Assessment of the targeting specificity of a fluorescent albumin conceived as a preclinical agent of the liver function.

In the context of increasing liver diseases, no contrast agent is currently available in Europe and the United States to directly assess the liver function. Only neolactosylated human serum albumin is being clinically used in Asia. In order to perform preclinical studies in the context of liver diseases, we conceived a fluorescent lactosylated albumin for the quantification of liver functional cells (l-Cyal). Precise characterization was achieved in order to determine the amounts of lactose and Cyanine 5 (Cy5) coupled to the albumin. In addition, potential aggregation was characterized by asymmetrical flow field-flow fractionation hyphenated to multi-angle light scattering (AF4-MALS). The optimal functionalized albumin exhibited a mass greater than 87 kDa which corresponds to the addition of 34 lactose moieties per protein and 1-2 Cy5 labels. Also, no significant formation of aggregates could be identified due to the modification of the native albumin. In healthy mice, the accumulation of l-Cyal in the liver and its selectivity for hepatocyte cells were shown by optical imaging and flow cytometry. Administration of l-Cyal to mice bearing liver metastases showed a reduced signal in the liver related to a decrease in the number of hepatocytes. The l-Cyal bioimaging contrast agent could be particularly useful for assessing the state of liver related diseases.

Pre-treatment of cells with pluronic L64 increases DNA transfection mediated by electrotransfer.

Gene transfer into muscle cells is a key issue in biomedical research. Indeed, it is important for the development of new therapy for many genetic disorders affecting this tissue and for the use of muscle tissue as a secretion platform of therapeutic proteins. Electrotransfer is a promising method to achieve gene expression in muscles. However, this method can lead to some tissue damage especially on pathologic muscles. Therefore there is a need for the development of new and less deleterious methods. Triblock copolymers as pluronic L64 are starting to be used to improve gene transfer mediated by several agents into muscle tissue. Their mechanism of action is still under investigation. The combination of electrotransfer and triblock copolymers, in allowing softening electric field conditions leading to efficient DNA transfection, could potentially represent a milder and more secure transfection method. In the present study, we addressed the possible synergy that could be obtained by combining the copolymer triblock L64 and electroporation. We have found that a pre-treatment of cells with L64 could improve the transfection efficiency. This pre-treatment was shown to increase cell viability and this is partly responsible for the improvement of transfection efficiency. We have then labelled the plasmid DNA and the pluronic L64 in order to gain some insights into the mechanism of transfection of the combined physical and chemical methods. These experiences allowed us to exclude an action of L64 either on membrane permeabilization or on DNA/membrane interaction. Using plasmids containing or not binding sequences for NF-κB and an inhibitor of NF-κB pathway activation we have shown that this beneficial effect was rather related to the NF-κB signalling pathway, as it is described for other pluronics. Finally we address here some mechanistic issues on electrically mediated transfection, L64 mediated membrane permeabilization and the combination of both for gene transfer.

Amphiphilic perfluoroalkyl carbohydrates as new tools for liver imaging.

The synthesis of three molecules containing a fluorocarbon chain (either C(6)F(13), C(8)F(17) or C(10)F(21)), a sugar moiety (derived from lactobionic acid) and a chelate (derived from DTPA) is reported. These molecules (C(6)F(13)-Gal-DTPA, C(8)F(17)-Gal-DTPA or C(10)F(21)-Gal-DTPA) have been dispersed in water and their critical micellar concentration (CMC) as well as their size were determined. Their interaction with serum was weak as evaluated by time resolved fluorimetry of europium complexes. The presence of sugar on the surface of the nanoparticles was confirmed by the agglutination test using ricin. Conditions of pH and concentrations were optimised for in vivo studies. Finally, the nanoparticles formed with C(10)F(21)-Gal-DTPA have been complexed with (99m)Tc and injected to rats in order to follow their biodistribution by scintigraphy while following their stability by transmission electronic microscopy. A majority of the compound was found in the liver post-bolus injection.

Anionic pH-sensitive pegylated lipoplexes to deliver DNA to tumors.

Anionic pegylated lipoplexes have been prepared from the combined formulation of cationic lipoplexes and pegylated anionic liposomes. To this end, two original (bis- and tetra-) carboxylated cholesterol derivatives have been synthesised. Titration of the particle surface charge was realised to determine the ratio between anionic and cationic lipids that would give pH-sensitive complexes. This ratio has been optimised to form particles sensitive to pH change in the range 5.5-6.5. Compaction of DNA into these newly formed anionic complexes was checked by DNA accessibility to picogreen and DNA electrophoresis on an agarose gel. Gene expression of the formulated gene was similar for the cationic formulation taken as a control and the anionic formulations prepared. The pH-sensitive properties of these formulations was shown in vitro using bafilomycin, a vacuolar H(+)ATPase inhibitor. The efficiency of the new formulations to deliver DNA to the tumor was compared with cholesteryl hemisuccinate (CHEMS) formulations. The tetracarboxylated compound gave the most efficient formulations for tumor delivery in vivo.

Neutral postgrafted colloidal particles for gene delivery.

Surface modification of cationic lipoplexes has been carried out by means of a postgrafting reaction. The original lipoplexes described comprise a cationic lipid, a neutral lipid, poly(ethylene glycol)-cholesterol (with or without a targeting ligand) and DNA. Modifying their surface via a chemical, postgrafting reaction did not alter their size (approximately 100 nm) nor their ability to compact DNA, but did give a reduced zeta potential (approximately 0 mV) to afford surface neutral particles. With the modified lipoplexes nonspecific NIH3T3 cell surface binding in vitro was inhibited. Intravenous injection of the neutralized lipoplexes in mice showed decreased accumulation of the particles in the lung as compared to PEGylated cationic lipoplexes. Tumor targeting was also achieved in vivo by the addition of an RGD-PEG-Cholesterol as a lipid-ligand in the postgrafted lipoplex formulation.

Synthesis and biological properties of new glycosidic cationic lipids for DNA delivery.

Lipidic glycosides with amino alkyl pendent groups were synthesized and evaluated for in vitro DNA transfection activity. The first representative of this new class of cationic lipids showed good gene delivery and low toxicity to HeLa and 3T3 cells.

Non-electrostatic complexes with DNA: towards novel synthetic gene delivery systems.

We have developed new DNA complexing amphiphile based on Hoechst 33258 interaction with DNA grooves. The synthesis and physicochemical characterisation of lipid/DNA complexes, as compared to that of classical lipopolyamine for gene delivery, are described and discussed.

Inhibition of cathepsin D by tripeptides containing statine analogs.

Various analogs of statine, a remarkable amino acid component of the protease inhibitor pepstatine, were synthesized and evaluated as tripeptide derivatives for their activity against cathepsin D and HIV-1 protease.

Application of lipids and plasmid design for gene delivery to mammalian cells.

Cationic lipids are widely used for in vitro gene transfer due to their efficiency. The major challenges for the improvement of in vivo cationic lipid-mediated gene delivery reside in the design of more biocompatible lipoplexes mimicking viral-mediated gene delivery and in understanding the fate of the lipoplexes within the cells.

[Quantitation of iodine by analytical ion microscopy (AIM)]. / Quantification de l'iode par Microscopie Ionique Analytique (MIA).

AIM allows imaging and quantification of all elements on a biological tissue section. However quantification is limited by physical parameters which are difficult to assess and which intervene for a given element in the relation between its concentration and the measured signal. Thus, to quantify iodine in thyroid follicles, an iodine standard was prepared. Homogeneity in its atomic iodine distribution was tested by surface and depth analyses. Then, a standard curve was generated to be used as a reference for quantification of iodine on histological preparation by AIM.