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Nucleic acid substance carrier containing degradable imine linkage as well as preparation method and application thereof

An imine bond and nucleic acid technology, which can be applied to other methods of inserting foreign genetic materials, genetic material components, medical preparations of non-effective ingredients, etc., can solve the problems of reduced transfection efficiency, escape of nucleic acid substances, slow degradation rate, etc. , to achieve the effect of simple vector structure, low cytotoxicity and high transfection activity

Inactive Publication Date: 2013-08-14
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0004] Sung Wan Kim et al. of the University of Utah published "Polyethyleneimine with acid-labile linkages as a biodegradable gene carrier" in the Journal of Controlled Release (Journal of Controlled Release 2005,103:209-219) Carrier) article, which uses glutaraldehyde as a linker to cross-link with low molecular weight PEI (1800Da) to generate characteristic degradable polyethyleneimines containing imine bonds. The structural characteristics of this class are: After the complex formed by the gene is taken up by the cell, under the acidic conditions of the endosome and lysosome of the condensed gene in the cell, the imine bond directly connected with the low molecular weight PEI is broken, and a non-cytotoxic low-weight PEI is generated. , but the polymeric structure containing imine bonds cannot ensure that the nucleic acid carried is stable enough before entering the cell, and its transfection activity is also average (compared to PEI25KDa)
[0005] Jin et al. applied for a PCT patent (WO2009 / 100645A1) entitled "Polycationic gene carriers formed of endogenous amino group-bearing monomers" (endogenous amino group monomers formed of polycationic gene carriers), which used The small molecular monomer spermine of human endogenous full-time professional condensation gene is condensed with three linking agents such as 1,4-butanediol dichloroformate, 1,4-succinoyl chloride and glyoxal, respectively, to obtain Polyspermine cations such as urethane bonds, amide bonds, and imine bonds, which exhibit good nucleic acid delivery capabilities, and the polymer can return to the initial state of spermine endogenous monomers through bioresponsive degradation, specifically It is characterized by good biocompatibility, but the defect of this technology is: the degradation rate of urethane bonds and amide bonds is relatively slow, resulting in the inability of nucleic acid substances to escape from endosomes in time, which affects the efficiency of nucleic acid delivery; The degradation rate is relatively fast, but the instability of the carrier causes the nucleic acid material delivered by it to be released from the extracellular space before reaching the cytoplasm, and the transfection efficiency is greatly reduced

Method used

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  • Nucleic acid substance carrier containing degradable imine linkage as well as preparation method and application thereof
  • Nucleic acid substance carrier containing degradable imine linkage as well as preparation method and application thereof
  • Nucleic acid substance carrier containing degradable imine linkage as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0049] Preparation of carrier (PPI) containing degradable imine bond nucleic acid material

[0050] Synthetic route such as figure 1 Shown. It includes the following steps:

[0051] (1) The reaction solvent is anhydrous ethanol. After adding anhydrous magnesium sulfate to the anhydrous ethanol, let it stand for 48 hours, and then distill to obtain fresh anhydrous ethanol for use; the whole reaction is carried out in an anhydrous, oxygen-free (high-purity nitrogen protection) environment , Take the molar ratio of small molecule PEI (Mw=800Da) to imidazole dialdehyde 1:2.5, dissolve small molecule PEI and imidazole dialdehyde with absolute ethanol, and make 20mL and 50mL solutions respectively;

[0052] (2) Add the imidazole dialdehyde solution to the small molecule PEI solution drop by drop with a constant pressure dropping funnel, and stir the reaction system at room temperature to allow condensation reaction to occur for 24 hours;

[0053] (3) After the reaction is stopped, first u...

Embodiment 2

[0067] Preparation of PPI and pDNA complex

[0068] Weigh a certain amount of polymer PPI and dissolve it in ultrapure water with a concentration of 2.0mg / mL and filter it with a 0.45μm microporous membrane for use; draw a certain amount of pDNA solution and dilute it with ultrapure water to a 20μg / mL pDNA stock solution . When preparing the PPI and pDNA complex, dilute the PPI solution to the corresponding concentration according to the set mass ratio of a series of PPI and pDNA, and then quickly add it to the same volume and constant concentration of pDNA solution (the final concentration of pDNA is 2μg / mL ), and finally, gently pipet, mix evenly, and incubate at room temperature for 20-30 minutes to obtain a series of complex solutions with different mass ratios for further physical and chemical characterization.

Embodiment 3

[0070] Agarose gel electrophoresis of PPI and pDNA complex

[0071] Weigh 1.0g of agarose, add 100mL of 1×TAE buffer, heat it in a microwave oven to dissolve, wait until the temperature drops to 65℃, add 4μL of ethidium bromide (EB) to prepare 1.0% agarose solution (containing 0.5μg / mL Ethidium bromide), pour it into the glue making tank, insert the sample comb, and place it at room temperature for 0.5-1 hour to solidify into glue. Then, pull out the sample comb, add TAE buffer to the electrophoresis tank to slightly cover the gel, and wait for the sample to be loaded. Then, according to the preparation method of the compound, compound solutions with different mass ratios are prepared, and the compound mass ratios are 0, 0.5, 1, 3, 5, 10, 20, 30, 50 in sequence. Marker chooses DS TM 5000 (100-5000bp), loading 5μL; 6× loading buffer (bromophenol blue-glycerol indicator, containing 0.25% bromophenol blue, 40% glycerol) 1μL mixed with 5μL of complex solution, loading 6μL. Electr...

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Abstract

The invention discloses a nucleic acid substance carrier containing a degradable imine linkage. The structural formula of the nucleic acid substance carrier containing the degradable imine linkage is described in the specification, wherein x is 1-20, y is 1-20, and n is 1-20. The invention also discloses a preparation method of the nucleic acid substance carrier containing the degradable imine linkage, an application in nucleic acid medicine delivery and a complex. Compared with the prior art, the nucleic acid substance carrier which contains an imine linkage structure and can degrade low-molecular-weight PEI (polyether imide) derivatives is simple in structure and easy to synthesize, shows higher transfection activity in different cell strains and has lower cell toxicity, so that the nucleic acid substance carrier is a high-efficiency and low-toxicity gene delivery carrier.

Description

Technical field [0001] The present invention relates to a carrier of low molecular weight PEI derivative nucleic acid material containing degradable imine bond, its preparation method, application and complex in the delivery of nucleic acid drugs (DNA and RNA). Background technique [0002] Gene therapy is to introduce exogenous genetic material (DNA or RNA) into cells to promote or inhibit the expression of specific proteins, or replace or repair problematic genes, so as to achieve the purpose of disease treatment. Gene therapy has encountered a series of technical bottlenecks in its development. One of the most important bottlenecks is the safe and effective delivery of genetic materials in vivo. [0003] Currently commonly used gene delivery vectors can be divided into viral vectors and non-viral vectors. Although viral vectors have high transfection efficiency, their mutations can cause potential disease hazards, and the surface components of the virus can cause human immune r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C08G73/04A61K48/00A61K47/34
Inventor 袁伟恩金拓吴飞苏靖吕楠陈顺
Owner SHANGHAI JIAO TONG UNIV
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