RESUMO
We evaluated the transfection efficiency of five different cationic liposome/plasmid DNA complexes, during the in vitro gene transfer into human epithelial tracheal cell lines. A dramatic correlation between the transfection efficiency and the charge ratio (positive charge of liposome to negative charge of DNA) has been found. DC-Chol-DOPE was found to be the most effective liposome formulation. Therefore, a morphological and structural analysis of DC-Chol-DOPE liposomes and DC-Chol-DOPE/DNA complexes, has been performed by transmission electron microscopy (TEM) and by confocal laser scanning microscopy (CLSM), respectively. The process of interaction between DC-Chol-DOPE/DNA complexes and human epithelial tracheal cells has been studied by CLSM. These results raise some issues for in vivo gene therapy.
Assuntos
Colesterol/análogos & derivados , DNA/química , Compostos Orgânicos , Fosfatidiletanolaminas/química , Transfecção/métodos , Benzotiazóis , Linhagem Celular Transformada , Colesterol/química , DNA/farmacologia , Diaminas , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Corantes Fluorescentes , Terapia Genética , Humanos , Lipossomos/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica , Quinolinas , Traqueia/metabolismoRESUMO
Large phosphatydilcholine unilamellar vesicles appear to be suitable controlled and protective delivery systems of beta-galactosidase. Kinetic measurements carried out on intact loaded liposomes show that most of the enzyme is entrapped inside the liposomes and its activity is latent. Nevertheless, intact liposomes also show significant activity, which can be controlled by addition of detergent. At sublytic detergent concentrations, liposome enzymatic activity reaches values two or three times greater than those of intact liposomes. This increase seems to be due to membrane structure modification that also enhances the substrate permeability across the bilayer. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 261-266, 1997.
RESUMO
The cellular uptake and distribution of cationic liposomes Dc-Chol/DOPECFTR gene complexes were assessed by electronic and confocal laser scanner microscopy (CLSM) for the CFTR gene transfer to human adenocarcinoma and tracheal epithelial cell lines. Cationic lipid forms unilamellar and multilamellar vesicles capable of rapid and efficient transport of gene into target cells. The number of fluorescent complexes was increasing with time in cells up to 6 hours showing a punctate and homogeneous DNA distribution in the cytoplasmatic and nuclear compartments, including the nucleolus. No significant difference in the biochemical and cellular behavior was observed between the investigated system and other systems previously tested. This study adds new insights into the CFTR cationic liposome-mediated gene delivery.