Chantal Pichon

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.

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.

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENETM6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5–7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

Synthesis and Transfection Activity of New Cationic Phosphoramidate Lipids: High Efficiency of an Imidazolium Derivative

In an effort to enhance the gene‐transfer efficiencies of cationic lipids and to decrease their toxicities, a series of new phosphoramidate lipids with chemical similarity to cell membrane phospholipids was synthesised. These lipids contained various cationic headgroups, such as arginine methyl ester, lysine methyl ester, homoarginine methyl ester, ethylenediamine, diaminopropane, guanidinium and imidazolium. Their transfection abilities, either alone or with the co‐lipid DOPE, were evaluated in HEK293–T7 cells. We found that imidazolium lipophosphoramidate 7 a/DOPE lipoplexes gave the most efficient transfection with low toxicity (15 %). The luciferase activity was 100 times higher than that obtained with DOTAP/DOPE lipoplexes. The size, ζ potential, pDNA–liposome interactions and cellular uptakes of the lipoplexes were determined. No definitive correlation between the ζ potential values and the transfection efficiencies could be established, but the uptake of lipoplexes by the cells was correlated with their final transfection efficiencies. Our results show that imidazolium phosphoramidate lipids constitute a potential new class of cationic lipids for gene transfer.

Nuclear delivery of NFκB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging

Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized κB motif to favour NFκB-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Förster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was ∼1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NFκB activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NFκB. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the gene-expression efficiency.