.3S demonstrated both cylinder and sphere structures. The results of dynamic light scattering (DLS) indicated that incorporating helper lipids reduced the size of self-assembled carriers. Structures inferred from TEM images were evaluated by small-angle X-ray scattering (SAXS) fitting. The results indicated that ILs formed reverse micelles in core-shell cylindrical structures and aggregated to each other as bundles. The incorporation of helper lipids resulted in vesicle formation. Among them, ILs0.3E and ILs0.3C exhibited similar membrane thickness, whereas ILs0.3S vesicles demonstrated thicker membrane thickness. We speculated that stronger intermolecular forces between DSPE molecules due to their saturated hydrocarbon chain hindered their dispersion with ILs molecules in vesicle membranes. Some ILs cylinders were also retained without the addition of DSPE, so two different structures appeared in TEM images. Ethidium bromide (EtBr) displacement and electrophoresis analysis showed that smaller vesicles formed due to the incorporation of helper lipids demonstrated superior DNA complexation capacity even at low concentrations. Flow cytometry examination showed that ILs0.3E and ILs0.3C exhibited higher cellular uptake efficiency than those of ILs0.3S and ILs, and thus these two groups also demonstrated higher transgene expression. Interestingly, although the internalization efficiency of ILs0.3E and ILs0.3C were comparable, the transfection efficiency of ILs0.3E was higher than that of ILs0.3C. Therefore, we used fluorescent labeling to track DNA and carrier molecules during transfection, which were analyzed by confocal microscopy to determine their transportation pathways. All carriers can be internalized through endocytosis, but only ILs0.3E possessed an additional membrane fusion pathway. Different from endocytic DNA, which has to be released from endosomes, the fusion pathway allows DNA to directly enter cytosol for transgene expression, and thus ILs0.3E demonstrated the best transfection efficiency. Through this study, we demonstrated that peptides with helper lipid incorporation not only altered self-assembly structure but also promoted gene encapsulation and cell uptake, which eventually regulated transportation pathway and determined the transfection efficiency. These results provided useful information for peptide-lipid combo-vector development. | en_US |