Contribution to the biophysical characterization of a dna/cationic liposome complex

Doctorat en sciences pharmaceutiquesinfo:eu-repo/semantics/nonPublishe

Calorimetry of cationic liposome-DNA complex and intracellular visualization of the complexes

SCOPUS: ar.kinfo:eu-repo/semantics/publishe

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.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Effet promoteur de l'anastomose portocave dans l'hépatocarcinogenèse expérimentale.

A promoting effect of portocaval anastomosis in a triphasic model of rat hepatocarcinogenesis is demonstrated since after initiation and selection premalignant lesions and hepatocellular carcinomas increase. This demonstrates that chronic administration of an exogenous compound is not the only way to promote cancer development

Promoting effect of portocaval anastomosis in rat hepatocarcinogenesis.

The effect of a surgical intervention, portocaval anastomosis (PCA) has been investigated in three different phases of a triphasic protocol of rat hepatocarcinogenesis. This protocol consists of the i.p. injection of 200 mg/kg of diethylnitrosamine (initiation) followed 2 weeks later by a 2 weeks diet with 2-acetylaminofluorene (2-AAF) and in the middle a necrogenic dose of CC14 (selection). One week later, a promoter such as phenobarbital (PB) is given chronically (promotion). PCA or a sham operation is performed 5 months before the end of the experiment. PCA alone does not induce hyperplastic nodules, nor does it when rats are initiated 5 weeks before. However, PCA alters the evolution of established nodules: it can act as a promoter like PB. Indeed, 5 months after initiation and selection, the number of rats bearing hepatocarcinomas is zero out of seven with a sham operation and six out of seven with PCA performed one week after the end of the selection. The combination of PCA and PB is more potent than PB or PCB alone since six out of six, five out of eight and six out of seven rats, respectively, bear malignant liver tumours. Thus PCA, which induces many systemic perturbations, has a promoting effect. This suggests that the chronic administration of a xenobiotic is not the only way to promote cancer development

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.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

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.Journal Articleinfo:eu-repo/semantics/publishe

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.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe