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Carrier of nucleic acid substance containing degradable imine bond, its preparation method and application

A technology of imine bonds and nucleic acid drugs, applied in other methods of inserting foreign genetic materials, genetic material components, pharmaceutical formulations, etc., can solve problems such as reduced transfection efficiency, general transfection activity, and escape of nucleic acid substances, and achieve vector The effect of simple structure, low cytotoxicity and high transfection activity

Inactive Publication Date: 2016-02-10
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] SungWanKim et al. from the University of Utah published an article "Polyethyleneiminewithacid-labilelinkagesasabiodegradablegenecarrier" (using acid-degradable linker polyPEI as a gene carrier) in the Journal of Controlled Release (JournalofControlledRelease2005, 103:209-219), which uses glutaraldehyde as a linker Cross-linked with low molecular weight PEI (1800Da) to generate characteristic degradable polyethyleneimines containing imine bonds. Under the acidic conditions of endosomes and lysosomes in the cell, the imine bond directly connected to the low molecular weight PEI is broken to generate a low molecular weight PEI without cytotoxicity, but the polymer structure containing the imine bond cannot guarantee this carrying capacity. The nucleic acid is stable enough before entering the cell, and its transfection activity is average (compared to PEI25KDa)
[0005] Jin et al. applied for a PCT patent (WO2009 / 100645A1) entitled "Polycationicgenecarriersformedofendogenousaminogroup-bearingmonomers" (monomers of endogenous amino groups form polycationic gene carriers). The small molecular monomer spermine is condensed with three linking agents such as 1,4-butanediol dichloroformate, 1,4-succinoyl chloride and glyoxal respectively to obtain a Polyspermine cations such as amine bonds, these exhibit good nucleic acid delivery capabilities, and the polymer is able to return to the initial state of the endogenous monomer of spermine through bioresponsive degradation, specifically characterized by good biocompatibility , but the defect of this technology is: the degradation rate of the urethane bond and amide bond is relatively slow, resulting in the inability of the nucleic acid substance to escape from the endosome in time, which affects the efficiency of nucleic acid delivery; the degradation rate of the imine bond is relatively fast, but the carrier The instability causes the nucleic acid material delivered by it to be released from the outside of the cell before it reaches the cytoplasm, and the transfection efficiency is greatly reduced

Method used

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  • Carrier of nucleic acid substance containing degradable imine bond, its preparation method and application
  • Carrier of nucleic acid substance containing degradable imine bond, its preparation method and application
  • Carrier of nucleic acid substance containing degradable imine bond, its preparation method and application

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Comparison scheme
Effect test

Embodiment 1

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

[0050] Synthetic route such as figure 1 shown. Specifically include the following steps:

[0051] (1) The reaction solvent is absolute ethanol, add anhydrous magnesium sulfate to absolute ethanol and let stand for 48 hours, then distill to obtain fresh absolute ethanol for later use; the whole reaction is carried out in an anhydrous and oxygen-free (high-purity nitrogen protection) environment , take the molar ratio of small molecule PEI (Mw=800Da) and imidazole dialdehyde as 1:2.5, dissolve small molecule PEI and imidazole dialdehyde respectively 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 a condensation reaction to occur for 24 hours;

[0053] (3) After ...

Embodiment 2

[0067] Preparation of PPI and pDNA complexes

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

Embodiment 3

[0070] Agarose gel electrophoresis of complexes of PPI and pDNA

[0071] Weigh 1.0 g of agarose, add 100 mL of 1×TAE buffer solution, heat and dissolve in a microwave oven, wait until the temperature drops to 65 °C, add 4 μL of ethidium bromide (EB) to prepare a 1.0% agarose solution (containing 0.5 μg / mL ethidium bromide), pour it into the glue tank, insert the sample comb, and leave 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 submerge the gel slightly, and wait for the sample to be loaded. Next, compound solutions with different mass ratios were prepared according to the preparation method of the compound, and the compound mass ratios were 0, 0.5, 1, 3, 5, 10, 20, 30, and 50 in sequence. Marker chooses DS TM 5000 (100–5000bp), load 5 μL of sample; 1 μL of 6× sample buffer (bromophenol blue-glycerol indicator, containing 0.25% bromophenol blue, 40% glycerol) is evenly mixed with...

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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 substance containing degradable imine bonds, its preparation method, application and compound 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 during its development, one of the most important bottlenecks is the safe and effective delivery of genetic material 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, virus mutations can cause potential risk of disease, and viral surface components can cause human immune...

Claims

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Application Information

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Patent Type & Authority Patents(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 JIAOTONG UNIV
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