Galactosylated DNA lipid nanocapsules for efficient hepatocyte targeting

The main objective of gene therapy via a systemic pathway is the development of a stable and non-toxic gene vector that can encapsulate and deliver foreign genetic materials into specific cell types with the transfection efficiency of viral vectors. With this objective, DNA complexed with cationic lipids of DOTAP/DOPE was encapsulated into lipid nanocapsules (LNCs) forming nanocarriers (DNA LNCs) with a size suitable for systemic injection (109+/-6 nm). With the goal of increasing systemic delivery, LNCs were stabilised with long chains of poly(ethylene glycol) (PEG), either from a PEG lipid derivative (DSPE-mPEG(2000)) or from an amphiphilic block copolymer (F108). In order to overcome internalisation difficulties encountered with PEG shield, a specific ligand (galactose) was covalently added at the distal end of the PEG chains, in order to provide active targeting of the asialoglycoprotein-receptor present on hepatocytes. This study showed that DNA LNCs were as efficient as positively charged DOTAP/DOPE lipoplexes for transfection. In primary hepatocytes, when non-galactosylated, the two polymers significantly decreased the transfection, probably by creating a barrier around the DNA LNCs. Interestingly, galactosylated F108 coated DNA LNCs led to a 18-fold increase in luciferase expression compared to non-galactosylated ones

Amphiphilic block copolymers enhance the cellular uptake of DNA molecules through a facilitated plasma membrane transport

Amphiphilic block copolymers have been developed recently for their efficient, in vivo transfection activities in various tissues. Surprisingly, we observed that amphiphilic block copolymers such as Lutrol® do not allow the transfection of cultured cells in vitro, suggesting that the cell environment is strongly involved in their mechanism of action. In an in vitro model mimicking the in vivo situation we showed that pre-treatment of cells with Lutrol®, prior to their incubation with DNA molecules in the presence of cationic lipid, resulted in higher levels of reporter gene expression. We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter. Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®. Microscopic examination of transfected cells pre-treated with Lutrol® confirmed that more plasmid DNA copies were internalized. Absence of cationic lipid did not impair Lutrol®-mediated DNA internalization, but critically impaired endosomal escape. Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways

Probing the in vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale

Cationic lipids are used for delivering nucleic acids (lipoplexes) into cells for both therapeutic and biological applications. A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy. Here, we developed a labelling protocol using aminated nanoparticles as markers for plasmid DNA to examine the intracellular route of lipoplexes in cell lines using transmission electron microscopy. Morphological changes of lipoplexes, membrane reorganizations and endosomal membrane ruptures were observed allowing the understanding of the lipoplex mechanism until the endosomal escape mediated by cationic lipids. The study carried out on two cationic lipids, bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) and dioleyl succinyl paramomycin (DOSP), showed two pathways of endosomal escape that could explain their different transfection efficiencies. For BGTC, a partial or complete dissociation of DNA from cationic lipids occurred before endosomal escape while for DOSP, lipoplexes remained visible within ruptured vesicles suggesting a more direct pathway for DNA release and endosome escape. In addition, the formation of new multilamellar lipid assemblies was noted, which could result from the interaction between cationic lipids and cellular compounds. These results provide new insights into DNA transfer pathways and possible implications of cationic lipids in lipid metabolism

Glycosylation-mediated targeting of carriers

For safe and effective therapy, drugs should be delivered selectively to their target tissues or cells at an optimal rate. Drug delivery system technology maximizes the therapeutic efficacy and minimizes unfavorable drug actions by controlling their distribution profiles. Ligand-receptor binding is a typical example of specific recognition mechanisms in the body; therefore, ligand-modified drug carriers have been developed for active targeting based on receptor-mediated endocytosis. Among the various ligands reported thus far, sugar recognition is a promising approach for active targeting because of their high affinity and expression. Glycosylation has been applied for both macromolecular and liposomal carriers for cell-selective drug targeting. Recently, the combination of ultrasound exposure and glycosylated bubble liposomes has been developed. In this review, recent advances of glycosylation-mediated targeted drug delivery systems are discussed

Transports intracellulaires ciblés de macromolécules biologiques

Le développement de nouveaux systèmes capables de transporter spécifiquement in vitro et in vivo des acides nucléiques dans des cellules cibles représente aujourd'hui un enjeu majeur pour le traitement de pathologies héréditaires ou acquises. Les vecteurs synthétiques cationiques utilisés jusqu'à présent s'avèrent très efficaces pour le transfert de gène in vitro mais la forte densité de charges positives présentes à la surface des complexes vecteurs/ADN conduit à une transfection non spécifique dans un type cellulaire donné. Ainsi un système alternatif a été développé pour s affranchir des interactions non spécifiques avec les membranes cellulaires. Ce système correspond à un assemblage supramoléculaire proche de l électroneutralité constitué d un cœur d ADN condensé et d une couronne périphérique de stabilisateurs stériques ioniques ou non ioniques fonctionnalisés par des résidus galactose afin de cibler spécifiquement le récepteur aux asialoglycoprotéines présent à la surface des hépatocytes. La présence des résidus galactose permet à ce système d exprimer spécifiquement un transgène dans les hépatocytes primaires alors que le système multimodulaire non galactosylé est incapable de transfecter ces cellules. Dans une deuxième partie, une nouvelle génération de vecteurs synthétiques a été développée pour le transfert de gène dans le tissu pulmonaire ce qui constitue une alternative indispensable aux lipides cationiques couramment utilisés lors des essais cliniques qui induisent une forte toxicité se traduisant par une importante inflammation au niveau des voies aériennes. Ce nouveau système de vectorisation est constitué de copolymères à blocs amphiphiles fonctionnalisés par un ligand de type glycosidique qui permet d'améliorer l'efficacité de transfection des copolymères à blocs natifs en transfectant un plus grand nombre de cellules épithéliales pulmonaires.Targeted gene transfer is a promising approach to treat various acquired or hereditary diseases. Numerous synthetic cationic vectors have been synthetized and are successfully used for in vitro gene transfer but an excess of positive charges lead to cytotoxicity and does not enable specific transfection. Therefore, we decided to design alternative molecular gene delivery systems to target cellular internalization through the receptor-mediated endocytosis pathway instead of the regular non-specific electrostatic interactions with cell membranes. This alternative system corresponds to a new neutral supramolecular assembly of lipoplexes equipped with galactose residues to specific target asialoglycoprotein receptors located on hepatocytes. The presence of galactose residues on this alternative molecular system led to specific transfection of primary hepatocytes through a specific endocytosis whereas ungalactosylated multimodular system did not transfect at all. Then, a novel class of galactosylated amphiphilic block copolymers were developed for pulmonary gene transfer as an essential alternativ to cationic lipids which lead to an important inflammation in the airways. The grafting of glycosyl residues onto the distal ends of block copolymers led to a significantly higher transfection efficiency in the lungs than unsubstituted block copolymers. Indeed, galactosylated block copolymers allows transgene expression in more epithelial pulmonary cells.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

Galactosylated multimodular lipoplexes for specific gene transfer into primary hepatocytes.

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