Common Non-Viral Gene Vectors
Gene therapy is the transfection of genes (plasmid DNA, siRNA and miRNA, etc.) into specific cells to promote or inhibit the expression of the target protein to achieve the purpose of treating human diseases. Because RNA and DNA gene fragments are negatively charged and are easily degraded by nucleases, it is difficult to pass through negatively charged cell membranes. Therefore, selecting appropriate gene carriers to protect and transport gene fragments into cells is an urgent problem to be solved. Viral vectors and non-viral vectors are the two most commonly used gene transfer vectors. Although viral vectors have high transfection efficiency, their shortcomings such as immunogenicity, tumorigenicity, and difficulty in mass production limit their clinical applications. Natural polysaccharide polymers, cationic polymers, liposomes and other non-viral vectors have the advantages of simple structure and weak immunogenicity, and are a class of gene carriers with very promising applications.
Natural Polysaccharide Polymer
Currently, chitosan, glucan, cyclodextrin, mannan, hyaluronic acid, etc. are the most common gene carriers for natural polysaccharides. Polysaccharides are composed of multiple repeating units and groups, which determine their solubility, surface charge, and ease of chemical modification, and ultimately affect the transfection efficiency.
Chitosan is a natural biological macromolecule formed by deacetylation of chitin. It has good biocompatibility, degradability and low immunogenicity. It can interact with negatively charged DNA, siRNA and miRNA, etc. through electrostatic attraction, and form a nano-polymer. It can not only protect these genes from nuclease degradation, but also help cells to adhere and swallow.
Dextran is a polysaccharide polymer composed of multiple repeating glucose units and has good biocompatibility. The structure of many hydroxyl groups of dextran is easy to be chemically modified, and it has been widely used in the research of gene transfection and therapy. Scholars often modify dextran to a certain extent, and use dextran derivatives to carry genes.
- Cyclodextrin
Cyclodextrin is a general term for a series of cyclic oligosaccharides, usually containing 6-12 D-glucopyranose units, and is produced by cyclodextrin glucosyltransferase enzymatically hydrolyzing amylose. As a natural biological material, cyclodextrin has no immunogenicity and good biocompatibility. The introduction of modifiers to modify the physical and chemical properties of cyclodextrin has become one of the key research directions of scholars. Studies have shown that the modification of cyclodextrin with polyethyleneimine (PEI) can significantly improve the transfection efficiency.
- Mannan
Mannan is a highly branched polymer with mannose as a monomer, which is widely present in many life forms. It can be derived from natural products such as konjac, aloe, seaweed, yeast, etc. Researchers connect spermine to the side chain of mannan, the grafting rate is about 12%, and then this mannan-spermine is loaded with DNA and transfected In macrophages with high expression of mannose receptor, the transfection efficiency was found to be 28.5 times that of spermine pullulan, 11.5 times that of PEI and 3.0 times that of liposomes 2,000. It is proved that the mannan-spermine transfection system is a stable specific transfection system mediated by macrophage mannose receptor.
In vitro, liposomes are one of the most commonly used gene transfection reagents. Cationic liposomes are positively charged on the surface and can be combined with negatively charged siRNA, miRNA and other nucleic acids. By encapsulating the nucleic acid, it protects it from degradation by nucleases in the blood and maintains its integrity and biological activity. However, due to the high toxicity of liposomes, non-specific cellular uptake and unnecessary immune response and other shortcomings, it is difficult to achieve safe and effective application in the body. The surface of the cationic liposome/nucleic acid complex is positively charged, and can be combined with the negatively charged cell membrane through electrostatic adsorption, and then the nucleic acid is introduced into the cell nucleus through cell membrane fusion.
Poly (lactide-co-glycolide) (PLGA)
PLGA polymer is a kind of water-insoluble high molecular organic polymer, which has the advantages of good biocompatibility, low toxicity and low immunogenicity. Many previous studies have focused on the efficiency of PLGA polymer-loaded miRNA and the process of non-specific transfection into cells. Recent studies have shown that PLGA also has the characteristics of controlled release miRNA. Cationic polymers (such as PEI, chitosan) modified PLGA can stabilize the zeta potential on the surface and increase the adsorption efficiency of miRNAs. PLGA polymers can easily graft other active molecules and form microparticles and nanoparticles. When PLGA nanoparticles are loaded with nucleotides such as DNA, miRNA, and siRNA, they have high drug loading capacity and can resist nucleic acid degradation.
Polyethyleneimine (PEI)
PEI is a widely studied polymer gene carrier, which is easy to synthesize and low in price. In a physiological environment, amine protonation makes it positively charged and can combine with nucleic acid to form a polymer. The cationic complex formed by PEI and nucleic acid usually retains a net positive charge, which can bind non-specifically to the negatively charged glycoproteins and proteoglycans on the cell surface. Once the complex is bound to the cell surface, it then enters the cytoplasm through endocytosis. The release process of PEI/nucleic acid polymer in the cytoplasm is mainly to promote the release of nucleic acid into the cytoplasm through a so-called “proton sponge” effect. The transfection efficiency of PEI largely depends on its own physical and chemical properties (such as relative molecular mass, degree of branching, and cationic charge density), the characteristics of nucleic acid-forming complexes (such as particle size, interface potential), and specific experimental conditions (such as incubation time, polymer concentration). Using PEI as a carrier, loading DNA and siRNA has been successfully transfected in animal models.
Inorganic Nano Carrier
With the widespread application of bio-nanotechnology in medicine, the application of inorganic nanocarriers in gene non-viral transport vectors has gradually become a hot research field. Currently commonly used non-viral gene carriers mainly include the following: metal nanoparticles, such as gold nanoparticles, magnetic nanoparticles; inorganic non-metallic nanoparticles, such as silica, calcium phosphate, and hydroxyapatite; biodegradable polymers Nanoparticles, such as polylactic acid microspheres, nanogels, and biological particles, such as proteins, glycoproteins, etc. These nanoparticle carriers have the advantages of low toxicity, no immunogenicity, easy modification, monodisperse particle size and long metabolic time in the body. Gold nanoparticles have a variety of excellent biological properties, such as easy surface functionalization, strong gene loading capacity, low cytotoxicity, and high transfection efficiency.
Hydrogel Carrier
Hydrogel is formed by cross-linking or self-assembly of a variety of natural or synthetic hydrophilic polymers. It has a certain degree of sustained release for genes, proteins, anti-tumor drugs, etc., has a high encapsulation rate and is convenient to load drugs. It is very suitable as a carrier and storage library for nucleic acids such as miRNA, and can continuously release gene drugs.