Mathieu Mével

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.

Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line.

Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy. We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.

Novel neutral imidazole-lipophosphoramides for transfection assays

New helper lipids, possessing an imidazole polar head, have been synthesized and included in formulations for transfection assays; these new helper lipids can improve the transfection by a factor of up to 100 compared to the use of DOPE as co-lipid.

Selective gene delivery in dendritic cells with mannosylated and histidylated lipopolyplexes

We report for the first time preparation of mannosylated and histidylated lipopolyplexes (Man-LPD100) with uptake and transfection selectivity for dendritic cells (DCs). Man-LPD100 were prepared by addition of mannosylated and histidylated liposomes (Man-Lip100) on preformed PEGylated histidylated polylysine/DNA polyplexes. Man-Lip100 comprised a cationic [O,O-dioleyl-N-(3N-(N-methylimidazolium iodide)propylene) phosphoramidate)] lipid, a neutral [O,O-dioleyl-N-histamine Phosphoramidate] co-lipid and β-D-mannopyranosyl-N-dodecylhexadecanamide (Man-lipid). At the best, Man-Lip100 containing 11 mol % Man-lipid was obtained. We found that dialysis of liposomes completely abolished cytotoxicity. We showed that the uptake of Man11-LPD100 by the murine DC line (DC2.4 cells) was at least 10-fold higher than that of Lac6-LPD100. A confocal microscopy study with DC2.4 cells expressing Rab5-EGFP or Rab7-EGFP, revealed that DNA uptake occurred through clathrin-mediated endocytosis. The transfection of DC2.4 cells with Man11-LPD100 containing DNA encoding luciferase gene gave luciferase activity two to three times higher (9 × 105 RLU/mg protein) than with non-mannosylated LPD100. In contrast to the latter, it was inhibited by 90% in the presence of mannose. Overall, the results indicate that mannosylated and histidylated LPD is a promising system for a selective DNA delivery in DCs.

Liposome-based Formulation for Intracellular Delivery of Functional Proteins

The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

Paromomycin and neomycin B derived cationic lipids: Synthesis and transfection studies

Cationic lipid-based nonviral gene delivery is an attractive approach for therapeutic gene transfer. Basically, gene transfection can be achieved by using synthetic vectors that compact DNA, forming cationic lipoplexes which can interact with the cell plasma membrane by electrostatic interactions. Among the basic components of any cationic lipid, the type of cationic headgroup has been shown to have a major role in transfection efficiency. We have previously reported the DNA transfection potential of vectors characterized by a kanamycin A headgroup. The encouraging transfection results obtained with these compounds prompted us to evaluate the potential of cationic lipids bearing headgroups based on other aminoglycosides. Thus, we herein report the synthesis and gene transfection properties of novel cationic lipids consisting of cholesteryl or dioleyl moieties linked, via various spacers, to paromomycin or neomycin B headgroups. Our results confirm that these new aminoglycoside-based cationic lipids are efficient for gene transfection both in vitro and into the mouse airways in vivo. We also investigated physico-chemical properties of the DNA complexes formed by this particular type of synthetic vectors in order to better understand their structure-activity relationships.

DNA/amphiphilic block copolymer nanospheres reduce asthmatic response in a mouse model of allergic asthma.

Asthma is a chronic, inflammatory, respiratory disease caused by an abnormal reactivity against allergens. The most promising treatments for asthma are based on specific immunotherapies, but they lack efficiency and can induce deleterious side effects. Among new modalities of immunotherapy currently in development, DNA vaccination presents a promising approach, as it enables targeted immunotherapy in association with reduced allergenicity. We have developed an innovative, DNA-based vaccine against Dermatophagoides farinae 1 allergen (Der f 1), one of the allergens most commonly encountered by asthma patients in Europe. Intramuscular administration of a Der f 1-encoding plasmid formulated with the block copolymer 704 in healthy mice induced a strong humoral and cellular response with a pro-helper T cell type 1 bias. Administration of the same formulation in asthmatic mice, according to an early vaccination protocol, led to a reduction of airway hyperresponsiveness and a significant decrease in the level of inflammatory cytokines in the bronchoalveolar lavage of Der f 1-vaccinated mice.

DNA nanocarriers for systemic administration: characterization and in vivo bioimaging in healthy mice.

We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS), containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP), or bis (guanidinium)-tren-cholesterol (DNA MMS BGTC), and DNA lipid nanocapsules (DNA LNCs). Active targeting of the asialoglycoprotein receptor (ASGP-R) using galactose as a ligand for DNA MMS (GAL DNA MMS) and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs) should improve the properties of these DNA nanocarriers. All systems were characterized via physicochemical methods and the DNA payload of DNA LNCs was quantified for the first time. Afterwards, their biodistribution in healthy mice was analyzed after encapsulation of a fluorescent dye via in vivo biofluorescence imaging (BFI), revealing various distribution profiles depending on the cationic lipid used and their surface characteristics. Furthermore, the two vectors with the best prolonged circulation profile were administered twice in healthy mice revealing that the new DNA MMS DOSP vectors showed no toxicity and the same distribution profile for both injections, contrary to PEG DNA LNCs which showed a rapid clearance after the second injection, certainly due to the accelerated blood clearance phenomenon.

Important role of phosphoramido linkage in imidazole-based dioleyl helper lipids for liposome stability and primary cell transfection.

To optimize synthetic gene delivery systems, there is a need to develop more efficient lipid formulations. Most cationic lipid formulations contain ‘helper’ neutral lipids because of their ability to increase DNA delivery, in particular by improving endosomal escape of DNA molecules via the pH‐buffering effect of protonatable groups and/or fusion with the lipid bilayer of endosomes.

Treatment efficacy of DNA lipid nanocapsules and DNA multimodular systems after systemic administration in a human glioma model.

We previously developed different types of DNA nanocarriers for systemic administration. Recently, the biodistribution profiles of these intravenously administered nanocarriers, DNA lipid nanocapsules (LNCs) and different multimodular systems (MMS), were analysed in healthy mice using in vivo biofluorescence imaging.