Jean-Serge Remy

Chitosan-Based Vector/DNA Complexes for Gene Delivery: Biophysical Characteristics and Transfection Ability

AbstractPurpose. Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. Methods. Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. Results. In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50−100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time, from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. Conclusions. Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.

The Magnetofection Method: Using Magnetic Force to Enhance Gene Delivery

Abstract In order to enhance and target gene delivery we have previously established a novel method, termed magnetofection, which uses magnetic force acting on gene vectors that are associated with magnetic particles. Here we review the benefits, the mechanism and the potential of the method with regard to overcoming physical limitations to gene delivery. Magnetic particle chemistry and physics are discussed, followed by a detailed presentation of vector formulation and optimization work. While magnetofection does not necessarily improve the overall performance of any given standard gene transfer method in vitro, its major potential lies in the extraordinarily rapid and efficient transfection at low vector doses and the possibility of remotely controlled vector targeting in vivo.

Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse†

Several nonviral vectors including linear polyethylenimine(L‐PEI) confer a pronounced lung tropism to plasmid DNA when injected into the mouse tail vein in a nonionic solution.

Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI)

The ideal non‐viral vector should be cell‐type directed and form complexes with DNA that are physically stable, small and electrically neutral.

A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin

Cell transfection requires cationic DNA complexes and heparan sulfate proteoglycans (HSPGs) at the cell surface. Syndecans are transmembrane HSPGs that are ubiquitously expressed on adherent cells. Their polyanionic heparan sulfate moieties are bound at the distal end of their ectodomain, thus facilitating interaction with large cationic particles.

Gene transfer with lipospermines and polyethylenimines

It is an obvious and basic principle that to be efficient, gene therapy requires effective gene transfer followed by adequate gene expression. However, getting DNA, a pro-drug, into the cell and into the nucleus, remains a crucially limiting factor. Even recombinant viral methods still show poor performances in clinical situations and non-viral methods are considered classically to be of yet lower efficiency. Here, we consider the mode of action, the nature of the complexes formed with DNA and the transfection potentials of two categories of inert, cationic vectors, the lipospermines and polyethylenimine. Both are among the best vectors currently available for in vitro work. Moreover, polyethylenimine is proving to be a versatile and effective carrier for different in vivo situations, especially for delivering genes into the mammalian brain.

Proton Sponge Trick for pH-Sensitive Disassembly of Polyethylenimine-Based siRNA Delivery Systems

Small interfering RNAs offer novel opportunities to inhibit gene expression in a highly selective and efficient manner but depend on cytosolic translocation with synthetic delivery systems. The polyethylenimine (PEI) is widely used for plasmid DNA transfection. However, the water-soluble PEI does not form siRNA polyplexes stable enough in extracellular media for effective delivery. We previously showed that rendering PEI insoluble in physiological media, without modifying drastically its overall cationic charge density, by simple conjugation with natural hydrophobic alpha-amino acids, can lead to effective siRNA delivery in mammalian cells. In here, we comprehensively investigated the mechanism behind the excellent efficacy of the leading PEIY vector. Our data revealed that the underlining proton sponge property is key to the effectiveness of the tyrosine-polyethylenimine conjugate as it may allow both endosomal rupture and siRNA liberation via an optimal pH-sensitive dissolution of the PEIY self-aggregates. Altogether, these results should facilitate the development of novel and more sophisticated siRNA delivery systems.

Effective polyethylenimine-mediated gene transfer into human endothelial cells.

The major advantage in choosing non‐viral vectors such as cationic polymers for in vitro and in vivo transfection is their higher biosafety than viral ones. Among the cationic polymers, polyethylenimines (PEIs) are promising molecules for gene delivery to a variety of cells. Efficient transfection of primary endothelial cells using PEIs could be regarded as an interesting strategy of treatment in some ischemic cardiovascular diseases.

Genuine DNA/polyethylenimine (PEI) Complexes Improve Transfection Properties and Cell Survival

Polyethylenimine (PEI) has been described as one of the most efficient cationic polymers for in vitro gene delivery. Systemic delivery of PEI/DNA polyplexes leads to a lung-expression tropism. Selective in vivo gene transfer would require targeting and stealth particles. Here, we describe two strategies for chemically coupling polyethylene glycol (PEG) to PEI, to form protected ligand-bearing particles. Pre-grafted PEG–PEI polymers lost their DNA condensing property, hence their poor performances. Coupling PEG to pre-formed PEI/DNA particles led to the expected physical properties. However, low transfection efficacies raised the question of the fate of excess free polymer in solution. We have developed a straightforward a purification assay, which uses centrifugation-based ultrafiltration. Crude polyplexes were purified, with up to 60% of the initial PEI dose being removed. The resulting purified and unshielded PEI/DNA polyplexes are more efficient for transfection and less toxic to cells in culture than the crude ones. Moreover, the in vivo toxicity of the polyplexes was greatly reduced, without affecting their efficacy.

Polycationic Pillar(5)arene Derivatives: Interaction with DNA and Biological Applications

Dendritic pillar[5]arene derivatives have been efficiently prepared by grafting dendrons with peripheral Boc-protected amine subunits onto a preconstructed pillar[5]arene scaffold. Upon cleavage of the Boc-protected groups, water-soluble pillar[5]arene derivatives with 20 (13) and 40 (14) peripheral ammonium groups have been obtained. The capability of these compounds to form stable nanoparticles with plasmid DNA has been demonstrated by gel electrophoresis, transmission electron microscopy (TEM), and dynamic light scattering (DLS) investigations. Transfection efficiencies of the self-assembled 13/pCMV-Luc and 14/pCMV-Luc polyplexes have been evaluated in vitro with HeLa cells. The transfection efficiencies found for both compounds are good, and pillar[5]arenes 13 and 14 show very low toxicity if any.