DISEÑO Y SÍNTESIS DE LÍPIDOS CATIÓNICOS DERIVADOS DE LA D-LISINA Y D-ORNITINA PARA LA TRANSFERENCIA DE GENES

Abstract

Gene therapy is a therapeutic strategy based on modification of the gene repertoire of cells through the administration of nucleic acids. To achieve a specific biological effect in gene therapy, the genetic material of interest needs to be inserted efficiently in the target cell and attain expression. An innocuous genetic vector is therefore necessary, and the most promising seem to be cationic liposomes. These are formed by synthetic cationic lipids which due to their positive charge, interact electrostatically with DNA to form a stable complex (lipoplex) that permits the efficient delivery of genetic material to the cell. Commercial cationic lipids have proved effective in transfection, but they are expensive and a certain degree of toxicity is associated with them limiting their use in vivo. Innocuous cost-effective cationic lipids need consequently to be obtained. Therefore, in this study, lipids designed for gene transfer were obteinde from the amino acids D-lysine and D-ornithine as well as octadecanol (D-lysyl-octadecanol and D-ornithine-octadecanol), none of which could be cytotoxic since they are composed of molecules found normally in the cell and they are also inexpensive and easy to use. These cationic lipids were synthesized by esterification of polar amino acids and purified by silica gel prep chromatography and differential solubility. Characterization was performed with thin layer chromatography, infrared spectroscopy, and 1H and 13C nuclear magnetic resonance (NMR). The plasmid pRSVgal, into which the Lac Z gene (β-galactosidase) has been cloned as a reporter gene, was used as the genetic material. The alkaline lysis method was used to purify the plasmid, and its integrity and purity were verified by electrophoresis and absorption spectrometry. Lipoplexes were formed with cationic lipids and cholesterol or DOPE as helper lipids and plasmid DNA, in order to transfect the C-33 A cell line which was evaluated by the enzymatic activity of β-galactosidase. Cytotoxicity was determined by supravital staining with 0.2% trypan blue. Finally, lipoplexes were characterized by transmission electron microscopy and flow cytometry. The cationic lipids D-lysyl-octadecanol and D-ornithine-octadecanol were synthesized and purified, with yields of 43 and 54% respectively. These compounds were identified by thin layer chromatography as amphipathic lipids with an Rf of 0.53. In the IR spectrum, bands for the characteristic bonds of each lipid were observed, particularly those of lengthening of the C=O bond at about 1740 cm-1 and lengthening of the C-O bond near 1050 cm-1, corresponding both to the ester bond. The 1H NMR spectrum displayed hydrogen of the methylene group of the ester bond signal at 4.2 ppm while the signal for the carbonyl of the ester bond was observed at 169 ppm in the 13C NMR spectrum. These data provide certainty as to the successful synthesis of both lipids. DNA with a high degree of purity was obtained from the plasmid pRSVgal with an A260/A280 ratio of 1.9 at a concentration of 28g/mL. Up to 25% transfection efficiency was achieved with D-lysyl-octadecanol and <2% with D-ornithine-octadecanol. Neither lipid showed cytotoxicity nor did their enantiomers L-lysyl-octadecanol and L-ornithine-octadecanol. Electron microscopy showed liposomes of D-lysyl-octadecanol with DOPE as nanospheres 30 to 80 nm in diameter, while lipoplexes displayed so-called ―beads-on-string‖ structures. Flow cytometry analysis revealed no significant differences between liposomes and lipoplexes. We conclude that D-lysyl-octadecanol carries out gene transfer without inducing toxicity in cell cultures, and therefore suggest this lipid can be used with a high degree of safety in test animals and potentially in humans.