Roger Mayer

Glycoconjugates as carriers for specific delivery of therapeutic drugs and genes

Abstract Cell surface receptors are good candidates to selectively target drugs, oligonucleotides or even genes by making use of their specific ligands. A large number of mammalian cells express cell surface sugar-binding proteins, also called “membrane lectins”. Therefore, sugars may be used as specific recognition signals to specifically deliver biological active components. Tens of membrane lectins with different sugar specificities have been characterized; some of them actively carry their ligands to intracellular compartments, including endsomes, lysosomes and, in some cases, Golgi apparatus. In this review, we summarize the main properties of neoglycoproteins and glycosylated polymers; they have been developed to study the properties of endogenous lectins and to carry various drugs. Glycoconjugates have been successfully used to carry biological response modifiers such as N -acetylmuramyldipeptide. N -Acetylmuramyldipeptide is, in vitro, hundreds of times more efficient in rendering macrophages tumoricidal when it is bound to this type of carrier. In vivo, the N -acetylmuramyldipeptide bound to glycoconjugates containing mannose in a terminal non-reducing position, induces the eradication of lung metastases, occurring when treatment is started, in 70% of mice; free N -acetylmuramyldipeptide is strictly inactive. Similarly, N -acetylmuramyldipeptide bound to the same glycoconjugates induces an active antiviral effect. Glycoconjugates are also suitable for carrying antisense oligonucleotides specific for viral sequences. Antisense oligonucleotides protected at both ends and linked through a disulfide bridge to the glycoconjugates are 10 times more efficient than the corresponding free oligonucleotides. Poly- l -lysine containing about 190 lysine residues has been substituted by three components: sugars as recognition signal, antiviral (or antiparasite) agents as therapeutic elements and gluconoic acid as neutralizing and solubilizing agent. This type of neutral, highly water-soluble glycosylated polymer is a very efficient carrier to deliver drugs in infected cells according to the nature of the sugar borne on the polymer and to the specificity of the lectin present at the surface of the infected cells. Finally, poly- l -lysine (190 residues) partially substituted with sugars (60 units) is a polycationic glycosylated polymer which easily makes complexes with plasmids. These complexes are very efficient in transfecting cells in a sugar-dependent manner. The expression of reporter gene is greatly enhanced when cells are incubated with the plasmid-glycosylated poly- l -lysine complex in the presence of either 100 μM chloroquine or 10 μM fusogenic docosapeptide. Furthermore, this transfection method leads to a much larger number of stable transfectants than the classical method using calcium phosphate precipitate. The general properties of glycosylated proteins and of glycosylated polymers are presented and their efficiency in targeting genes in comparison with that of other available targeted transfection methods is discussed.

Muramyl dipeptide bound to poly-l-lysine substituted with mannose and gluconoyl residues as macrophage activators

Poly-l-lysine modified with mannose derivatives, the residual cationic charges of which being neutralized byN-acylation, were synthesized and used as carriers of a macrophage activator (N-acetylmuramyl dipeptide, MDP). The influence of the acylating agent on the targeting efficiency was investigated: a hydrosolubilizing group such as a gluconoyl moiety led to very efficient carrier conjugates, while an acetyl group did not. The effect of sugar and acyl content of the polymers was assessed using these compounds as inhibitors of red blood cell agglutination by Concanavalin A. The binding and specific endocytosis of poly-l-lysine substituted with several mannose derivatives and gluconoyl residues (GlcAx-, Many-PLK) have been determined by a quantitative flow cytometry analysis. MDP bound to these conjugates was much more efficientin vitro than free MDP in macrophage cytostasis assays.

Membrane permeabilization by α-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.

Purification and identification of a neutral endopeptidase inPlasmodium falciparum schizonts and merozoites

An endopeptidase specific to thePlasmodium falciparum erythrocytic schizont stage and to free merozoites was detected using the fluorogenic GlcA-Val-Leu-Gly-Lys(or Arg)-AEC substrate. The enzyme was purified by high performance liquid chromatography (HPLC); its optimal activity was around pH 7.5 and its isoelectric point was 4.4. The molecular weight of the enzyme was about 68000, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The endopeptidase was strongly inhibited by thiol proteinase inhibitors, leupeptin, and antipain. The possible involvement of this neutral endopeptidase in the reinvasion process is discussed.

Neutral proteases involved in the reinvasion of erythrocytes by Plasmodium merozoites

Neutral proteases of Plasmodium sp erythrocytic stages were studied by means of a sensitive fluorogenic method and gelatin‐SDS‐PAGE. The substrates gluconoyl‐Val‐Leu‐Gly‐Lys(or Arg)‐3‐amido‐9‐ethylcarbazole were selectively hydrolyzed by an endopeptidase from rodent Plasmodium berghei (Pb) and Plasmodium chabaudi (Pc) and from human Plasmodium falciparum (Pf) parasites. These endopeptidases were purified from 100,000‐g soluble schizont extract by high pressure liquid chromatography; they have a similar Mr of 68,000 in SDS‐PAGE, and an optimal activity at pH 7.4. The Pb 68 and Pf 68 endopeptidases were localized in shizonts and also in merozoites as shown by indirect immunofluorescence on Pb merozoites and by the identification of the Pf68 endopeptidase activity in free viable merozoites. The Pb68 and Pf68 endopeptidases belong to the class of cysteine proteases. Analysis by gelatin‐SDS‐PAGE of a Pb 68 endopeptidase‐enriched fraction showed a reproducible 95,000 proteolytic band. The initial extracts showed a similar 95,000 proteolytic band, and also 2 other 90,000 and 85,000 major bands. During reinvasion experiments, it was possible to recover a 95,000 and a 40,000 protease band from supernates of cultures grown in a semidefined medium without serum. Hydrophilic peptide derivatives related to the substrate of Pf 68 endopeptidase are shown to be potential inhibitors of the Pf reinvasion process in vitro.

Detection and characterization of a selective endopeptidase from Plasmodium berghei by using fluorogenic peptidyl substrates

By using fluorogenic peptidyl-3-amino-9-ethyl-carbazole a highly selective endopeptidase for the Val-Leu-Gly-Arg sequence was demonstrated in endoerythrocytic stages of Plasmodium berghei. Val-Leu-Gly-Arg-endopeptidase showed a maximum activity in pH range 7.0-8.0; it was completely inhibited by 1 mM leupeptin and 1 mM antipain. A complete inhibition was also obtained by 15 mM chloroquine. This trypsin-like activity was negligible in uninfected red blood cells. The high sensitive fluorogenic procedure could be performed on cell fractions, cell lysates as well as supernatants.

Delivery of oligonucleotides into mammalian cells by anionic peptides: comparison between monomeric and dimeric peptides.

The use of antisense oligonucleotides as putative therapeutic agents is limited by their poor delivery into the cytosol and/or the nucleus because they are not able to efficiently cross lipid bilayers. To circumvent this pitfall, anionic amphipathic peptides derived from the influenza virus fusogenic peptide have been used to destabilize membranes in an acidic environment. In this paper, we compare the ability of a monomeric and a dimeric peptide to introduce oligonucleotides into the cytosol and nuclei of several types of cultured cells. Cells incubated at pH 6.2 or at a slightly lower pH in the presence of the monomeric peptide but not the dimeric peptide were efficiently permeabilized. The location of fluorescent derivatives of peptides and of oligonucleotides was assessed by confocal microscopy. Both the peptides and oligonucleotides remained entrapped in vesicular compartments at neutral pH; at acidic pH, oligonucleotides in the presence of the monomeric peptide were mainly in the nucleus, while in the presence of the dimeric peptide they co-localized with the peptide into vesicles. The data are interpreted on the basis of the spectroscopic behaviour of monomeric and dimeric peptides in relation to the environmental pH.

Plasmodium berghei and Plasmodium chabaudi: A neutral endopeptidase in parasite extracts and plasma of infected animals

By using a sensitive fluorometric method with Val-Leu-Gly-Arg-3-amino-9-ethylcarbazole (VLGR-AEC) as a substrate, two endopeptidase activities were identified in two fractions of Sephacryl S-200 gel filtration from soluble P. berghei and P. chabaudi extracts. Controls with normal mouse erythrocytes, with leukocytes, and with reticulocyte enriched blood and different washing procedures during the preparation of soluble P. berghei extracts showed that the MW greater than 200 kDa fraction was a contaminant from erythrocytes and exhibited an optimal pH activity of 8.2. In contrast, the fraction 130 kDa was related to P. berghei and P. chabaudi and exhibited an optimal pH activity of 7.4. The two enzyme activities were compared with eight different substrates. The parasite endopeptidase showed a strong activity with Val-Leu-Gly-Lys-AEC (VLGK-AEC) and Ser-Gly-Lys-AEC (SGK-AEC) as substrates; in contrast, the mouse host endopeptidase poorly cleaved the VLGK-AEC and did not cleave SGK-AEC. Presence of the hydrophobic benzyl group on serine reduced the hydrolizing properties of P. berghei endopeptidase: the reverse was observed with host endopeptidase. The hydrolysis of the N-polyhydroxyalcanoyl-VLGK-AEC substrate by the parasite neutral endopeptidase strongly increased with the schizogonic stage, as shown with synchronized P. chabaudi in mice. By its physiological pH and specificity the release of this enzyme in mouse plasma during the infection could be of interest in a peptidyl-drug strategy.

Glycofection: The Ubiquitous Roles of Sugar Bound on Glycoplexes

Glycofection (transfection by using sugar-substituted polylysine) was assessed in order to provide an alternative to viral vectors for the transfer of genes into vascular smooth muscle cells. A rabbit vascular smooth muscle cell line (Rb-1 cells) was selectively transfected by using glycoplexes (glycosylated polylysine/pSV2LUC complexes) in the presence of 10 mu M of the fusogenic peptide GALA. A sugar-specific transfection was obtained when the glycofection was conducted for 1 h with glycoplexes containing either alpha Gal, alpha -Glc, alpha -GalNAc, beta -GlcNAc, or beta -GalNAc residues. The gene expression was high after transfection, with glycoplexes bearing alpha Gal, alpha -GalNAc, or beta -GalNAc residues that were weakly internalized, and low with glycoplexes carrying Lact or Rha residues that were well taken up by cells. These results suggest that 1) glycofection can be a good approach for a selective transfer of genes intovascular smooth muscle cells, 2) an efficient uptake of the glycoplexes i...

Uptake of Dimannoside Clusters and Oligomannosides by Human Dendritic Cells

Knowing that human blood monocyte-derived dendritic cells express cell-surface mannose-specific lectins, we prepared various mannoses containing glycoconjugates with the aim of developing highly specific synthetic carriers of oligonucleotides and genes. Conjugates were prepared from oligosaccharides obtained by hydrazinolysis of Saccharomyces cerevisiae invertase glycopeptides. The reducing saccharides were converted into glycosynthons, i.e., into glyco-amino acids. Fluorescein derivatives were obtained by coupling the free carboxyl group of oligosaccharyl-pyroglutamate to the α-amino group of ε-fluoresceinyl-thiocarbamyl lysine methyl ester. It has been shown by others that glycosylated linear oligolysines containing up to six α-D-mannopyranosylphenylthiocarbamyl units have a high affinity for the human mannose receptor. In order to obtain fully biodegradable clusters and to improve both the specificity and the selectivity, disaccharides transformed into glycosynthons were coupled to pentalysine carriers (Lys5-Ala-Cys-NH2). Glycosylated pentalysyl cysteine conjugates were made fluorescent upon substitution of the cysteine thiol group with fluorescein iodoacetamide. As shown by flow cytofluorimetry, both the dimannoside clusters and yeast oligomannosides were very efficiently taken up by DC, conversely lactoside clusters were not.