RESUMO
Dendritic poly(L-lysines) (DGL) constitute promising nanomaterials applicable as a nonviral gene-delivery vector. In this study, we evaluate the transfection abilities of four DGL generations with special emphasis on the systematic description of the relationship of how generation (i.e., molecule size) affects the transfection efficacy. Using Hep2 cells, we demonstrated that the capability of unmodified DGL to deliver plasmid is of a magnitude lower than that of jetPEI. On the other hand, employing the Hep2 cell line stably transduced with eGFP, we observed that DGL G5 delivers the siRNA oligonucleotide with the same efficiency as Lipofectamine 2000. In further experiments, it was shown that DGL affords excellent ability to bind DNA, protect it against DNase I attack, and internalize it into cells.
Assuntos
Polilisina/química , Polilisina/farmacocinética , Transfecção/métodos , Sequência de Bases , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Desoxirribonuclease I/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Lipídeos , Dados de Sequência Molecular , Oligonucleotídeos/metabolismo , Oligonucleotídeos/farmacocinética , Plasmídeos/farmacocinética , RNA Interferente Pequeno , Relação Estrutura-AtividadeRESUMO
Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported. We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5' end. All ONs were 3' labeled with fluorescent Cy 5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization. The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 microM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours.