Hagit Eliyahu

Synthesis of Cationic Polysaccharides and Use for In Vitro Transfection

Polycations are effective nonviral carriers for gene delivery systems. These carriers vary in molecular weight, polymer structure, polymer:DNA ratio, molecular architecture, and the ability to introduce target-specific moieties. Polycations are capable of complexing various plasmids and transfecting them into different cells to produce a high yield of a desired protein. Cationic polysaccharides are attractive candidates for gene delivery. They are natural or semi-natural, nontoxic, biodegradable, and biocompatible materials that can be modified for improved physicochemical properties. Cationic polysaccharides are synthesized by conjugation of various oligoamines to oxidized polysaccharides via reductive amination. These conjugates have been rigorously tested for gene delivery in cultured cells and in animals. From more than 300 polysaccharide-oligoamine derivatives tested, only dextran-spermine (D-SPM) was found to be highly effective in gene transfection, both in vitro and in vivo. This protocol describes the synthesis of cationic polysaccharides and their use for DNA transfection in vitro.

Cationic Lipid-Nucleic Acid Complexes (Lipoplexes): From Physicochemical Properties to In Vitro and In Vivo Transfection Kits

Lipoplexes are complexes formed spontaneously upon mixing of negatively charged nucleic acids (or other polyelectrolytes such as proteins) with positively-charged lipid assemblies [1]( for definitions see [2]). The relevant nucleic acids include plasmid DNA (pDNA), linear DNA, single chain DNA, oligonucleotide (ODN), messenger RNA, and silencing double stranded short RNA (siRNA) [1, 2, 3] The lipoplex-mediated nucleic acid delivery appears to be a promising system for a broad spectrum of both in vitro and in vivo applications in life sciences, biotechnology, medicine, and agriculture [1]. These applications are referred to as “lipofection”, namely, lipoplex- mediated transfection [2]. Among all currently available nonviral delivery systems, lipoplexes are one of the most popular and most versatile. This “popularity” is explained by the following: