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P Midoux

Publications and source records attributed to P Midoux.

At least 19 recordsLinked to original sources
PubMed2001

Histidine-rich peptides and polymers for nucleic acids delivery.

Nucleic acids transfer into mammalian cells requires devices to improve their escape from endocytic vesicles where they are mainly confined following cellular uptake. In this review, we describe histidine-rich molecules that enable the transfer of plasmid and oligonucleotides (ODN) in human and non-human cultured cells. An histidine-rich peptide which permeabilizes biological membrane at pH 6.4, favored the transfection mediated by lactosylated polylysine/pDNA complexes. Histidylated polylysine forms cationic particles of 100 nm with a plasmid and yielded a transfection of 3-4.5 orders of magnitude higher than polylysine. The biological activity of antisense ODN was increased more than 20-fold when it was complexed with highly histidylated oligolysine into small cationic spherical particles of 35 nm. Evidence that imidazole protonation mediates the effect of these molecules in endosomes are provided. We also describe a disulfide-containing polylysine conjugate capable of mediating DNA unpackaging in a reductive medium and to increase the transfection efficiency. Overall, these molecules constitute interesting devices for developing non-viral gene delivery systems.

Animals↗
PubMed2000

Histidylated oligolysines increase the transmembrane passage and the biological activity of antisense oligonucleotides.

We have designed histidylated oligolysines which increase the uptake, the cytosolic delivery and the nuclear accumulation of antisense oligonucleotides (ODN). Flow cytometry analysis showed a 10-fold enhancement of the ODN uptake in the presence of histidylated oligolysines. The intracellular localizations of fluorescein-labeled ODN and of rhodamine-labeled histidylated oligolysines were investigated by confocal microscopy. Histidylated oligolysines favor the cyto-solic delivery of ODN from endosomes and increase their nuclear accumulation. In contrast, in their absence fluorescent ODN were not observed inside the nucleus but were distributed overwhelmingly within the vesicles in the cytosol. In addition, histidylated oligolysines yielded a more than 20-fold enhancement of the biological activity of antisense ODN towards the inhibition of transient as well as constitutive gene expression. Prevention of endosome lumen acidification using bafilomycin A(1)abolished the effect of histidylated oligolysines, suggesting that protonation of the histidyl residues was involved in the transmembrane passage of ODN.

Amino Acid Sequence↗
PubMed2000

Spacer length dependence on the efficiency of dimeric anionic peptides in gene transfer by glycosylated polylysine/plasmid complexes.

Amphiphilic anionic peptides have been used to enhance the efficiency of transfection by helping plasmids to escape from endosomes to the cytosol. It has been shown that efficiency of an eicosamers containing five glutamyl residues (E5), can be considerably enhanced either by transforming it into a dimer or by adding a tripeptide WYG in a C-terminal position (E5WYG). The dimerization of the peptide E5WYG leads to a more efficient tool when the dimerization device includes the tripeptide WYG unit and a longer spacer arm made of Gly-betaAla-betaAla residues, but to a 10-fold less efficient tool when the dimerization device includes a shorter spacer, a glycyl residue. Both dimers are taken up by the cells to a similar extent. Both dimers seem to be surrounded similarly as far as the environmental pH is concerned. In contrast, we found a correlation between the propensity of the peptides to adopt a helical structure at neutral pH and the gene transfer efficiency.

Amino Acid Sequence↗
PubMed2000

Efficient gene transfer into human normal and cystic fibrosis tracheal gland serous cells with synthetic vectors.

Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of reporter gene transfer into immortalized normal (MM-39) and CF (CF-KM4) human airway epithelial gland serous cells using various synthetic vectors: glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using alpha-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using alpha-glycosylated polylysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane. The presence of membrane lectins, i. e., cell surface receptors binding oligosaccharides, was also examined on MM-39 and CF-KM4 cells by assessing the binding and uptake of fluorescein-labeled neoglycoproteins and fluorescein-labeled glycoplexes (glycofectins complexed to plasmid DNA). Among all the neoglycoproteins and glycoplexes tested, those bearing alpha-mannosylated derivatives were most efficiently taken up by both normal and CF gland serous cells. However, alpha-mannosylated polylysine was quite inefficient for gene transfer, indicating that the efficiency of gene transfer is determined both by the uptake of the complexes and also by their intracellular trafficking. Moreover, our results show that an efficient in vitro gene transfer was achieved in human airway gland serous cells with the same synthetic vectors described to efficiently transfect human airway surface epithelial cells.

Cell Line, Transformed↗
PubMed1999

Sugar-mediated uptake of glycosylated polylysines and gene transfer into normal and cystic fibrosis airway epithelial cells.

We have examined the membrane lectin expressed by immortalized normal and cystic fibrosis (CF) airway epithelial cells, using fluorescein-labeled neoglycoproteins; the uptake of plasmid DNA using fluoresceinylated glycoplexes (plasmid/glycosylated polylysine complexes); and the efficiency of gene transfer when glycosylated polylysines and glycosylated, partially gluconoylated polylysines were used as vectors. The most efficient uptake of neoglycoproteins by normal and CF cells was obtained with mannosylated BSA (bovine serum albumin). Similarly, the most efficient uptake of plasmid DNA was obtained with glycoplexes bearing alpha-D-Man residues. Surprisingly, glycoplexes bearing alpha-D-Man residues were poorly efficient for gene transfer into normal and CF cells. The highest luciferase activity was achieved with lactosylated polylysine- and beta-D-GlcNAc-substituted gluconoylated polylysine as vectors. Gene transfer efficiency obtained with gluconoylated polylysine bearing beta-D-GlcNAc residues was similar to that observed with polyethylenimine (PEI; 25 and 800 kDa) and 10-fold higher than that observed with lipofectin and LipofectAMINE. These results suggest that the transfection efficiency with glycoplexes is not determined only by the specificity of the lectin expressed at the cell surface membrane but also by intracellular trafficking of the glycoplexes, which could be mediated by lectins present inside the cells.

Bronchi↗
PubMed1999

Glycotargeting: influence of the sugar moiety on both the uptake and the intracellular trafficking of nucleic acid carried by glycosylated polymers.

Nucleic acids (plasmids as well as oligonucleotides) used to specifically express or modulate the expression of a gene, must reach the cytosol and/or the nucleus. Several systems have been developed to increase their uptake and their efficiency. Glycosylated polylysines have been shown to specifically help nucleic acids to be taken up in cells expressing a given cell surface membrane lectin. However, it appeared that the efficiency of the imported nucleic acid was not directly related to the extent of the uptake. Indeed, some glycosylated polylysines bearing sugar moities which are poor ligands of the cell surface lectins of a given cell were found to be more efficient than those bearing better sugar ligands. The interpretation of this paradoxal result is discussed with regards to the nature of the compartment allowing the nucleic acid to cross the membrane and to be delivered in the cytosol on the one hand, and to the presence of intracellular lectins on the other hand.

Animals↗
PubMed1998

Glycotargeting: the preparation of glyco-amino acids and derivatives from unprotected reducing sugars.

Lectins are present on the surface of many cells. Many lectins actively recycle from membrane to endosomes and efficiently take up glycoconjugates in a sugar-dependent manner. On this basis, glycoconjugates, specially those obtained by chemical means, are good candidates as carriers of drugs, oligonucleotides or genes. In this paper, we present a panel of methods suitable to transform unprotected reducing oligosaccharides into glycosynthons designed to be easily linked to therapeutic agents. All the glycosynthons presented here are glycosylamines or derivatives, mainly glyco-amino acids or glycopeptides. Glycosylamines are easy to obtain, but they are very labile in slightly acidic or neutral medium; they must be stabilized, by acylation for instance. The coupling efficiency of a reducing sugar with ammonia as well as an alkylamine or an arylamine is higher at high temperature, however, because of the Amadori rearrangement, special conditions have to be selected to prepare the expected glycosylamine derivative with a high yield. Glycosylamines are easily acylated by N-protected amino acids, or by halogeno acids which can then be transformed into amino acids. Alternatively, unprotected reducing oligosaccharides may very efficiently be transformed into N-glycosyl-amino acids and then protected by N-acylation. With a glutamyl derivative having both the alpha-amino and the gamma-carboxylic groups free, the coupling and the acylation, which is intramolecular, are roughly quantitative. N-oligosaccharyl-amino acid derivatives are interesting glycosynthons, because their sugar moiety bears the specificity towards membrane lectins while the amino acid part has the capacity to easily substitute a therapeutic agent.

Amines↗
PubMed1997

The reduction of the positive charges of polylysine by partial gluconoylation increases the transfection efficiency of polylysine/DNA complexes.

A polylysine partially substituted with polyhydroxyalkanoyl residues and specially with gluconoyl residues was developed in order to increase the transfection efficiency by decreasing the strength of the electrostatic interactions between the DNA and the cationic polymer. Partially gluconoylated polylysine/DNA complexes were more easily dissociated in solution and their transfection efficiency in the presence of chloroquine, evaluated with HepG2 cells, a human hepatocarcinoma line, was higher when 43 +/- 4% of the epsilon-amino groups of polylysine were blocked with gluconoyl residues. Partially gluconoylated polylysine/plasmid complexes were efficient in transfecting different adherent as well as non-adherent cell lines. Partially gluconoylated polylysine formed highly soluble (above 100 micrograms/ml in DNA) complexes with DNA plasmids. In addition, partially gluconoylated polylysine bearing few lactosyl residues increased the transfection efficiency of HepG2 cells which express a galactose-specific membrane lectin.

Acylation↗
PubMed1997

Cytosolic and nuclear delivery of oligonucleotides mediated by an amphiphilic anionic peptide.

Antisense oligonucleotides (ODN) were easily introduced into the cytosol of mammalian cells on permeabilization of the plasma membrane by an amphiphilic anionic peptide. The E5CA peptide (GLFEAIAEFIEGGWEGLIEGCA) is an E5 peptide analog derived from the N-terminal segment of the HA2 subunit of influenza virus hemagglutinin. This peptide undergoes a conformational change when the pH shifts from neutral to around 6.0, inducing a transient permeabilization of the plasma membrane. In the presence of the E5CA peptide at pH close to 6.0, fluoresceinylated ODN were rapidly taken up by cells and diffused into the nucleus. The uptake of ODN was dependent on the E5CA peptide concentration and on the duration of the incubation at low pH, as shown by confocal microscopy and flow cytometry analyses. This procedure is suitable for loading adherent cells as well as nonadherent cells with single-stranded or double-stranded ODN. Under optimal conditions, a high percentage of cells were nuclei loaded, and the viability was not affected. This method makes use of a well-defined chemical product without the requirement of any special equipment. It will be useful to study the interactions of single-stranded or double-stranded ODN used as antisense, antigenes, or decoys.

Amino Acid Sequence↗
PubMed1996

Gluconoylated and glycosylated polylysines as vectors for gene transfer into cystic fibrosis airway epithelial cells.

To provide an alternative to viral vectors for the transfer of genes into airway epithelial cells in cystic fibrosis (CF), a novel set of substituted polylysines were employed. Polylysine was partially neutralized by blocking a number of positively charged residues with gluconoyl groups. In addition, polylysine was substituted with sugar residues on a specified number of amino groups. Using the gluconoylated polylysine as vector, the pCMVLuc plasmid gave high expression of the reporter gene luciferase in immortalized CF/T43 cells. The luciferase activity was 75-fold greater in the presence of 100 microM chloroquine. Luciferase gene expression persisted at high levels for up to at least 120 hr following transfection. Glycosylated polylysines/pCMVLuc complexes were compared to the gluconoylated polylysine/pCMVLuc complex and beta-Gal-, alpha-Glc-, and Lac-substituted polylysines gave 320%, 300%, and 290%, respectively, higher expression of the reporter gene luciferase. Luciferase expression ranged from 35 to 2 ng of luciferase per milligram of cell protein in the order: beta-Gal = alpha-Glc = Lac > alpha-Gal = Rha = Man > beta-GalNAc > alpha-GalNAc = alpha-Fuc, suggesting that the transfection efficiency is sugar dependent. Most importantly, in primary cultures of both CF and non-CF airway epithelial cells grown from tracheal tissue explants, lactosylated polylysine gave uniformly high expression of luciferase. The glycosylated polylysines provide an attractive nonviral approach for the transfer of genes into airway epithelial cells.

Cell Count↗
PubMed1996

Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes.

Chloroquine improves drastically the transfection of cells upon exposure to plasmid DNA/glycosylated polylysine complexes. So far the mechanism of action of chloroquine is not well understood. In this paper, the effect of chloroquine was investigated by measuring the transfection efficiency of a human hepatocarcinoma (HepG2 cells) by pSV2LUC/lactosylated polylysine complexes involving their internalization via the galactose-specific membrane lectin of these cells. The luciferase activity in the transfected cells was maximal when the transfection was performed for 3 or 4 h in the presence of 100 microM chloroquine. The luciferase activity was also enhanced in the presence of primaquine, a chloroquine analogue, but was not increased when transfection was performed in the presence of ammonium chloride, methylamine, spermine, or monensin, compounds known to neutralize the pH of the endocytotic vesicle lumen as chloroquine does. Chloroquine enters cells and accumulates in vesicular compartments; the overall intracellular concentration increases to 9 mM, which means that in the vesicular compartment, the chloroquine concentration is still higher. At such high concentrations, chloroquine induces the dissociation of plasmid DNA/lactosylated polylysine complexes, as shown in acellular experiments.

Carcinoma, Hepatocellular↗
PubMed1996

Gene transfer by DNA/glycosylated polylysine complexes into human blood monocyte-derived macrophages.

Macrophages are putative target cells for expressing an exogenous gene with therapeutical effects. Knowing that macrophages express membrane lectins mediating endocytosis of their ligands, DNA/glycosylated polylysine complexes were used to transfect human blood monocyte-derived macrophages. Monocytes from human peripheral blood were matured in culture for 7 days to differentiate into macrophage-like cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Adherent cells, which displayed characteristic macrophage markers, CD 14, CD 11b, HLA-DR, and HLA-ABC antigens and mannose receptor, were transfected by DNA/glycosylated polylysine complexes in the presence of chloroquine. The luciferase reporter gene expression was maximal 24 hr after transfection with a DNA/mannosylated polylysine complex and by using plasmids in which the promoters (either the long terminal repeat of the human immunodeficiency virus or the human cytomegalovirus) drove the luciferase gene expression. Luciferase gene expression was lower when the promoter was the early region of the large T antigen of SV40 virus. Transfection mediated by DNA/mannosylated polylysine complexes was much more efficient than with DEAE-dextran or lipofectin. The possibility of transferring and expressing an exogenous gene into macrophage-like cells by using a nonimmunogenic synthetic vector as a DNA carrier opens new ways to develop nonviral gene therapy strategies.

Cell Count↗
PubMed1996

Expression of a mannose/fucose membrane lectin on human dendritic cells.

Dendritic cells (CD) are the most efficient antigen presenting cells for T lymphocytes. CD1a+ CD14- CD with high antigen-presenting capacities can now be obtained easily from adherent peripheral blood monocytes by culture in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4 (Sallusto et al., J. Exp. Med. 1994. 179: 1109). Human macrophages express a membrane lectin, or sugar-specific receptor, which specifically mediates the binding and endocytosis of mannose- and fucose-terminated glycoproteins and is involved in the phagocytosis of pathogens. A similar lectin activity was sought on cultured human DC using flow cytometry and confocal microscopy to detect binding and internalization of fluoresceinated neoglycoproteins [bovine serum albumin (BSA) substituted with sugar residues]. Several neoglycoproteins, especially alpha-L-fucosyl-, alpha-D-mannosyl-, N,N'-di-acetyl-beta-chitobiosyl- and beta-D-glucosyl-BSA, were endocytosed by cultured human CD1a+ DC as well as by CD1a- CD14- cells which were also obtained in the culture. Fuc-BSA and Man-BSA had the same number of binding sites (1.7 x 10(6)/cell) on CD1a+ DC, and bound with an affinity constant close to 10(7) 1/mol. Inhibition experiments indicated that these two neoglycoproteins bound to the same membrane lectin. CD1a+ and CD1a- cells were both labeled by an antiserum specific for the human macrophage mannose receptor. The membrane lectin specific for mannose and fucose that is evidenced in these experiments on cultured DC may be similar to the macrophage membrane lectin or may share functional and structural properties with it.

Antigens, Surface↗
PubMed1996

Enhanced biological activity of antisense oligonucleotides complexed with glycosylated poly-L-lysine.

We sought to exploit glycosylated poly-L-lysine (pLK) to increase the uptake and biological antisense activity of a phosphorothioate oligonucleotide (pt-odn) [pt-odn complementary to the 3' noncoding region of intercellular adhesion molecule-1 (ICAM-1) (odn(ICAM-1))] complementary to the 3'-noncoding region of ICAM-1 in A549 cells. Dose-dependent inhibition of ICAM-1 expression was obtained (IC50 = 500 nM) through treatment of cells with odn(ICAM-1) complexed with pLK carrying fucose residues in the presence of 100 microM chloroquine. Alteration in the charge ratio between fucosylated pLK and pt-odn had a significant effect on the efficacy of inhibition (optimal conditions, charge ratio = 1.1). This effect was also dependent on the number of fucose moieties per pLK. Free pt-odn or pt-odn complexed with nonglycosylated pLK gave no inhibition at concentrations of < or = 2 microM. Two control pt-odn (one was targeted against an unrelated gene not present in these cells, gag(HIV), and the other had a randomized sequence) gave no inhibition of ICAM-1 expression in the presence or absence of pLK carrying fucose residues at concentrations of < or = 2 microM. When complexed with pLK carrying 100 fucose residues, the amount of cell-associated pt-odn was increased by 15-fold compared with the free pt-odn. Nongycosylated pLK also increased the amount of cell-associated pt-odn by >10 fold but did not alter the biological activity. These results demonstrate clearly the potential of glycosylated pLK as a pt-odn transporter.

Chloroquine↗
PubMed1995

Membrane permeabilization by alpha-helical peptides: a flow cytometry study.

The permeabilization by alpha-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluorescent and poorly membrane permeant molecule, becomes strongly fluorescent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by alpha-helical peptides such as melittin, succinylated melittin and anionic peptides derived from the N-terminus of HA2 subunit of the influenza virus hemagglutinin, was measured. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) caused a rapid (< 5 min) and dose-dependent (ED50 = 0.5 microM) permeabilization of HL60 cells at neutral pH, whereas the succinylated derivative induced cell permeabilization only at pH below 4.5 with an ED50 = 18 microM. The permeabilization by the anionic E5CA peptide (GLFEAIAEFIEGGWEGLIEGCA) containing 5 glutamic residues occurred (ED50 = 11 microM) at pH ranging from 6.5 to 6.0; replacing the tryptophan residue in position 14 by a phenylalanine residue decreased by about 1 unit the pH at which membrane permeabilization was effective. The membrane permeabilization activity of the E5CA peptide was reversibly abolished when the peptide was linked to a protein carrier. These results show that alpha-helical peptide-induced membrane permeabilization can be easily monitored by using flow cytometry in the presence of a non permeant dye. This method allows a rapid screening and an efficient mean of selection of peptides suitable to induce membrane permeabilization.

Amino Acid Sequence↗
PubMed1993

Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.

Amino Acid Sequence↗