# Cationic Lipid Derived from a Basic Amino Acid: Design and Synthesis
## 4 Discussion  
Genetic carriers enable the transport and protection of genetic material for transfection, as the uptake of naked genetic material by eukaryotic cells is challenging and inefficient [35; 36]. Therefore, genetic carriers that facilitate the transfection process are commonly required. Currently, the most common and safe genetic carriers are cationic liposomes; however, their main drawback is low transfection efficiency [37; 38; 39]. In this study, a CL derived from cholesterol, cadaverine linked by a carbamate bond, and lysine linked by an amide bond were synthesized to enhance transfection efficiency and avoid cytotoxicity. These molecules were chosen as they are normally part of cells, making them potentially innocuous for in vitro and in vivo applications. It is important to note that CLs derived from cholesterol have been previously used for gene delivery to eukaryotic cells with acceptable transfection efficiencies. However, most of them exhibited some degree of cytotoxicity due to the polar region they contain, preventing their establishment as effective non-viral gene delivery vectors [40; 41]. Studies have shown that cells can hydrolyze CLs used for transfection, with units linked by carbamate bonds, without causing cytotoxicity [41; 42; 43]. Additionally, the amide bond formed in this newly synthesized CL is highly recognized by the cell, as it links the amino acids constituting peptides and proteins, making its hydrolysis non-problematic for the cell. The purification of the CholCadLys lipid was achieved through differential solubility using liquid-liquid extractions with chloroform and water. In the aqueous phase, excess polar molecules (pyridine, cadaverine, and lysine) were separated, while the complete lipids and cholesterol chloroform remained in the organic phase. However, preparative chromatography was also employed to obtain a purer lipid, separating it completely from excess raw material. The yield of the reaction was around 60%, which is considered acceptable [44]. However, this could be improved by modifying certain parameters, such as temperature and the amount of raw material used, favoring the formation of the final product. Increasing the amount of raw material is a viable option due to the low synthesis cost, the use of economical reagents, and the lack of requirement for any sophisticated equipment, both for synthesis and purification, which are considered advantageous. Monitoring the synthesis and identifying the CholCadLys lipid were carried out through thin-layer chromatography on silica gel (TLC), which is essential for the analysis of lipids and other small molecules like amino acids, nucleotides, and vitamins, among others [45]. TLC is a sensitive and precise identification tool, reliable for initial correct identification. It does not require complex instrumentation, making it simple to use, providing advantages for its application. For the elution of the chromatographic plate, a solvent mixture suitable for amphipathic molecules was used as the mobile phase. Therefore, an Rf value between the point of application and the solvent front (0.8 \(>\) Rf \(>\) 0.3) indicates the presence of amphipathic molecules, such as cationic lipids. It was observed that cholesteryl chloroform, which is highly soluble in organic solvents, migrates with the mobile phase on the chromatographic plate. In contrast, L-lysine, due to its two functional groups capable of forming hydrogen bonds (the amino and hydroxyl groups, both acting as hydrogen bond donors and acceptors) and its short hydrocarbon chain, remained at the application point. Furthermore, the majority of lysine was eliminated in the aqueous phase. The presence of an intermediate spot with an Rf of 0.8 confirmed the presence of an amphipathic compound, corresponding to the cationic lipid. This chromatography also allowed a rough assessment of the lipid's purity after chromatographic elution.