Double-targeted ternary complex nucleic acid transfer and release system and preparation method thereof

A ternary compound and compound technology, which is applied in the field of biomedicine, can solve the problems of low serum stability, high toxicity, and body toxicity.

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A ternary compound and compound technology, which is applied in the field of biomedicine, can solve the problems of low serum stability, high toxicity, and body toxicity.

CN106902354AActive Publication Date: 2017-06-30FUDAN UNIV

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Embodiment 1

[0051] Embodiment 1: the method for synthesizing dendritic cationic polymer polyaminoamine (RHB)

[0052] Accurately weigh CBA (0.260g, 1.0mmol), MBA (0.308g, 2.0mmol), add AEPZ (0.193g, 1.5mmol) methanol / water mixture (3.5mL, 7 / 3, v / v) In a round bottom flask, magnetically stirred, protected by N2, protected from light, reacted in an oil bath at 50°C for 3 days to obtain a viscous solution. Add a small amount of 4-amino-1-butanol, and continue stirring at 50°C for 12h. The product solution was dialyzed against HCL aqueous solution (pH=3) for 1 d, distilled water for 2 d, freeze-dried, and weighed to obtain RHB.

Embodiment 2

[0053] Embodiment 2: the method for synthesizing RIF7 modified HA (RIF7-HA)

[0054]Accurately weigh HA (7.5kD, 50mg, 0.005mmol) and dissolve it in PBS (5ml, pH7.4) with magnetic stirring until clarification, add EDC·HCL (20mg, 0.1mmol) and HOBT (15mg, 0.1mmol), and continue stirring for 2h . Dissolve 11.8 mg of RIF7-related peptide in 5 ml of triple-distilled water and add dropwise to the activated HA solution, add an appropriate amount of NaCl, and add 140 μl of N-methylmorpholine to adjust the pH to 7.4. Stir overnight, dialyze with a dialysis bag with a molecular weight of 3500, remove unreacted polypeptide, and lyophilize.

Embodiment 3

[0055] Embodiment 3: the method for preparing RHB / DNA complex nanoparticle

[0056] Cationic polymer RHB and DNA self-assemble to form nanoparticles; prepare 1 μg / μl polymer RHB aqueous solution and 1 μg / μl DNA aqueous solution respectively, and take corresponding volume of mother solution according to the mass ratio (w / w) of polymer RHB to DNA Disperse in PBS with pH 7.4, then mix with an equal volume of DNA solution, vortex for 5 s and incubate at room temperature for 5 min to prepare RHB / DNA binary complex nanoparticles.

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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...

Claims

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Application Information

Patent Timeline

30 Jun 2017

Publication

CN106902354A

IPC: A61K47/59; A61K47/36; A61K31/7088; A61K31/713; A61P35/00; C12N15/85; C12N15/87

CPC: A61K31/7088; A61K31/713; A61K47/36; C12N15/85; C12N15/87; C12N2800/107

Inventors: 沙先谊; 陈昕怡