Two-stage targeted ternary complex nucleic acid delivery system and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to a D type IF-7 polypeptide-modified reductive dendrimer-like macromolecule (RHB) self-assembled double-targeted nucleic acid transfer and release system and a preparation method thereof. The preparation method comprises the following steps of automatically compressing plasmid DNA (deoxyribonucleic acid) by reductive dendrimer-like cationic polymer polyamino ammonia (RHB), so as to form a binary complex; using an inverted sequence D type technique to transform an IF7 peptide of a targeted tumor blood vessel endothelial cell surface specificity receptor Anxal, and synthesizing RIF7 polypeptide; performing amidation reaction to combine the RIF7 polypeptide onto an HA molecular structure of a CD44 receptor on the surface of a targeted tumor cell, and synthesizing a double-targeted coating material; under the electrostatic action, coating the formed RHB / DNA binary complex, so as to form a targeted ternary complex nucleic acid transfer and release system. The preparation method has the advantages that the transfer and release system can effectively compress and coat the plasmid DNA; by utilizing the double-targeted action, the complex can cross tumor blood vessel barriers and tumor cytomembrane barriers step by step, the transfection efficiency of a gene is improved, and the toxicity of a carrier system is decreased.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a non-viral nucleic acid delivery system and a preparation method thereof, in particular to a reducing dendritic macromolecule (RHB) modified by a D-type IF-7 polypeptide. ) self-assembled dual targeting ternary complex nucleic acid delivery system and preparation method thereof. Background technique [0002] Gene therapy is a hot spot in the field of current tumor therapy research; the key to the application of this technology is the need for a safe and efficient gene delivery carrier system. Compared with viral vectors, non-viral vectors have the characteristics of low toxicity, easy synthesis, low immune response, and targetable modification. Cationic polymer is a kind of non-viral gene delivery carrier that has been researched more at present, commonly used are polyethyleneimine (Polyethyleneimine, PEI), chitosan and its derivatives, polylysine (Poly(L-lysine), PLL), poly...

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Problems solved by technology

[0004] ①High toxicity: the surface of the cationic polymer has a large amount of positive charges, and it is easy to electrostatically interact with the negatively charged polysaccharide-protein complex on the cell membrane, causing cytotoxicity; in addition, the cationic polymer is prone to aggregation, Cause erythrocyte agglutination, cause body toxicity, and reduce in vivo transfection efficiency; some studies have shown that after some cationic polymers (such as PEI) release nucleic acids in cells, the polymer skeleton still stays and accumulates in the body, resulting in cell and body toxicity ;

[0005] ② Poor stability: The complex formed by the compression of the cationic polymer and nucleic acid is easily electrostatically combined with negatively charged proteins in the body such as enzymes, immunoglobulins, albumin, etc., which dissociates the formed complex, which not only reduces the complex The in vivo transfection efficiency of the drug will also affect the normal physiological function;

[0006] ③ Poor specificity: the complex formed by the cationic polymer and nucleic acid is non-specific to tumors, and can also strongly interact with normal cells, thereby causing liver toxicity, nephrotoxicity, etc.; therefore, common cationic polymers are non-viral The carrier cannot efficiently deliver the therapeutic gene to the target tissue cells after intravenous administration to play a role in disease treatment. It is very necessary to study a non-viral gene delivery system with high efficiency, low toxicity, good targeting and safety

[0007] Studies have shown that in polymer carrier materials, dendrimers are highly branched polymers with monodisperse and completely uniform molecular structures; some studies have disclosed that a reduced polyaminoamine (RHB) is a class of Bisacrylamide and triamine monomer undergo Michael addition polymerization to obtain a dendritic polymer. The topology of the polymer can be controlled by the polymerization temperature. When the reaction temperature is raised above 48°C, it can lead to the formation of hyperdendrimer At present, RHB is only used for in vitro transfection, and there is no report of in vivo transfection; the reason is that the surface of RHB / nucleic acid complex has a large number of positive charges, which has poor tumor targeting, low serum stability, and long circulation time in vivo. Short, long-term stable gene expression is difficult; therefore, enhancing the tumor targeting of RHB / nucleic acid complexes and reducing the positive surface charge are key issues for the in vivo application of non-viral gene delivery vectors

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