ENDOCYTOSIS AND INTRACELLULAR PROCESSING ACCOMPANYING TRANSFECTION MEDIATED BY CATIONIC LIPOSOMES

Cationic liposomes mediate efficient transfection of mammalian cells, but the manner in which cells internalize and process cationic liposom e-DNA complexes has not been well characterized. We exposed several ce ll types, including human and murine erythroleukemia cells, African gr een monkey kidney cells (CV-1), isolated rat alveolar type II cells an d alveolar macrophages to DNA-cationic liposome complexes containing N -(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and Dioleylph osphatidylethanolamine (DOPE). The morphology of liposome-cell interac tions was assessed by electron microscopy. Liposome preparations were complexed to colloidal gold particles or to both plasmid DNA and gold particles. Cells treated with DOTMA liposome-DNA complexes demonstrate d endocytosis of the liposome-DNA complexes in coated pits, which were seen in early endosomes, late endosomes, and lysosomes. In isolated a lveolar type II cells, the gold-labelled DOTMA lipid apparently mixed with the contents of lamellar bodies. In most cells, gold particles we re dispersed throughout the cytoplasmic matrix. In a small proportion of CV-I and U937 cells, a membrane system resembling the endoplasmic r eticulum developed within the nucleus. This novel structure was also p resent in nuclei after they were isolated from CV-1 cells and then mix ed with DOTMA-containing liposomes. Membranes which form after exposur e to DOTMA-containing liposomes were 10 nm in thickness as compared to the approx. 8 nm thickness of endogenous cellular membranes. Based on these morphologic observations, we propose that the main route of ent ry of cationic liposomes into cells is by endocytosis. In some instanc es, the endosomal compartment releases its cationic liposome-DNA conte nts into the cytoplasmic matrix. Occasionally, liposomes may enter the nucleus by fusion with the nuclear envelope, creating vesicular and r eticular intranuclear membranes. It is not clear at present which, if any of these morphological observations correlates with transfection m ediated by cationic liposomes.