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.

The enzyme-like catalytic activity of cerium oxide nanoparticles and its dependency on Ce3+ surface area concentration.

Cerium oxide nanoparticles are known to catalyze the decomposition of reactive oxygen species such as the superoxide radical and hydrogen peroxide. Herein, we examine the superoxide dismutase (SOD) and catalase (CAT) mimetic catalytic activities of nanoceria and demonstrate the existence of generic behaviors. For particles of sizes 4.5, 7.8, 23 and 28 nm, the SOD and CAT catalytic activities exhibit the characteristic shape of a Langmuir isotherm as a function of cerium concentration. The results show that the catalytic effects are enhanced for smaller particles and for the particles with the largest Ce3+ fraction. The SOD-like activity obtained from the different samples is found to superimpose on a single master curve using the Ce3+ surface area concentration as a new variable, indicating the existence of particle independent redox mechanisms. For the CAT assays, the adsorption of H2O2 molecules at the particle surface modulates the efficacy of the decomposition process and must be taken into account. We design an amperometry-based experiment to evaluate the H2O2 adsorption at nanoceria surfaces, leading to the renormalization of the particle specific area. Depending on the particle type the amount of adsorbed H2O2 molecules varies from 2 to 20 nm-2. The proposed scalings are predictive and allow the determination of the SOD and CAT catalytic properties of cerium oxide solely from physicochemical features.

NF-kB related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer.

BACKGROUND: When activated, NF-κB can promote the nuclear import and transcription of DNA possessing NF-κB consensus sequences. Here, we investigated whether NF-κB is involved in the plasmid electrotransfer process. METHODS: Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-κB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBα were studied as a marker of NF-κB activation. RESULTS: Intra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-κB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IκBα was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IκBα and thus of free NF-κB in the absence of any stimulation. GENERAL SIGNIFICANCE: pDNA electrotransfer can increase transgene expression independently of NF-κB. The insertion of NF-κB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-κB in muscle might be sufficient to enhance the activity of pDNA bearing NF-κB consensus sequences.

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.

Interactions between sub-10-nm iron and cerium oxide nanoparticles and 3T3 fibroblasts: the role of the coating and aggregation state.

Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of cerium and iron oxide sub-10-nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), M(W) = 2000 g mol(-1)). Electrostatically adsorbed on the surfaces, the organic moieties provide a negatively charged coating in physiological conditions. We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. Only the bare and the citrate-coated nanoceria exhibit a slight decrease in mitochondrial activity at very high cerium concentrations (>1 g l(-1)). We also observe that the citrate-coated particles are internalized/adsorbed by the cells in large amounts, typically 250 pg/cell after 24 h incubation for iron oxide. In contrast, the polymer-coated particles are taken up at much lower rates (<30 pg/cell). The strong uptake shown by the citrated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes. In conclusion, we show that the uptake of nanomaterials by living cells depends on the coating of the particles and on its ability to preserve the colloidal nature of the dispersions.

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 of new cationic lipids from an unsaturated glycoside scaffold.

[see structure]. We report the synthesis of new cationic lipids. These amphiphiles present a hydrophobic domain connected to a guanidinium entity by an unsaturated glycoside scaffold. The synthetic strategy using amide or acetal linkage led to various mono- and bicatenar derivatives. Investigation of their physicochemical properties indicated that these new compounds compact DNA.

Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR.

The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Taq polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by XbaI, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity.

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with TAQ polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.

The spherulites(TM): a promising carrier for oligonucleotide delivery.

Concentric multilamellar microvesicles, named spherulites(TM), were evaluated as an oligonucleotide carrier. Up to 80% oligonucleotide was encapsulated in these vesicles. The study was carried out on two different spherulite(TM) formulations. The spherulite(TM) size and stability characteristics are presented. Delivery of encapsulated oligonucleotide was performed on a rat hepatocarcinoma and on a lymphoblastoid T cell line, both expressing the luciferase gene. We showed that spherulites(TM) were able to transfect both adherent and suspension cell lines and deliver the oligonucleotide to the nucleus. Moreover, 48-62% luciferase inhibition was obtained in the rat hepatocarcinoma cell line when the antisense oligonucleotide targeted to the luciferase coding region was encapsulated at 500 nM concentration in spherulites(TM) of different compositions.

Zwitterionic oligodeoxyribonucleotide N3'-->P5' phosphoramidates: synthesis and properties.

Zwitterionic, net neutral oligonucleotides containing alternating negatively charged N3'-->P5' phosphoramidate monoester and positively charged phosphoramidate diester groups were synthesized. The ability of zwitterionic phosphoramidates to form complexes with complementary DNA and RNA was evaluated. Stoichiometry and salt dependency of these complexes were determined as a function of the nature of the heterocyclic bases of the zwitterionic compounds. Unlike the melting temperatures of the natural phosphodiester-containing oligomers, the T m of the duplexes formed with the zwitterionic oligothymidylates was salt concentration independent. The thermal stability of these duplexes was much higher with Delta T m values of 20-35 degrees C relatively to phosphodiester counterparts at low salt concentrations. The zwitterionic oligoadenylate formed only 2Py:1Pu triplexes with complementary poly(U) or poly(dT) strands. The thermal stability of these complexes was dependent on salt concentration. Also, the T m values of the complexes formed by the zwitterionic oligoadenylate with poly(U) were 6-41 degrees C higher than for the natural phosphodiester counterpart. Triplexes of this compound with poly(dT) were also more stable with a Delta T m value of 22 degrees C at low salt concentrations. Complexes formed by the zwitterionic oligonucleotides with complementary RNAs were not substrates for RNase H. Surprisingly, the duplex formed by the all anionic alternating N3'-->P5'phosphoramidate-phosphodiester oligothymidylate and poly(A) was a good substrate for RNase H.