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Cation microvesicle and preparation method thereof

A cationic and microbubble technology, which is applied in pharmaceutical formulations, medical preparations of non-active ingredients, gene therapy, etc., can solve the problems of small charged groups, small positively charged phospholipids, and low ability to carry naked plasmids.

Active Publication Date: 2012-07-11
广州康臣药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 2. When preparing microbubbles, mix positively charged phospholipids on the shell of microbubbles, and then adsorb DNA by electrostatic adsorption, but the positively charged phospholipids have a small charge and weak adsorption force
[0009] 5. Modified sulfhydryl groups on 25KDa PEI, and then covalently coupled to microbubbles with maleimide groups, transfected to a better effect, but the steps are complicated and the cost of materials is high
[0010] Traditional ultrasound contrast agent microbubbles have many advantages as a non-viral carrier, but the existing membrane materials have small charged groups, relatively small charge, weak DNA binding, and due to their single-layer membrane structure, the ability to carry naked plasmids is relatively low. Small, after intravenous injection, due to the shear force of blood flow, mononuclear macrophage system and enzyme degradation, etc., it is quickly cleared, and the transfection efficiency is not high

Method used

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  • Cation microvesicle and preparation method thereof
  • Cation microvesicle and preparation method thereof
  • Cation microvesicle and preparation method thereof

Examples

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preparation example Construction

[0059] Also provide a kind of preparation method of above-mentioned cationic microbubble, such as figure 1 Shown, the preparation method of this cationic microbubble comprises the steps:

[0060] S10, phospholipids with 12 to 22 carbon atoms, polyethyleneimine modified with hydrophobic segments, and phospholipids with 12 to 22 carbon atoms modified with polyethylene glycol are 70 to 90: 1 to 20 in molar percentage : 1~10 dissolved in an organic solvent and mixed.

[0061] The phospholipids with 12 to 22 carbon atoms can be dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylcholine At least one of alkali (DMPC), dimyristoylphosphatidylethanolamine (DMPE), di(eicosyl)phosphatidylcholine and polyethylene glycol monostearate.

[0062] The hydrophobic segment can be a fatty acid with 12-22 carbon atoms or its corresponding fatty acid polyethylene glycol monoester.

[0063] Hydrophob...

Embodiment 1

[0084] (1) Modification of polyethyleneimine PEI600.

[0085] Carboxyl terminal activation of stearic acid: According to the molar ratio of stearic acid to N,N'-carbonyldiimidazole of 1:1.2, stearic acid and N,N'-carbonyldiimidazole were dissolved in chloroform solvent respectively. The chloroform solution of stearic acid was added dropwise to the chloroform solution of N,N'-carbonyldiimidazole under magnetic stirring. The reactor was reacted at 30° C. for 24 hours under an argon atmosphere. Then quickly wash the extraction with saturated sodium chloride solution to prepare carboxyl-activated stearic acid. The reaction equation is as follows:

[0086]

[0087] According to the molar ratio of polyethyleneimine and carboxyl-activated stearic acid of 1:1, dry hyperbranched polyethyleneimine with a molecular weight of about 600 and carboxyl-activated stearic acid were dissolved in trichloro in methane. The chloroform solution of stearic acid activated by the carboxyl group ...

Embodiment 2

[0105] (1) Modification of polyethyleneimine PEI25000.

[0106] Referring to Example 1, replace polyethyleneimine PEI600 with PEI25000.

[0107] (2) Design and manufacture of polyethyleneimine (stearic-PEI600) microbubbles modified with 10% stearic acid.

[0108] According to the molar ratio of 85:1:14, 1,2-distearoylglyceryl phosphatidylcholine (DSPC), 1,2-distearoylglyceryl phosphatidylethanolamine (DSPC) of polyethylene glycol 2000 DSPE-PEG) and stearic acid-modified polyethyleneimine (Stearic-PEI) were dissolved in chloroform and mixed on a vortex mixer. The chloroform was removed with a dry nitrogen flow to form a uniform film of the dissolved material on the wall of the test tube, and dried in a vacuum oven at 45°C for more than 2 hours. Add a certain volume of buffer solution (0.1M PBS phosphate buffer) with a pH of 7.6 to hydrate the film of the phospholipid mixture, and heat it above the phase transition temperature of the mixture to make it fall off the wall of the...

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Abstract

The invention discloses a cation microvesicle, which has a microvesicle structure formed by a lipid material. The lipid material comprises phospholipid, hydrophobic segment-modified polymine and polyethylene glycol-modified phospholipid, wherein the phospholipid is taken as a main body material, contains 12-22 carbon atoms and accounts for 70-90 percent by mole of the ester material; the hydrophobic segment-modified polymine is taken as a modified material and accounts for 1-20 percent by mole of the lipid material; and the polyethylene glycol-modified phospholipid contains 12-22 carbon atomsand accounts for 1-10 percent by mole of the lipid material. The cation microvesicle serving as a novel gene transfection reagent is used for combining the advantages of an ultrasonic microvesicle and polymine and overcoming defects thereof, has the advantages of an ultrasonic developing agent microvesicle, can be applied to gene transfection under the guidance of an ultrasonic image, and can be used for performing fixed-point targeted transfection on a microvesicle under ultrasonic control. The invention further provides a preparation method of the cation microvesicle.

Description

【Technical field】 [0001] The invention relates to the technical fields of transgene carrier and contrast-enhanced ultrasound, in particular to a cationic microbubble and a preparation method thereof. 【Background technique】 [0002] Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate diseases caused by gene defects and abnormalities and achieve therapeutic purposes. Isolated or modified exogenous genes and nucleic acid sequences cannot replicate by themselves, and need vectors to carry them to appropriate cells for replication and expression. As a tool for gene introduction into cells for gene therapy, gene carrier can deliver the target gene into the target cells, so as to exert the specific function of the target gene. Gene vectors can be divided into two categories: viral vectors and non-viral vectors. Viral vectors mainly include retrovirus, adenovirus, adeno-associated virus and herpes simplex virus, etc., which ...

Claims

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Application Information

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IPC IPC(8): A61K49/22A61K48/00A61K47/34A61K9/00
Inventor 郑海荣邱本胜靳巧锋王志勇
Owner 广州康臣药业有限公司