Michel Monsigny

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.

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.

Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives

To investigate the nuclear import mechanism of plasmid/polyethylenimine (PEI) derivative complexes and the putative nuclear targeting of therapeutic genes by the use of oligosaccharides, we have studied the nuclear import of plasmid DNA complexed either with PEI or with lactosylated PEI (Lac–PEI) in cystic fibrosis human airway epithelial cells (∑CFTE29o‐ cells).

Potocytosis and cellular exit of complexes as cellular pathways for gene delivery by polycations.

Although polycations are among the most efficient nonviral vectors for gene transfer, the gene expression they allow is still too low for in vivo applications. To engineer more potent polycationic vectors, the factors governing the intracellular trafficking of a plasmid complexed with current polycations need to be identified.

Lactosylated polyethylenimine for gene transfer into airway epithelial cells: role of the sugar moiety in cell delivery and intracellular trafficking of the complexes.

As we have previously shown that lactosylated polyethylenimine (PEI) is the most efficient glycosylated PEI for gene transfer into human airway epithelial cells in primary culture, we have studied here the role of the lactose residue in the enhancement of gene transfer efficiency observed with lactosylated PEI as compared with unsubstituted PEI in immortalized (ΣCFTE29o‐ cells) and primary human airway epithelial cells.

Uptake of plasmid/glycosylated polymer complexes and gene transfer efficiency in differentiated airway epithelial cells.

We have studied gene transfer efficiency of glycosylated polylysines and glycosylated polyethylenimines as vectors in immortalized differentiated airway gland serous cells and primary cultures of human airway surface epithelial cells.

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.

Tumoricidal activation of murine alveolar macrophages by muramyldipeptide substituted mannosylated serum albumin

Rat and mouse alveolar macrophages have almost no spontaneous tumoricidal activity and are only slightly activated by muramyldipeptide (MDP). When MDP was carried by serum albumin, the activation was higher than with free MDP but only at high concentration. When MDP was bound to a neoglycoprotein (mannosylated serum albumin) - which binds to the sugar binding receptor at the macrophage cell surface and is actively endocytosed - the activation of rat or mouse alveolar macrophages is dramatically enhanced even at very low concentration of neoglycoprotein -bound MDP. Furthermore, neoglycoprotein -bound MDP injected i.v. or i.p. was found to be able to activate alveolar macrophages, the activity of which was maximal after 48 hours in mice and 72 hours in rats. Such conjugates have so potential values as new immunostimulant agents in cancer and parasite therapy.

Membrane lectins on human monocytes: Maturation-dependent modulation of 6-phosphomannose and mannose receptors

Freshly isolated human monocytes, which do not contain cell‐surface mannose‐specific receptors, bind mannose 6‐phosphate and actively endocytose mannose 6‐phosphate‐bearing neoglycoproteins (6‐P‐Man‐F‐BSA). Three days after isolation, human monocytes endocytose very actively 6‐P‐Man‐F‐BSA as well as Man‐F‐BSA, and the endocytosed neoglycoproteins are rapidly degraded. These results were obtained in quantitative flow cytofluorometry by using a panel of fluoresceinylated sugar‐substituted serum albumins (neoglycoproteins). Thus, in contrast to mannose receptors which appear only after maturation, mannose 6‐phosphate receptors are already present on freshly isolated human monocytes.

Histidylated polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells.

We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes.