RESUMO
Cationic liposomes are promising candidates for the delivery of various therapeutic nucleic acids. Here, we report a convenient synthesis of carbamate-type cationic lipids with various hydrophobic domains (tetradecanol, dialkylglycerol, cholesterol) and positively charged head-groups (pyridinium, N-methylimidazolium, N-methylmorpholinium) and data on the structure-transfection activity relationships. It was found that single-chain lipids possess high surface activity, which correlates with high cytotoxicity due to their ability to disrupt the cellular membrane by combined hydrophobic and electrostatic interactions. Liposomes containing these lipids also display high cytotoxicity with respect to all cell lines. Irrespective of chemical structures, all cationic lipids form liposomes with similar sizes and surface potentials. The characteristics of complexes composed of cationic liposomes and nucleic acids depend mostly on the type of nucleic acid and P/N ratios. In the case of oligodeoxyribonucleotide delivery, the transfection activity depends on the type of cationic head-group regardless of the type of hydrophobic domain: all types of cationic liposomes mediate efficient oligonucleotide transfer into 80-90% of the eukaryotic cells, and liposomes based on lipids with N-methylmorpholinium cationic head-group display the highest transfection activity. In the case of plasmid DNA and siRNA, the type of hydrophobic domain determines the transfection activity: liposomes composed of cholesterol-based lipids were the most efficient in DNA transfer, while liposomes containing glycerol-based lipids exhibited reasonable activity in siRNA delivery under serum-free conditions.
Assuntos
Antineoplásicos/farmacologia , DNA/administração & dosagem , Portadores de Fármacos/farmacologia , Compostos Heterocíclicos/farmacologia , Lipídeos/farmacologia , Lipossomos/farmacologia , RNA Interferente Pequeno/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Carbamatos/química , Cátions/administração & dosagem , Cátions/química , Cátions/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Relação Dose-Resposta a Droga , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Compostos Heterocíclicos/administração & dosagem , Compostos Heterocíclicos/química , Humanos , Lipídeos/administração & dosagem , Lipídeos/química , Lipossomos/administração & dosagem , Lipossomos/química , Estrutura Molecular , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/química , Oligodesoxirribonucleotídeos Antissenso/genética , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
New polycationic lipids corresponding to the two different classes of amphiphiles ("head-tail" and "gemini") were designed and used as components of non-viral gene delivery systems. The hydrophobic domain of lipids is based on the cholesterol residue and the hydrophilic one--on the naturally occurring polyamine--spermine. Ester and carbamate linker groups as well as oligomethylene spacers of various lengths were used to connect cholesterol and spermine motifs in order to estimate the structure-activity relationships of novel polycationic lipids and to determine an effective and safe transfectant suitable for the delivery of different nucleic acids. The cationic liposomes composed of the synthesized polycationic lipids and DOPE provided delivery of FITC-labeled oligonucleotide, plasmid DNA and siRNA into HEK293 cells with an efficiency significantly higher than that of Lipofectamine 2000. We found that the transfection activity of polycationic lipids is influenced by a linker type, a spacer length and the amount of cholesterol residues. The lipid containing two cholesterol units, carbamate linker and spacer of six methylene groups demonstrated the best in vitro transfection results among other analogues tested and was defined as a promising candidate for further transfection studies to be hold in vivo.
Assuntos
Colesterol/química , DNA/administração & dosagem , Portadores de Fármacos/química , Técnicas de Transferência de Genes , RNA Interferente Pequeno/administração & dosagem , Espermina/química , Animais , Sobrevivência Celular , Colesterol/análogos & derivados , Cricetinae , DNA/genética , Portadores de Fármacos/síntese química , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Lipossomos , Estrutura Molecular , Tamanho da Partícula , Fosfatidiletanolaminas/química , Plasmídeos , RNA Interferente Pequeno/genética , Espermina/análogos & derivados , Relação Estrutura-Atividade , Propriedades de Superfície , Tensoativos/química , TransfecçãoRESUMO
Cholesterol amphiphiles containing positively charged groups (pyridinium, N-methylimidazolium, N-methylmorpholinium, and N-methylpiperidinium) linked via ß-glucosyl spacer were prepared by alkylation of the corresponding bases with 6-Ð-mesyl-ß-D-cholesteryl glucopyranoside. IC(50) values were in the range 20-35µM for the series of compounds and liposomal formulations with DOPE (1:1) were significantly less toxic. The liposomal formulations provided the accumulation of FITC-labeled oligonucleotide in cells, and the efficiency of this process was comparable to that of Lipofectamine 2000. Cationic liposomes were able to deliver siRNA into the cells, and the liposomal formulation 7d/DOPE provided the most pronounced down-regulation of EGFP expression both in the presence and in the absence of serum (up to 30%).