Dimerizable Cationic Detergents with a Low cmc Condense Plasmid DNA into Nanometric Particles and Transfect Cells in Culture

Abstract

The size of condensed DNA particles is a key determinant for in vivo diffusion and gene delivery to cells. Gene molecules can be individually compacted by cationic thiol detergents into nanometric particles that are stabilized by oxidative conversion of the detergent into a gemini lipid. To reach the other goal, gene delivery, a series of cationic thiol detergents with various chain lengths (C(12)-C(16)) and headgroups (ornithine or spermine) was prepared, using a versatile polymer-supported synthetic strategy. Critical micelle concentrations and thiol oxidation rates of the detergents were measured. The formation and stability of complexes formed with plasmid DNA, as well as the size, xi-potential, morphology, and transfection efficiency of the particles were investigated. Using the tetradecane/ornithine detergent, a solution of 5.5 Kpb plasmid DNA molecules was converted into a homogeneous population of 35 nm particles. The same detergent, once oxidized, exhibited a typical lipid phase internal structure and was capable of effective cell transfection. The particle size did not increase with time. Surprisingly, the gel electrophoretic mobility of the DNA complexes was found to be higher than that of plasmid DNA itself. Favorable in vivo diffusion and intracellular trafficking properties may thus be expected for these complexes.

Gene Therapy Progress and Prospects: Nonviral vectors

“…Dauty et al 35 reported a dimerizable cationic detergent, which contains free thiol, amine and alkyl groups. This alkylated ornithinyl cysteine derivative forms a complex with plasmid DNA.…”

Actin Cytoskeleton as the Principal Determinant of Size-dependent DNA Mobility in Cytoplasm

“…To overcome the skeletal barrier, some viruses such as SV40 activate tyrosine kinase-induced signaling cascades that lead to the local dissociation of filamentous actin. Because macromolecule-sized solutes with a gyration radius (R G ) up to 30 nm are freely diffusible in the cytoplasm (7), strategies to increase DNA mobility, such as DNA compaction into spherical 30 nm-particles (37) and active transport of the DNA toward the nucleus, are predicted to enhance the efficiency of transgene delivery (38,39). In summary, using the complementary fluorescence techniques of FCS and photobleaching, we have established a quantitative mechanism for the reduced translational diffusion of large, non-complexed DNAs in cell cytoplasm and identified the actin cytoskeleton as a new barrier for non-viral gene delivery.…”

Development of plasmid and oligonucleotide nanometric particles

“…The large transfection increase observed between decane, dodecane and tetradecane gemini lipids tended to culminate for tetra-and hexadecane lipids. 10 In order to further explore the structureactivity relationship, we introduced some variations in the structure of the detergent, using another DNA-binding head group (spermine; C 14 CSper) or an unstable ester bond in place of an amide (C 14 oCOrn). The highest transfection activity was found with the C 14 oCOrn derivative (Table 1) and correlates with the apparition of a supramolecular organization similar to that of Transfectam.…”

“…C 14 COrn, C 14 CSper and C 16 COrn were synthesized as previously described. 10 The syntheses of C 14 oCOrn and DPPE-PEG 3400 (lipid-PEG) will be reported elsewhere. Lissamine rhodamine B-DPPE (lipid-Rhodamine) was from Molecular Probes (Eugene, OR, USA).…”

“…The tetradecane-based detergent exhibited both monomolecular DNA condensation and efficient cell transfection properties. 10 Here, we present the most prominent features of these nanoparticles together with ongoing efforts to increase their bioavailability.…”