PURPOSE: We have demonstrated that a heteromolecular complex consisting of cationic lipids and DNA can be prepared and isolated (1). Cationic lipids bind DNA through electrostatic interactions. However, when sufficient lipids are bound to DNA the physical and chemical properties of the complex are governed by hydrophobic effects. Here we describe an approach where this hydrophobic complex is used as an intermediate in the preparation of lipid-DNA particles (LDPs). METHODS: The approach relies on the generation of mixed micelles containing the detergent, n-octyl beta-D-glucopyranoside (OGP), the cationic lipid, N-N-dioleoyl-N, N-dimethylammonium chloride (DODAC), and selected zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or egg sphingomyelin (SM). RESULTS: When these micelles were prepared at low detergent concentrations (20 mM OGP) and combined with pCMV beta DNA, LDPs spontaneously formed. The mean diameter of these particles as measured by quasielastic light scattering was 55-70 nm, a result that was confirmed by negative stain electron microscopy. Further characterization of these LDPs showed that DNA within the particles was inaccessible to the small fluorochrome TO-PRO-1 and protected against DNase I degradation. LDPs could also be prepared in high concentrations of OGP (100 mM), however particles formed only after removal of OGP by dialysis. Particles formed in this manner were large (> 2000 nm) and mediated efficient transfection of Chinese hamster ovary cells. Transfection activity was greater when the lipid composition used consisted of SM/ DODAC. Small particles (< 100 nm) prepared of SM/DODAC were, however, inefficient transfecting agents. CONCLUSIONS: We believe that LDP formation is a consequence of the molecular forces that promote optimal hydrocarbon-hydrocarbon interactions and elimination of the hydrocarbon-water interface.
PURPOSE: We have demonstrated that a heteromolecular complex consisting of cationic lipids and DNA can be prepared and isolated (1). Cationic lipids bind DNA through electrostatic interactions. However, when sufficient lipids are bound to DNA the physical and chemical properties of the complex are governed by hydrophobic effects. Here we describe an approach where this hydrophobic complex is used as an intermediate in the preparation of lipid-DNA particles (LDPs). METHODS: The approach relies on the generation of mixed micelles containing the detergent, n-octyl beta-D-glucopyranoside (OGP), the cationic lipid, N-N-dioleoyl-N, N-dimethylammonium chloride (DODAC), and selected zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or egg sphingomyelin (SM). RESULTS: When these micelles were prepared at low detergent concentrations (20 mM OGP) and combined with pCMV beta DNA, LDPs spontaneously formed. The mean diameter of these particles as measured by quasielastic light scattering was 55-70 nm, a result that was confirmed by negative stain electron microscopy. Further characterization of these LDPs showed that DNA within the particles was inaccessible to the small fluorochrome TO-PRO-1 and protected against DNase I degradation. LDPs could also be prepared in high concentrations of OGP (100 mM), however particles formed only after removal of OGP by dialysis. Particles formed in this manner were large (> 2000 nm) and mediated efficient transfection of Chinese hamster ovary cells. Transfection activity was greater when the lipid composition used consisted of SM/ DODAC. Small particles (< 100 nm) prepared of SM/DODAC were, however, inefficient transfecting agents. CONCLUSIONS: We believe that LDP formation is a consequence of the molecular forces that promote optimal hydrocarbon-hydrocarbon interactions and elimination of the hydrocarbon-water interface.
Authors: Sean Patrick Gordon; Svitlana Berezhna; Dag Scherfeld; Nicoletta Kahya; Petra Schwille Journal: Biophys J Date: 2004-10-29 Impact factor: 4.033
Authors: Catarina Madeira; Luís M S Loura; M Raquel Aires-Barros; Aleksander Fedorov; Manuel Prieto Journal: Biophys J Date: 2003-11 Impact factor: 4.033