# Cationic Lipid Derived from a Basic Amino Acid: Design and Synthesis
## 4 Discussion
Note that two developing reactions were used to identify the molecules, as they indicate two different characteristics: the ninhydrin development indicated the presence of free amino groups, which provide the positive charge to the cationic lipid and form its hydrophilic part; the sulfuric acid development indicated the presence of organic matter in the hydrophobic region and the general presence of organic contaminants, both in the crude reaction mixture and after purification. To confirm the presence of the CholCadLys lipid, other techniques, such as infrared (IR) spectroscopy and mass spectrometry, were necessary. IR spectroscopy identified thefunctional groups of the molecules, confirming the nucleophilic substitution between the cholesterol and the cadaverine and the amidation between the cadaverine and the lysine. Mass spectrometry identified the exact mass of the compounds and indirectly indicated their purity, showing the signal of the synthesized lipid along with other signals that could correspond to unreacted compounds or secondary compounds formed during the synthesis, such as a lysine molecule attached to a cholesteryl chloroformate molecule, a cadaverine molecule attached to a cholesteryl chloroformate molecule, and two lysine molecules (polymerized) attached to a cholesteryl chloroformate molecule. This suggests that the synthesized lipid is not completely pure. However, it is also known that the same method used for analysis can fragment the evaluated molecule, which could account for the signals detected prior to those of the molecule of interest. Cationic liposomes were formed from amphipathic CLs mixed with colipids, mainly providing stability in the liposome's lipid bilayer. The most used helper lipids are DOPE, cholesterol, or dioleoylphosphatidylcholine (DOPC). In this study, liposome stability was tested using the synthesized CL and helper lipids solubilized in PBS. Not all lipid mixtures tested formed stable liposomes. Liposome stability depends on the molecular structure of the CLs and helper lipids for the lipid bilayer to form properly. This study found that the CL formed liposomes with both cholesterol and DOPE. We observed the formation of a distinct turbid suspension, indicative of vesicles that efficiently diffract light [46]. This confirms that the lipoplexes are soluble in PBS and remain stable for up to 15 days in this medium. Such prolonged stability in PBS suggests that these lipoplexes could be well-suited for in vivo administration, as they may support a more consistent dosing regimen and reduce the need for frequent preparation. Additionally, their stability in aqueous environments enhances compatibility with biological systems, potentially improving bioavailability and efficacy in therapeutic applications. The lipid size, determined by the size of the hydrophobic and hydrophilic parts, is the major parameter for liposome stability. The geometry of the synthesized CL appears to be a thin cylinder, and DOPE's molecular geometry is conical [47; 48]. Despite DOPE being a neutral lipid, its interaction with the synthesized CL could provide stabilization through electrostatic interactions, making liposomes more stable when formed with DOPE as a helper lipid [49; 50]. With stable cationic liposomes, lipoplexes were prepared for transfecting the Hek-293 FT cell line. To perform transfection, the first step involved obtaining pDNA with high integrity and purity to ensure proper gene expression and avoid cytotoxicity due to bacterial contaminants (RNA and proteins), focusing solely on liposome-induced cytotoxicity. Lipoplexes result from the electrostatic interaction of the positive charges of the hydrophilic region of CLs with the negative charges of the phosphate groups in the genetic material (pDNA). DNA-liposome binding is crucial for efficient GT, as the electrostatic interaction determines the release and intracellular delivery of genetic material [51; 52].