Liposome-base Transfection Reagents
The liposomes are cationic lipids that encapsulate the negatively charged nucleic acid sequences and are attracted by the cell membrane and integrated by endocytosis into the host cell. These vesicles are artificially created from phospholipids, mainly from mixtures containing both negatively charged and neutral lipids that are capable of spontaneously interacting with the nucleic acids, forming stable structures that enter the cells. The method is relatively not toxic and ensures the integration of large DNA fragments into the host cell, with a high efficacy. The endosome enzymes degrade the liposome, and the DNA fragment is released into the cytosol.
Polyethylene glycol –cholesterol (PEG-Ch) conjugates modified with targeting ligands and molecular conjugates are used for improving the efficacy of the liposome transfection. The Ch2 –PEGn can be obtained by esterification, using succinic anhydride cholesterol and polyethylene glycol dissolved in chloroform, concentrated under vacuum and purified on silica-gel. The cationic liposomes can be obtained by dissolving chloroform, Ch2 –PEGn, and DOTAP in chloroform and the removal of the solvent under a rotary evaporator that enhances the formation of a thin lipid film that can be further dried under vacuum, addition of 5% glucose solution and sonication.
Polymer-based Transfection Reagents
The cationic polymers bind to the negatively charged nucleic acid sequences and the positively charged complex is attracted by the cell membrane and integrated by endocytosis into the host cell. Polymers obtained from PLGA (poly-lactic-co-glycolic acid), a food grade polymer that can be safely used for transfection procedures, and PBAE (poly-β-amino ester), a polymer that is capable of protecting DNA molecules to be delivered inside the host cell, can be used for the safe and efficient delivery of DNA constructs into cultured cells. The double emulsion solvent removal method can be used for preparing the polymer microparticles, with dichloromethane as a solvent, and aqueous solution containing DNA plasmids or RNA-based constructs. Following homogenization and centrifugation, the lyophilization with mannitol can be performed to generate polymer particles used for DNA vaccine development.
The number of polyethylenimine-based polymers were developed and used for transient and stable transfection in several mammalian cell lines, as well as insect and bacterial cells.
Nanoparticle-based Transfection Reagents
Nanoparticles are non-viral vectors consisting of inorganic and organic particles and polymers, which can be used for gene delivery, avoiding the toxicity and compatibility problems if the natural biopolymers such as lipids, proteins, and sugars are used. These vectors ensure gene delivery into specific cells or into specific tissues, for cancer therapy and also for diabetes, autoimmune diseases and genetic deficiencies.
Nanoparticles consisting of polyethylenimide and tripolyphosphate can be developed using freeze-dry method . Scientists used nanoparticles for siRNA and plasmid transfection into cancer cell lines and primary cell cultures.
Cell Line Specific Transfection Reagents
Transfection by calcium phosphate was one of the earliest chemical based methods developed to deliver exogenous nucleic acids to cultured mammalian cells. Advances in the development of nanoparticles, liposomes, lipid and polymer-based carrier molecules capable of binding and encapsulating nucleic acids led to the development of new generation of transfection reagents – optimized and cell line specific transfection reagents that provide high transfection efficacy with minimal cytotoxicity. There are a number of commercially available pre-optimized transfection reagents and associated transfection protocols for both in votro and in vivo applications – see cell line-specific cell transfection kits and invivo transfection reagents. These reagents and transfection methods are widely used in in basic life sciences research and associated clinical studies.
Altogen Biosystems is a life sciences company dedicated to the development, marketing and manufacture of cell type specific transfection reagents. Efficient delivery of DNA, RNA, and siRNA enabled by advanced formulation of reagents and peculiar design of protocols. Altogen Biosystems offers a complete transfection system for a broad range of cell lines. All reagents are functionally tested to be highly reproducible, serum compatible, induce low toxicity, and can be used for co-transfection experiments, and high throughput applications.