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Carrier composition for transferring nucleic acid and application of carrier composition in preparation of small interfering RNA drugs

A composition and low-interference technology, which can be used in drug combinations, medical preparations without active ingredients, and medical preparations containing active ingredients, etc. Increase the consumption of cationic materials, etc., to achieve the effects of high binding efficiency, good stability, and simple preparation method

Active Publication Date: 2020-06-12
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A high NP ratio will increase the consumption of cationic materials, increase the positive charge on the surface of the drug delivery system and reduce the stability in vivo, making the carrier easier to be cleared by the immune system and lead to certain toxic effects

Method used

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  • Carrier composition for transferring nucleic acid and application of carrier composition in preparation of small interfering RNA drugs
  • Carrier composition for transferring nucleic acid and application of carrier composition in preparation of small interfering RNA drugs
  • Carrier composition for transferring nucleic acid and application of carrier composition in preparation of small interfering RNA drugs

Examples

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preparation example Construction

[0058] PCL-CDM was synthesized in the present invention. The synthesis method includes: dissolving CDM (2-propionic acid-3-methylmaleic anhydride, 60mg, 1.0eqv) in 4mL of anhydrous dichloromethane, then adding oxalyl chloride (52mg, 1.25eqv), N,N - Dimethylformamide (DMF, 80 μL). The above reaction was first placed in an ice-water bath for 10 min, and then transferred to room temperature for 2 h. Dichloromethane, N,N-dimethylformamide and excess oxalyl chloride were removed under vacuum to give the acid chlorided CDM intermediate. Dissolve CDM acid chloride in 4 mL of anhydrous dichloromethane, and dissolve PCL-OH (0.20 g, 0.33 eqv) that has been dried by azeotropic toluene in 3 mL of anhydrous dichloromethane, and place in a dry constant pressure The dropping funnel was slowly added in an ice-water bath, and after the dropwise addition was completed, it was transferred to room temperature to continue the reaction for 2 h. After adding saturated aqueous ammonium chloride so...

Embodiment 1

[0073] Example 1. Preparation and characterization of siRNA drug delivery system

[0074] The amphiphilic block copolymer PEG-PLGA and the amphiphilic polymer PAMAM-CDM-PCL were used to prepare siRNA-loaded nanoparticles by ultrasonic double emulsification.

[0075] 1. The binding ability of particles and siRNA under different NP ratios

[0076] Preparation of nanoparticles loaded with siRNA, the specific method is: polymer PEG-PLGA (2.25mg) and PAMAM-CDM-PCL of different quality (refer to Table 1) were dissolved in 0.5mL chloroform, add siRNA (0.025mL, 0.055 mg) solution under ultrasonication (ultrasonic power 80 watts, stop for two seconds every five seconds, ultrasonic for a total of one minute) to form the initial emulsion, the initial emulsion was added to 5mL DEPC water and phacoemulsified again (sonic power 80 watts, every ultrasonic Ten seconds to stop for two seconds, a total of one minute of ultrasonication), the organic solvent was evaporated under reduced pressure...

Embodiment 2

[0084] Example 2. Effect evaluation of this drug delivery system at the cellular level

[0085] When the NP ratio is 4, the nanoparticle (i.e. the carrier composition for nucleic acid transfer) can fully bind siRNA, and the material used is the least under this NP ratio, so the nanoparticle with the NP ratio of 4 is named NCN (Nano-confined Nanoparticle ), and illustrate the biological effect of this drug delivery system with this example ratio.

[0086] 1. Ability of nanoparticles to enter cells

[0087] The NCN loaded with Cy5-siRNA was prepared by the method described in Example 1 to study the cellular uptake of this drug delivery system.

[0088] The FAM-siRNA nanoparticles loaded (Cy5-siRNA final concentration is 100nM) and human breast cancer MDA-MB-231 cells (24-well plate, 5 × 10 4 cells / well) After co-cultivation at 37°C for 5 hours, the cells were washed 3 times with ice-cold PBS, and the cells were fixed with 4 wt% paraformaldehyde for 15 minutes. Remove 4wt% par...

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Abstract

The invention discloses a carrier composition for transferring nucleic acid and an application of the carrier composition in preparation of a small interfering RNA drug. The composition is prepared byuniformly mixing polyethylene glycol-polylactic acid and (polyamidoamine)-2-propionic acid-3-methyl maleic anhydride-polycaprolactone. According to the invention, siRNA is added into a carrier composition solution, and a complex of siRNA and nanoparticles is obtained by a double emulsification method, so that the small interfering RNA drug is obtained. According to the preparation method disclosed by the invention, the nanoparticles capable of being efficiently combined with the siRNA can be prepared, and the minimum NP ratio required by complete combination of the siRNA and the nanoparticlesis reduced. Moreover, the small interfering RNA drug can effectively enter cells, effectively silence the expression of pathogenic target genes and inhibit the growth of breast cancer in vivo. Therefore, a drug delivery system has a good prospect in drug delivery of siRNA and similar small nucleic acid drugs for disease treatment.

Description

technical field [0001] The invention belongs to the field of small interfering RNA medicines, and in particular relates to a carrier composition for nucleic acid transfer and its application in preparing small interfering RNA medicines. Background technique [0002] Small interfering RNA can simply and efficiently silence the expression of target genes, so it has become an important tool for studying gene function and disease treatment. Compared with small molecule drugs, siRNA can specifically down-regulate the expression of disease-causing genes without affecting the normal gene expression in cells, which has more therapeutic potential than traditional small molecule drugs. In August 2018, Alnylam's Onpattro (patisiran) was approved by the United States and the European Union for the treatment of hereditary ATTR (hATTR) amyloidosis polyneuropathy, becoming the first RNAi approved for marketing since the RNAi phenomenon was discovered 20 years ago drug, which marks a major...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/34A61K47/60A61K47/59A61K31/713A61K48/00A61P35/00A61P15/14
CPCA61K47/34A61K47/60A61K47/595A61K31/713A61K48/0041A61P35/00A61P15/14
Inventor 王均章望成杨显珠
Owner SOUTH CHINA UNIV OF TECH
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