[12] aneN3 cationic lipid containing targeted group and fluorescent group, transgenic vector, and preparation methods of cationic lipid and transgenic vector

A technology of cationic lipids and transgenic carriers, applied in other methods of inserting foreign genetic materials, gene therapy, organic chemistry, etc., can solve the problems of carcinogenicity, poor cell selectivity, lack of targeting groups, etc., and achieve good selectivity , simple preparation method and high transfection efficiency

Active Publication Date: 2016-05-04
BEIJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viral vector is a highly efficient transfection reagent, but it has disadvantages, such as immunogenicity and carcinogenicity, the size of the gene is easily limited by the size of the virus, and there is a possibility of mutation, so its safety is worrying
However, since most of the current gene vectors lack fluorescent labeling groups, the intracellular distribution and transport pathways of non-viral vectors are unclear, and the transmembrane level, internalization pathway, and endosome escape mechanism of non-viral vectors are also unclear. Not very clear, at the same time, because most of the current vectors lack targeting groups, the selectivity to cells is relatively poor and the transfection efficiency is low

Method used

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  • [12] aneN3 cationic lipid containing targeted group and fluorescent group, transgenic vector, and preparation methods of cationic lipid and transgenic vector
  • [12] aneN3 cationic lipid containing targeted group and fluorescent group, transgenic vector, and preparation methods of cationic lipid and transgenic vector
  • [12] aneN3 cationic lipid containing targeted group and fluorescent group, transgenic vector, and preparation methods of cationic lipid and transgenic vector

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example 1

[0046] A [12]aneN containing a targeting group and a fluorescent group 3 Class cationic lipid, the structural formula of described cationic lipid is as follows:

[0047]

[0048] The method for cationic lipid, its synthetic route is as follows:

[0049]

[0050] The specific steps of the method include:

[0051] Step 1. Weigh the compound of formula 3 (1.28g, 3.13mmol), compound of formula 2 (1.0g, 3.13mmol), 1-hydroxybenzotriazole catalyst (1.15g, 0.57mmol), 1-ethyl- (3-Dimethylaminopropyl) carbodiimide hydrochloride condensation agent (0.60g, 3.13mmol) and N,N-diisopropylethylamine (0.80g, 6.26mmol), add dimethyl sulfoxide Dissolve 40mL of solvent and react under nitrogen atmosphere for 8-10 hours. After the reaction, evaporate the solvent under reduced pressure, add 35mL of water, extract with dichloromethane (40mLx2), combine the organic phases, wash with saturated brine, and dry over anhydrous sodium sulfate , the solvent was removed, and silica gel column chroma...

Embodiment 1

[0082] The preparation of the transgenic carrier containing cationic lipid formula 1 compound in embodiment 1

[0083] The raw materials include cationic lipid compound 1 prepared in Embodiment 1, dioleoylphosphatidylethanolamine DOPE (dioleoylphosphotidylethanolamine) and anhydrous chloroform. In a high-temperature sterilized flask, the synthesized cationic lipid formula 1 compound (0.005mmol) and DOPE were dissolved in 2.5mL anhydrous chloroform at a molar ratio of 1:2, and after fully mixing and dissolving, the solvent was spin-dried under reduced pressure at room temperature to obtain Liposome membrane, the mixture was put into a vacuum drying oven for drying (drying time 12 hours, temperature 25°C) to remove residual chloroform, and the dried liposome membrane was preheated to 70°C in advance, trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) buffer (10 mM, pH 7.4) was mixed and added in such an amount that the final lipid concentration was 1.0 mM. Finally, the ...

Embodiment 2

[0085] The transgenic carrier prepared in embodiment 2 was tested in the following aspects:

[0086] (1) Agarose gel electrophoresis experiment of transgene vector and pUC18-DNA complex

[0087] Preparation of transgenic vector and pUC18-DNA complex

[0088] At room temperature, add the transgene carrier into the PE tube respectively. The concentration of the transgene carrier substance is 0, 1, 5, 10, 20, 50 μM, and the concentration of pUC18-DNA is 9 μg / mL. Add the corresponding volume of ultrapure water to make The final volume of the reaction solution was kept at 20 μL, and centrifuged to mix evenly. Put it in a constant temperature water bath at 37°C, and after incubating for 1 hour, add 2 μL of 10×LoadingBuffer loading buffer to terminate the reaction.

[0089] Preparation of agarose gel

[0090] Weigh 280 mg of agarose, add 40 mL of 1×TAE (Tris-acetic acid) buffer solution, and heat with microwave to completely dissolve the agarose particles to obtain a colorless tra...

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Abstract

The invention provides a [12] aneN3 cationic lipid containing a targeted group and a fluorescent group and a transgenic vector containing the same. The invention also discloses preparation methods of the cationic lipid and the transgenic vector. The cationic lipid provided by the invention contains the targeted group cholic acid and the fluorescent group naphthalimide simultaneously; the transgenic vector formed by the cationic lipid has lower cytotoxicity, has a certain targeting property on hepatoma cells, not only can monitor intracellular transport and release of vector/DNA complexes in a same cell but also has a certain selectivity on the hepatoma cells to promote the development of gene therapy, and also has higher transfection efficiency; the preparation methods of the cationic lipid and the transgenic vector are simple, mature, and easy to control.

Description

technical field [0001] The invention relates to a cationic lipid and a transgene carrier, in particular to a [12]aneN containing a targeting group and a fluorescent group 3 A cationic lipid, a transgenic carrier containing the cationic lipid and a preparation method thereof. Background technique [0002] Gene therapy is to introduce exogenous normal genes into target cells through certain methods to replace or compensate some defective or abnormal genes to achieve the purpose of treatment. Gene therapy provides the possibility for the treatment of many diseases, such as: cancer, diabetes, cystic fibrosis, AIDS, cardiovascular disease and so on. [0003] The key to gene therapy is to efficiently introduce therapeutic genes into cells. However, naked DNA generally exists in the form of a stretched linear helix with a large volume, and the DNA molecule itself is a negatively charged anion, which will interact with the cell membrane when it enters the cell. Electrostatic repul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D401/14C12N15/87A61K48/00
CPCA61K48/00C07D401/14C12N15/87
Inventor 卢忠林高永光唐权史幼荻张影
Owner BEIJING NORMAL UNIVERSITY
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