Patrick Midoux

Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers

DNA/cationic lipid (lipoplexes), DNA/cationic polymer (polyplexes) and DNA/cationic polymer/cationic lipid (lipopolyplexes) electrostatic complexes are proposed as non‐viral nucleic acids delivery systems. These DNA‐nanoparticles are taken up by the cells through endocytosis processes, but the low capacity of DNA to escape from endosomes is regarded as the major limitations of their transfection efficiency. Here, we present a current report on a particular class of carriers including the polymers, peptides and lipids, which is based on the exploitation of the imidazole ring as an endosome destabilization device to favour the nucleic acids delivery in the cytosol. The imidazole ring of histidine is a weak base that has the ability to acquire a cationic charge when the pH of the environment drops bellow 6. As it has been demonstrated for poly(histidine), this phenomena can induce membrane fusion and/or membrane permeation in an acidic medium. Moreover, the accumulation of histidine residues inside acidic vesicles can induce a proton sponge effect, which increases their osmolarity and their swelling. The proof of concept has been shown with polylysine partially substituted with histidine residues that has caused a dramatic increase by 3–4.5 orders of magnitude of the transfection efficiency of DNA/polylysine polyplexes. Then, several histidine‐rich polymers and peptides as well as lipids with imidazole, imidazolinium or imidazolium polar head have been reported to be efficient carriers to deliver nucleic acids including genes, mRNA or SiRNA in vitro and in vivo. More remarkable, histidylated carriers are often weakly cytotoxic, making them promising chemical vectors for nucleic acids delivery.

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.

Polymer-Based Gene Delivery: A Current Review on the Uptake and Intracellular Trafficking of Polyplexes

Lipoplexes and polyplexes, electrostatic complexes between a plasmid DNA and cationic lipids or polymers are chemical systems that are developed for gene delivery. Considerable efforts have been done to delineate the exact knowledge of their entry mechanisms and the intracellular routing of the plasmid DNA that are of major importance for the designing of these gene delivery systems. While the uptake of lipoplexes made with several types of cationic lipids proceeds mainly by the clathrin-dependent pathway, it appears that for polyplexes the uptake pathway is more dependent on the polymer and the cell types. So, after an overview of the current knowledge of different endocytic pathways, we present here a selection of current reports related to the entry mechanisms and intracellular routing of plasmid DNA complexed with select cationic polymers. The review includes the role of glycosaminoglycans, cell polarization and cell cycle in the polyplex uptake and their transfection efficiency. We also report current data showing that the insertion of specific kappaB motifs in the nucleic acid sequence provides an increase of the plasmid import into the nucleus. This has been demonstrated by fluorescence methods suitable to investigate the intracellular trafficking of pDNA. Overall, it appears that polyplex uptake proceeds both by the clathrin-dependent pathway and a clathrin-independent (cholesterol-dependent) pathway. These two entry mechanisms are not exclusive and can occur simultaneously in the same cell. Both of them lead to cell transfection but polyplexes still need improvements for clinical use.

Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA

UNLABELLED We report the preparation of mannosylated nanoparticles loaded with messenger RNA (mRNA) that enhance the transfection of dendritic cells (DCs) in vivo and the anti-B16F10 melanoma vaccination in mice. Mannosylated and histidylated lipopolyplexes (Man(11)-LPR100) were obtained by adding mannosylated and histidylated liposomes to mRNA-PEGylated histidylated polylysine polyplexes. Upon intravenous injection, ∼9% of the radioactivity of technetium 99 m-labeled lipopolyplexes measured in the liver, spleen, lungs, and kidneys was found in the spleen. We demonstrate that spleen from mice injected with enhanced green fluorescent protein (EGFP) mRNA-loaded Man(11)-LPR100 contained four times more DCs expressing EGFP than that from mice injected with sugar-free LPR100. This better transfection of DCs is correlated with a better inhibition of B16F10 melanoma growth and an increased survival time when mice were immunized with MART-1 mRNA-loaded Man(11)-LPR100. These results indicate that mannosylated and histidylated LPR is an efficient system for the delivery of tumor antigen mRNA in splenic DCs aiming to induce an anticancer immune response. FROM THE CLINICAL EDITOR This paper discusses the preparation of mannosylated nanoparticles loaded with messenger RNA that enhance the transfection of dendritic cells (DCs) in vivo and the anti-B16F10 melanoma vaccination in mice. The authors describe an efficient system for the delivery of tumor antigen mRNA in splenic DCs aiming to induce an anticancer immune response.

Chemical vectors for gene delivery: uptake and intracellular trafficking

Chemical vectors for non-viral gene delivery are based on engineered DNA nanoparticles produced with various range of macromolecules suitable to mimic some viral functions required for gene transfer. Many efforts have been undertaken these past years to identify cellular barriers that have to be passed for this issue. Here, we summarize the current status of knowledge on the uptake mechanism of DNA nanoparticles made with polymers and liposomes, their endosomal escape, cytosolic diffusion, and nuclear import of pDNA. Studies reported these past years regarding pDNA nanoparticles endocytosis indicated that there is no clear evident relationship between the ways of entry and the transfection efficiency. By contrast, the sequestration of pDNA in intracellular vesicles and the low number of pDNA close to the nuclear envelop are identified as the major intracellular barriers. So, intensive investigations to increase the cytosolic delivery of pDNA and its migration toward nuclear pores make sense to bring the transfection efficiency closer to that of viruses.

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.

Characterization and biological implications of membrane lectins in tumor, lymphoid and myeloid cells

Complex carbohydrates and sugar receptors at the surface of eukaryotic cells are involved in recognition phenomena. Membrane lectins have been characterized, using biochemical, biological and cytological methods. Their biological activities have been assessed using labeled glycoproteins or neoglycoproteins. Specific glycoproteins or neoglycoproteins have been used to inhibit their binding capacity in both in vitro and in vivo experiments. In adults, lymphoid and myeloid cells as well as tumor cells grow in a given organ and eventually migrate and home in another organ; these phenomena are known as the homing process or metastasis, respectively. In specific cases, membrane lectins of endothelial cells recognize cell surface glycoconjugates of lymphocytes or tumor cells, while membrane lectins of lymphocytes and of tumor cells recognize glycoconjugates of extracellular matrices or of non-migrating cells. Therefore, membrane lectins are involved in cell-cell recognition phenomena. Membrane lectins are also involved in endocytosis and intracellular traffic of glycoconjugates. This property has been demonstrated not only in hepatocytes, fibroblasts, macrophages and histiocytes but also in tumor cells, monocytes, thyrocytes, etc. Upon endocytosis, membrane lectins are present in endosomes, whose luminal pH rapidly decreases. In cells such as tumor cells or macrophages, endosomes fuse with lysosomes; it is therefore possible to target cytotoxic drugs or activators, by binding them to specific glycoconjugates or neoglycoproteins through a linkage specifically hydrolyzed by lysosomal enzymes. In cells such as monocytes, the delivery of glycoconjugates to lysosomes is not active; in this case, it would be preferable to use an acid-labile linkage. Cell surface membrane lectins are developmentally regulated; they are present at given stages of differentiation and of malignant transformation. Cell surface membrane lectins usually bind glycoconjugates at neutral pH but not in acidic medium: their ligand is released in acidic specialized organelles; the internalized ligand may be then delivered into lysosomes, while the membrane lectin is recycled. Some membrane lectins, however, do bind their ligand in relatively acidic medium as in the case of thyrocytes. The presence of cell surface membrane lectins which recognize specific sugar moieties opens the way to interesting applications: for instance, isolation of cell subpopulations such as human suppressor T cells, targeting of anti-tumor or anti-viral drugs, targeting of immunomodulators or biological response modifiers.

Sonoporation: Mechanistic insights and ongoing challenges for gene transfer

Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble-cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.

Accelerated Achilles tendon healing by PDGF gene delivery with mesoporous silica nanoparticles

We report the ability of amino- and carboxyl-modified MCM-41 mesoporous silica nanoparticles (MSN) to deliver gene in vivo in rat Achilles tendons, despite their inefficiency to transfect primary tenocytes in culture. We show that luciferase activity lasted for at least 2 weeks in tendons injected with these MSN and a plasmid DNA (pDNA) encoding the luciferase reporter gene. By contrast, in tendons injected with naked plasmid, the luciferase expression decreased as a function of time and became hardly detectable after 2 weeks. Interestingly, there were neither signs of inflammation nor necrosis in tendon, kidney, heart and liver of rat weekly injected with pDNA/MSN formulation during 1.5 months. Our main data concern the acceleration of Achilles tendons healing by PDGF-B gene transfer using MSN. Biomechanical properties and histological analyses clearly indicate that tendons treated with MSN and PDGF gene healed significantly faster than untreated tendons and those treated with pPDGF alone.

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.