# Cationic Lipid Derived from a Basic Amino Acid: Design and Synthesis
## 4 Discussion
Additionally, liposomes, besides acting as genetic carriers, allow the condensation of genetic material on their surface, preventing degradation by nucleases [53; 54], and participate in the interaction with target cells for the efficient internalization or delivery of genetic material [55; 56]. Using the lipoplexes formed from stable liposomes, the Hek-293 FT cell line was transfected. Clear differences in gene transfer efficiency were observed based on the helper lipid used, with higher transfection efficiency achieved when DOPE was employed. In vitro studies have shown that DOPE is more efficient for gene transfer [57; 58; 59], a finding confirmed by the results of transfections with CholCadLys lipid. Furthermore, in vitro transfection assays have shown that cholesterol is the most effective helper lipid for forming lipoplexes, likely due to its role in stabilizing lipid bilayers within cellular membranes [60; 61]; however, in this work, transfection efficiency significantly decreased when lipoplexes were formed with cholesterol as a helper lipid. This may be because as the hydrophobic portion of the synthesized CholCadLys lipid is a cholesterol molecule, it cannot interact effectively with cholesterol. Cholesterol stabilizes the phospholipids and glycolipids in the plasma membrane, but two cholesterol moleculescannot associate correctly in the lipid bilayer [62; 63], resulting in less stable and less efficient lipoplexes for genetic material transfer. Since both the CholCadLys and the CholCad lipids showed good transfection efficiency, a mixture of these two CLs with DOPE was prepared to assess if efficiency could be increased. The transfection efficiency was found to be up to 40%, which was very similar to that obtained with commercial liposomes LipofectamineTM 2000 (Thermo Fisher Scientific) (used as a comparison method). This outcome may be attributed to stabilizing the liposome bilayer surface through electrostatic interactions between DOPE and the synthesized CLs, leaving two positive charges on the surface, enhancing the electrostatic interaction with DNA, and improving the transfection process. Nevertheless, the 40% transfection efficiency of ChoCadLys is significantly higher than that reported for other cationic lipids. For instance, DOTAP has been reported to achieve transfection efficiencies ranging from 9% to 17%, and DOTMA has been reported to range from 8% to 16%, when mixed with DOPE as a helper lipid [64; 65]. When comparing ChoCadLys to cholesterol-based cationic lipids such as DC-Chol, our study shows a higher transfection percentage, as DC-Chol has reported transfection efficiencies of around 35% [40]. Thus, our results remain promising for efficiently delivering nucleic acids to eukaryotic cells, surpassing commercially available cationic lipids. Our viability assays revealed that cells showed no damage with any of the mixtures containing CholCadLys lipid. Therefore, it can be asserted that the synthesized CholCadLys lipid, being composed of molecules naturally found in the cell, is not cytotoxic. Once the lipoplex is taken up by the cell, and the pDNA can migrate to the cell nucleus, the next step would be the hydrolysis of the amide and carbamate bonds by nonspecific enzymes [66; 67; 68], generating cell-native molecules (lysine, cadaverine, and cholesterol). Lysine can be used for protein synthesis, or the cell can utilize it to oxidize it to CO2 and water plus ATP. The primary amine can be used in the cell for polyamine production, and the activated cholesterol can serve as a precursor for bile salts, steroid hormones, and vitamin D, and as a structural component of cell membranes [69]. Therefore, these CLs, lacking cytotoxicity, can be considered safe for gene transfer, both in vitro and in vivo. Characterizing the lipoplexes allowed the comparison of their structural and physicochemical characteristics with TEM using negative staining. All liposomes were observed to have spherical or ovoid shapes, with heterogeneous sizes ranging from 26.4 to 126 nm.