Biophysical and structural properties of DNA.diC(14)-amidine complexes. Influence of the DNA/lipid ratio.

Cationic liposomes are used as vectors for gene delivery both in vitro and in vivo. Comprehension of both DNA/liposome interactions on a molecular level and a description of structural modifications involved, are prerequisites to an optimization of the transfection protocol and, thus, successful application in therapy. Formation and stability of a DNA/cationic liposome complex were investigated here at different DNA:lipid molar ratios (rho). Isothermal titration calorimetry (ITC) of cationic liposomes with plasmid DNA was used to characterize the DNA-lipid interaction. Two processes were shown to be involved in the complex formation. A fast exothermic process was attributed to the electrostatic binding of DNA to the liposome surface. A subsequent slower endothermic reaction is likely to be caused by the fusion of the two components and their rearrangement into a new structure. Fluorescence and differential scanning calorimetry confirmed this interpretation. A kinetic model analyzes the ITC profile in terms of DNA/cationic liposome interactions.

Liposomes composed of a double-chain cationic amphiphile (vectamidine) induce their own encapsulation into human erythrocytes.

Vectamidine is a liposome-forming double-chain cationic amphiphile. The present work was aimed to microscopically study the interactions of Vectamidine liposomes with the human erythrocyte plasma membrane. Vectamidine rapidly induced stomatocytic shapes. Attachment of Vectamidine liposomes to the erythrocyte induced a strong local invagination of the membrane. This frequently resulted in a complete encapsulation of the liposome. Liposomes composed of phosphatidylcholine (neutral) or phosphatidylserine/phosphatidylcholine (anionic) did not perturb the erythrocyte shape. Our results indicate that besides an attraction of Vectamidine liposomes to the plasma membrane, there is a preference of Vectamidine for the inner bilayer leaflet. We suggest that cationic amphiphiles may transfer from membrane-attached liposomes to the plasma membrane and then translocate to the inner bilayer leaflet where they induce a strong local inward bending of the plasma membrane resulting in an encapsulation of the liposome.

Physico-chemical characterization of a double long-chain cationic amphiphile (Vectamidine) by microelectrophoresis.

We recently synthesized a novel cationic amphiphile (N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine or Vectamidine (previously described as diC14-amidine)) that associates with DNA and RNA and facilitates their entry and expression into eukaryotic cells. Among several parameters that have been shown to influence the transfection process, the surface charge density plays a key role. Quantitative information about that charge density associated to the cationic amphiphiles organized in liposomal structure is not yet available. We provide here evidence by titration and microelectrophoresis measurements that an evaluation of the intrinsic acidity constants, the surface pH and the counterion binding constants allows to determine the charge density at physiological pH of Vectamidine liposomes. The knowledge of this superficial charge is a prerequisite to a molecular understanding of the DNA-cationic amphiphile complex formation. The method described could be extended to any kind of cationic amphiphile.

The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids.

We used a 32P-labeled pCMV-CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV-CAT plasmid DNA to quantify the CAT activity after transfection of COS cells using each of the three following cationic compounds: [1] vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, and previously described as diC14-amidine [1]), [2] lipofectin (a 1:1 mixture of N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and dioleylphosphatidylethanolamine (DOPE)), and [3] DMRIE-C (a 1:1 mixture of N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide (DMRIE) and cholesterol). Surprisingly, a high CAT activity was observed with vectamidine although the DNA uptake efficiency was lower as compared to lipofectin and DMRIE-C. Transmission electron microscopy (TEM) revealed endocytosis as the major pathway of DNA-cationic lipid complex entry into COS cells for the three cationic lipids. However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE-C often exhibited a disrupted morphology. This disruption of endosomes was much less frequently observed with the DNA-lipofectin complexes. This comparison of the three compounds demonstrate that efficient transfection mediated by cationic lipids is not only correlated to their percentage of uptake but also to their ability to destabilize and escape from endosomes.

Double long-chain amidine liposome-mediated self replicating RNA transfection.

We present experimental evidence that a complex made of a double long chain cationic amphiphile and recombinant mRNA facilitates the entry and expression of genetic material into cells. Combining the properties of the self replicating recombinant mRNA driven by the Semliki Forest Virus (SFV) replicon and the transfection potentialities of a new cationic amphiphile (N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine) yields a highly efficient mRNA transfection system conferring up to 100% infectivity. The preparation and characterization of the long chain amidine cationic amphiphile-mRNA complex as well as the influence of the diC14-amidine/RNA ratio on the infective activity are described.