Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes.

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.

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.

A novel cationic amphiphile for transfection of mammalian cells.

We describe here a new cationic amphiphile, N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), which interacts with plasmid DNA and generates hydrophobic stable complexes resistant against DNase I. In partition experiments between two non-miscible phases, DNA was transferred into an organic phase upon complex formation with diC14-amidine-containing vesicles. Finally, vesicles made of a diC14-amidine and phosphatidylethanolamine (PE) (1:1, mol:mol) mixture or pure diC14-amidine were efficient in mediating transfection of adherent (CHO) and suspension (K562) cell lines, using the chloramphenicol acetyltransferase (CAT) gene as reporter.

Specific adsorption of serine proteases on coated silica beads substituted with amidine derivatives.

Amidine derivatives interact with serine proteases, the inhibition being due to interactions between amidine functions and the active sites of the enzymes. Five different types of amidine (substituted or unsubstituted) were coupled to coated silica beads, which had previously been coated with DEAE-dextran to minimize the non-specific interactions due to silanol groups. Coated silica functionalized with substituted amidines shows a strong affinity towards human plasmin. This affinity is probably due to hydrophobic interactions between the substituted amidine and the human plasmin structure. Coated silica grafted by p-aminobenzamide gives a specific interaction with human plasmin. The importance of ionic strength and the steric conformation of the ligand is discussed. This support was used to purify thrombin from crude preparations by high-performance affinity chromatography.

[Not Available]. / Détermination de constantes d'acidité d'amidines N-N'-substituées. Relations entre les pKA et les vibrations IR - I. Acétamidines et métacrylamidines.

Changes in the ultraviolet absorption spectra with pH in aqueous solution were used to determine the pKA values of amidines. A new mathematical procedure was used to study sparingly soluble compounds. In this technique, all the experimental data are used to solve for the absorbance of the insoluble basic species and for the pKA value by a 'complex method' of optimization, which is more general than the classical least-squares method. A relationship between a double bond vibration frequency and the acidity constants of these compounds has been demonstrated.