Annie-Claude Roche

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.

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.

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.

In vivo gene delivery in the mouse lung with lactosylated polyethylenimine, questioning the relevance of in vitro experiments.

Polyethylenimine (PEI) is an efficient vector for in vitro and in vivo gene transfer into respiratory cells. Glycosylated PEIs were shown to enhance in vitro gene transfer by favoring the complex entry into the airway cells. The aim of our study was to evaluate the in vivo efficiency of gene transfer mediated by glycosylated PEIs in the mouse lung and to determine the transfected cell type and the intracellular trafficking of the complexes. Upon nasal instillation in mice of complexes made with various glycosylated PEIs, a high luciferase activity was observed while the green fluorescent protein (GFP) expression was similar for all the vectors tested with few cells expressing GFP. Complexes made with lactosylated PEI were then labeled and their localization studied by confocal microscopy. In the lungs, large numbers of complexes were taken up by epithelial cell which were mostly alveolar cells. In the airways, complex uptake varied greatly, depending on the area observed. Eight hours upon nasal instillation and in contrast with the in vitro situation, a dissociation between the plasmid DNA and the lactosylated PEI was usually observed, leading to the plasmid mostly localized in lysosomes and the Lac-PEI localized in the nucleus. These results emphasize the need to engineer a plasmid able by itself to overcome the nuclear barrier and to quickly move to in vivo experiments to select the best carrier.

Preparation and biological properties of a covalent antitumor drug—arm—carrier (DAC conjugate)

Cancer chemotherapeutic agents are not selective in their action against cancer cells. Therefore, the possibility of using a macromolecule, such as an antibody molecule, as a specific carrier of antitumor drugs has attracted considerable attention [ 1,2]. For instance, daunorubicin was covalently linked to antibodies [3-61 or to Fab dimers [7] or to a lectin [8] by coupling methods using glutaraldehyde, carbodiimide and periodate oxidation of the drug. For technical reasons, and because the activity of a drug is partially or totally lost when it is substituted or chemically modified, we devised a spacer arm such that the drug-carrier conjugate is stable in serum and can be specifically split by lysosomal proteases leading to the free drug inside the target cells.

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.

Endocytosis of α1-acid glycoprotein variants and of neoglycoproteins containing mannose derivatives by a mouse hybridoma cell line (2C11–12). Comparison with mouse peritoneal macrophages

Macrophages from various origins are known to express membrane lectins that mediate the endocytosis of mannose-bearing glycoconjugates. Most macrophage tumor cell-lines lack such receptors. In this paper we show by flow cytometry analysis that a newly generated macrophage hybridoma (2C11–12), which displays several macrophage characteristics, also expresses mannose membrane lectins, resulting in the internalization of fluoresceinylated neoglycoproteins into acidic compartments.Thioglycolate elicited mouse peritoneal macrophages and the 2C11–12 hybridomas were compared by flow cytometry with regard to the binding and endocytosis of α1-acid glycoprotein (AGP) variants separated by affinity chromatography on immobilized concanavalin A. AGP C eluted specifically with methyl α-mannopyranoside, which contains two bi-antennary oligosaccharides, was endocytosed as mannosylated serum albumin (Man-BSA). In both types of macrophages, the fluoresceinylated ligands were internalized in acidic compartments as demonstrated by the fluorescence intensity increase upon monensin post-incubation. However the behaviour of the internalized ligands was found to be quite different. AGP C and Man-BSA were rapidly degraded by thioglycolate elicited peritoneal macrophages and excreted in the medium as small peptide fragments; conversely they remained a longer time in the 2C11–12 hybridoma.

Endocytosis of α1-acid glycoprotein variants by human monocytic lineage cells

Summary— Human α1‐acid glycoprotein (AGP or orosomucoid) is a major glycoprotein of plasma. AGP can be separated on immobilized concanavalin A into three variants bearing none (AGP A), one (AGP B) or two (AGP C) biantennary glycans. In this paper, we show, using flow cytometry and confocal microscopy, that AGP C which is eluted from concanavalin A with mannose, binds to human monocytes, monocyte‐derived macrophages as well as human promonocytic cell lines such as THP1 or U937. Conversely HL60, a promyelocytic cell line, does not express the surface AGP C binding protein. AGP C is internalized and degraded with an efficiency depending on the state of differentiation of these cells. In contrast, AGP A which is not recognized by concanavalin A, does not bind to any of these cells.