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Poly spermine cations, construction method thereof, and preparation method of nano-grade particles

A nanoparticle and construction method technology, applied in the field of degradable amide polyspermine cations and its construction, can solve the problems of slow degradation rate, affecting nucleic acid delivery efficiency, and reduced transfection efficiency

Inactive Publication Date: 2012-10-17
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The first object of the present invention is to provide a polyspermine cation to solve the problem that the polycations containing urethane bonds and amide bonds in the prior art have a relatively slow degradation rate when transporting nucleic acid substances, resulting in that the nucleic acid substances cannot be removed from the content in time. Escape in the body, affecting the efficiency of nucleic acid delivery; while polycations containing imine bonds degrade faster when transporting nucleic acid substances, but the instability of the carrier causes the nucleic acid substances transported by them to be released from the outside of the cell before they reach the cytoplasm out, resulting in a technical problem that greatly reduces the transfection efficiency

Method used

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  • Poly spermine cations, construction method thereof, and preparation method of nano-grade particles
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  • Poly spermine cations, construction method thereof, and preparation method of nano-grade particles

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1. Construction of Polyspermine Cationic PSIA

[0043] figure 1 It is a schematic diagram of the synthetic route of polyspermine cationic PSIA. The whole reaction was carried out in an anhydrous and oxygen-free environment. Weigh 100mg imidazole-4,5-dicarboxylic acid, 528mg dicyclohexylcarbodiimide, 295mg N-hydroxysuccinimide and 1mL trifluoroacetic acid and dissolved in 5mL N, In the N-dimethylformamide solution, stir at room temperature for 12 hours; then, weigh 130 mg of spermine and dissolve it in 5 mL of the N,N-dimethylformamide solution at -10~10°C (preferably 0°C), The above carboxyl activated imidazole-4,5-dicarboxylic acid was slowly added dropwise to the above spermine solution, and the reaction was stirred for 24 hours at room temperature. Vacuum filtration, evaporate the solvent, and re-dissolve the reaction solution residue in ultrapure water, dialyze for 12 to 48 hours, pre-freeze at -80 degrees for 4 hours, and then freeze-dry to obtain polyspermin...

Embodiment 2

[0045] Example 2. Preparation of PSIA and pDNA nanoparticles

[0046] Weigh a certain amount of polyspermine cation PSIA and dissolve it in water to a concentration of 2.0mg / ml, filter it through a 0.45μm water membrane for use; draw a certain amount of pDNA solution and dilute with water to a 20μg / ml pDNA stock solution. When preparing PSIA and pDNA nanoparticles, dilute the PSIA solution to the corresponding concentration according to the set mass ratio of a series of PSIA and pDNA, and then quickly add it to the pDNA solution of the same volume and fixed concentration, so that the final concentration of pDNA is 2μg / ml, finally gently pipetting, mixing uniformly, and incubating at room temperature for 20-30 minutes to obtain a series of nanoparticle solutions with different mass ratios of PSIA and pDNA for further physical and chemical characterization.

Embodiment 3

[0047] Example 3. Agarose gel electrophoresis of PSIA and pDNA nanoparticles

[0048] Weigh 1.0g agarose, add 100ml 1×TAE buffer solution, heat it in a microwave oven to dissolve it, wait until the temperature drops to 65℃, add ethidium bromide (EB) to make 1.0% agarose solution (containing 0.5μg / ml Ethidium bromide), pour it into the glue making tank, insert the sample comb, and leave it at room temperature for 0.5-1 hour until the glue solidifies. Then, pull out the sample comb, add TAE buffer to the electrophoresis tank to cover the gel, and wait for the sample to be loaded. Next, prepare nanoparticle solutions with different mass ratios according to the preparation method of nanoparticles. The mass ratios of PSIA and pDNA in the nanoparticle solution are 0, 1, 2, 3, 5, 7, 10, 15, 20, 30, 50, 70, 100. Marker chooses DS TM 5000 (100-5000bp), loading 2μl; 6x loading buffer (bromophenol blue-glycerin indicator, containing 0.25% bromophenol blue, 40% glycerol) 1μl and nanopart...

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Abstract

The invention relates to poly spermine cations, a construction method thereof, and a preparation method of nano-grade particles containing the poly cations. The invention specifically relates to a type of degradable amide poly spermine cations used for conveying nucleic acid medicaments (DNA and RNA), a construction method thereof, and a preparation method of nano-grade particles. The poly spermine cations provided by the invention have a structural formula represented below, wherein n is 10 to 1000. Compared with prior arts, the poly spermine cations provided by the invention can be used for agglomerating and conveying nucleic acid medicaments. During a process of internal circulation, both the requirements on the stability before the cations reach the target cells and the biological response after the cations enter the target cells are satisfied. Further, the poly spermine cations can independently accomplish non-toxic metabolism in vivo.

Description

Technical field [0001] The present invention relates to a polycation, a construction method and a preparation method of polycation-containing nanoparticles, in particular to a degradable amide polyspermine cation for the delivery of nucleic acid drugs (DNA and RNA), and a construction method and nanometer Preparation method of particles. Background technique [0002] Nucleic acid drugs (DNA and RNA) can achieve the purpose of treating diseases by regulating the expression or silencing of disease-related genes. At present, the main obstacle for nucleic acid drugs to become a new generation of therapeutic drugs is the lack of in vivo delivery technology. Among the reported nucleic acid drug delivery vectors, synthetic non-viral vectors have a series of advantages compared to viral vectors: no immunogenicity, no virus mutation, large gene load, simple preparation method, and low production cost But there are also disadvantages such as poor biocompatibility, no tissue or cell targe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08G69/26C08G69/28A61K47/34
Inventor 金拓袁伟恩吴飞段诗悦葛雪梅吕楠
Owner SHANGHAI JIAO TONG UNIV
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