Lipofection

Liposomal transfection delivery system

Lipofection is a lipid-based transfection technology which belongs to biochemical methods including also polymers, DEAE dextran and calcium phosphate. The main advantages of lipofection are its high efficiency, its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility and low toxicity. In addition this method is suitable for all transfection applications (transient, stable, co-transfection, reverse, sequential or multiple transfections…), high throughput screening assay and has also shown good efficiency in some in vivo models.

DNA transfection mechanisms

The lipid-based reagents used for lipofection are generally composed of synthetic cationic lipids that are often mixed with helper lipids such as DOPE (L-a-dioleoyl phosphatidyl-ethanolamine) or cholesterol. These lipids mixture assembles in liposomes or micelles with an overall positive charge at physiological pH and are able to form complexes (lipoplexes) with negatively charged nucleic acids through electrostatics interactions. The association of the lipid-based transfection reagent with nucleic acids results in a tight compaction and protection of the nucleic acids and these cationic complexes are mainly internalized by endocytosis. Once inside the cells two mechanisms leading to the nucleic acids release into the cytoplasm have been described. One relies on the endosomes buffering capacity of the polycationic residues (called “proton sponge effect”). The other describes the ability of cellular negatively charged lipids to neutralize the cationic residues of the transfection reagent leading to destabilization of endosomal membranes. Finally, the cellular and molecular events leading to the nuclear uptake of DNA (not required for siRNA) following by gene expression remain highly speculative. However, the significance of cell division on transfection efficiency favours the assumption that nuclear membrane disruption during the mitosis process promote DNA nuclear uptake. Nonetheless, transfections of primary cells (non-dividing) and in vivo are also achievable with lipofection demonstrating that DNA can make its way to the nucleus where gene expression takes place.

Tee-Technology

The cationic lipids (lipoplexes) and polymers (polyplexes) are the most employed non-viral gene delivery systems. The Tee-Technology (Triggered Endosomal Escape) combines and exploits the properties of both entities to achieve extremely efficient nucleic acids delivery into cells. Indeed, this new generation of lipopolyamines contains a lipophilic part, like lipids, and a charged polyamine moiety, like cationic polymers. These moieties act in synergy to ensure a tight nucleic acids compaction and protection and a very efficient destabilization of the endosomal membrane which allows the release of large nucleic acids amounts in the cytosol and DNA nuclear uptake. A particular focus on the synthesis of fully biodegradable entities was integrated. In this way, the transfection reagents do not interfere with cellular mechanisms, high cell viability is maintained in every experiment and any potential secondary effects are avoided.

The principal Tee-Technology advantages are:

• Compaction of DNA in nanoparticles efficiently internalized by cells

• Protection of nucleic acids against nucleases degradation

• Efficient membrane destabilization and DNA delivery

• Highly efficient even with low amounts of nucleic acids

• Biodegradability

Lipofection applications and results

Transfection efficiency combines with high transgene expression level or high gene silencing and minimize cytotoxicity depends on multiple critical parameters. Those factors include cell type, plasmid DNA characteristics (size, promoter, reporter gene) & purity, siRNA sequence & purity, cell culture conditions (medium with or without serum, cell number, absence of contaminations…), amount of nucleic acids and reagents, transgene assays to name a few. Consequently, transfection reagents need to be specifically designed according to the nucleic acids to be delivered (DNA, siRNA, mRNA, ODN, shRNA etc.) and the cell types used in order to achieve optimal efficiency. In this context, OZ Biosciences has developed several outstanding transfection reagents:

Transfection of various cell lines with DreamFect™ Gold

Transfection of Jurkat cells with DreamFect	Gene silencing in different cell lines with Lullaby ™

GFP inhibition in HeLa cells with Lullaby™