Nuclear delivery of NFκB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging

Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized κB motif to favour NFκB-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Förster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was ∼1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NFκB activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NFκB. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the gene-expression efficiency

Systematic Comparisons of Formulations of Linear Oligolysine Peptides with siRNA and Plasmid DNA

The effects of lysine peptide lengths on DNA and siRNA packaging and delivery were studied using four linear oligolysine peptides with 8 (K8), 16 (K16), 24 (K24) and 32 (K32) lysines. Oligolysine peptides with 16 lysines or longer were effective for stable monodisperse particle formation and optimal transfection efficiency with plasmid DNA (pDNA), but K8 formulations were less stable under anionic heparin challenge and consequently displayed poor transfection efficiency. However, here we show that the oligolysines were not able to package siRNA to form stable complexes, and consequently, siRNA transfection was unsuccessful. These results indicate that the physical structure and length of cationic peptides and their charge ratios are critical parameters for stable particle formation with pDNA and siRNA and that without packaging, delivery and transfection cannot be achieved

Synthèse de nouveaux vecteurs polyaminés : Application au transfert de gène

Les poly(éthylènimine)s (PEI) branchées et linéaires sont parmi les vecteurs synthétiques les plus efficaces pour la transfection d'ADN in vitro dans les cellules de mammifères. La forme linéaire, obtenue par hydrolyse totale de poly(alkyl ou phényloxazoline), possède une grande efficacité bien qu'elle soit peu soluble dans l'eau. Afin d'accroître sa solubilité, la PEI linéaire a été modifiée par du polyoxyde d'éthylène (POE). L'insertion de motifs éthyloxazoline par hydrolyse partielle de la PEtOXZ a également permis d'augmenter sa solubilité. Il a été montré que les amines tertiaires transfectaient moins bien que les secondaires. Enfin, la LPEI a été modifiée par des greffons éthanamine et N,N-diméthyléthanamine afin d'étudier l'influence de la classe et la proportion des amines présentent dans ces nouveaux vecteurs. Les résultats montrent que la macrostructure du polymère semble être un facteur plus important que la proportion des différentes amines.Polyethylenimines branched (BPEI) and linear (LPEI) are among the most efficient non-viral gene transfer agents. LPEI, obtained by acid hydrolysis of poly(alkyl-2-oxazoline)s, show high transfection efficiency, in spite of a low solubility in water. In order to increase this solubility, LPEI was modified with poly(ethylene oxide) (POE) and 2-ethyl-2-oxazoline (EtOXZ) units. The synthesis of new linear polymers containing N-propylethylenimine (NPEI) units was realized allowing the study of influence of the class of the amine function on the biological activity of the polymers. In the same vein, LPEI was modified with éthanamine and N,N-dimethylethanamine side groups in order to study the influence of the proportion of the various amine functions on the transfection efficiency. The results show that secondary amino groups are better suited to mediate gene transfer than tertiary ones and that the vector macrostructure is a factor more prominent than the proportion of the amine function.EVRY-BU (912282101) / SudocSudocFranceF

Tuning Macromolecular Structures of Synthetic Vectors for Gene Therapy

International audienceThe synthesis of triblocks poly(2‐methyl‐2‐oxazoline‐b‐tetrahydrofurane‐b‐2‐methyl‐2‐oxazoline) has been developed. It was shown that the technique of polymerization of the second block from the living species created on the two chain ends of poly(THF) is successful but makes the control of the size of the poly(THF) block difficult due a fast depolymerization upon the introduction of the second monomer. A purification technique was used to get rid of the possible homopoly(2‐methyl‐2‐oxazoline) formed. Various analytical techniques were used to characterize the behavior of the triblock and more particularly in the presence of DNA. Electrophoresis on agarose gels and neutron scattering, demonstrated that the neutral triblock does not appreciably interact with DNA. It was also shown that the triblock for which approximately half (47%) of the methyloxazoline units were transformed into ethylenimine units by hydrolysis gives only loose interactions with DNA. This result is assigned to the fact that charge density plays a major role in the interactions of positive polyelectrolytes with the negatively charged DNA. The triblock was shown being able to interact with bilayer lipid membranes mimicking cell membranes. The efficiency of the hydrolysed triblock was much higher, while the size of holes created in the membranes is not large enough to give passage to DNA

Traits de personnalité et virage de l'humeur sous antidépresseur (Etude chez 56 patients bipolaires)

Le virage de l'humeur pharmaco-induit chez les patients atteints de maladie maniaco-dépressive est un réel problème thérapeutique. Les antidépresseurs, en effet, sont responsables, dans environ 20% des cas, d'une inversion brutale de l'humeur ou virage et d'une accélération des cycles de maladie. De ce fait, les patients bipolaires sont le plus souvent exclus des essais thérapeutiques sur les antidépresseurs. Il est recommandé de ne prescrire ces traitements que très prudemment alors que la dépression du sujet bipolaire est souvent d'intensité plus importante que celle du sujet unipolaire, et que son risque suicidaire est plus marqué. Les études sur les facteurs prédictifs du virage maniaque sous antidépresseur ont proposé divers paramètres et souligné la grande variabilité de ce risque d'un sujet à l'autre. Le but de ce travail était de mettre en évidence les facteurs de risque de virage de l'humeur chez les patients bipolaires au regard des données de la littérature existante mais aussi, de mettre en évidence un lien entre l'induction d'un épisode maniaque ou hypomaniaque par les antidépresseurs et des traits de personnalité. Pour ce faire, nous avons réalisé une évaluation de la personnalité chez 56 patients bipolaires à l'aide du Temperament and Character inventory de Cloninger. La première partie de cet exposé est consacrée à la clinique du virage des patients bipolaires. Dans la deuxième partie, nous traiterons plus précisément des facteurs de risque socio-démographiques, cliniques et médicamenteux du virage de l'humeur des patients bipolaires . Dans la troisième et quatrième partie, nous nous intéresserons aux profils de personnalité des patients bipolaires ; en particulier, nous aborderons les liens possibles entre les dimensions évaluées par le questionnaire TCI (Temperament and Character Inventory) de Cloninger et le virage sous antidépresseur...PARIS7-Villemin (751102101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Preliminary investigations on a simple polyelectrolyte derived from (CH 2 CH 2 C(COOH) 2 ) n : Unexpected solubility-insolubility pattern controlled selectively by the nature of the alkali counterion

International audienceMonodisperse samples of alkali poly(trimethylene-1,1-dicarboxylate) polyelectrolytes of (CH2CH2C(COO(-))2)n structure and of low to moderate molecular weights have been synthesized and characterized. These polymers are densely and evenly decorated by carboxylate groups, with the anionic moieties extending symmetrically in transverse directions on every third carbon alongside the backbone. In sharp contrast to other polyelectrolytes, they easily precipitate with sodium and potassium ions in water. The precipitation is selective, the solubility varying with the nature of the alkali cation in the order: Li+ > K+ > Na+. Precipitation is fast and reversible, the solubility-insolubility transition being rapidly crossed in both directions when the proper alkali ion is added in excess to the solution/sus- pension. Experimental investigations (WAXS, FTIR) on the precipitated polymer and DFT theoretical calculations on model 1:1 complexes of malonate subunits with alkali cations have been carried out in order to cast some light on the above phenomena. In addition, a model molecule (CH3C(COOH)2CH2CH2C(COOH)2CH3) has been prepared, supporting the structure of the obtained polyelectrolyte as well as the formation of specific structures at half-deprotonation of the poly(carboxylic acid)

Synthesis, Characterization, and Solid-State Properties of (CH2–CH2–C(COOH)2)n : A Highly Symmetrical, Semicrystalline Carbon-Chain Poly(carboxylic acid)

International audienceThe synthesis and structural characterization of poly(trimethylene-1,1-dicarboxylic acid) (CH2–CH2–C(COOH)2)n, a carbon-chain polymer bearing two carboxylic acids on every third carbon alongside the backbone, are described for the first time. Solid-state properties are investigated by a variety of techniques (WAXS, FT-IR, CP-MAS, and TGA), in particular with respect to the semicrystalline character of the polymer and its affinity toward water. The observed experimental features are rationalized on the basis of the ability for the macromolecular chains to associate via the pendant carboxylic acids and of the high global symmetry obtained for this polyacid through the geminal placement of the side substituents