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Targeting liposome for interfering expression of Claudin3 gene

A technology targeting liposomes and lipids, applied in the field of medicine, can solve the problems of low interference efficiency of Claudin3

Inactive Publication Date: 2015-04-29
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of tumor selectivity of the existing RNAi and the low interference efficiency of Claudin3, the anti-tumor efficacy needs to be improved, so it is urgent to develop a new RNAi targeted delivery system

Method used

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  • Targeting liposome for interfering expression of Claudin3 gene
  • Targeting liposome for interfering expression of Claudin3 gene
  • Targeting liposome for interfering expression of Claudin3 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 RNA sequence design that interferes with Claudin3 gene expression

[0035] Use Invitrogen's BLOCK-iT? RNAi Designer online design software (http: / / rnaidesigner.invitrogen.com / rnaiexpress / ) to design the RNA sequence that interferes with the expression of Claudin3 gene, and then compare it with the GeneBank sequence to screen out the RNA sequence that does not exist with any other mRNA in the human body. The homologous sequence of SEQ1, the sense strand is: GCAACAUCAU CACGUCGCAT T, and the antisense strand is: UGCGACGUGA UGAUGUUGCT T.

[0036] Use the online design software (http: / / sidirect2.rnai.jp / ) to design the RNA sequence that interferes with the expression of Claudin3 gene, and then compare it with the GeneBank sequence to screen out the sequence SEQ2 that does not have homology with any other mRNA in the human body, the sense strand is: TCCCGCAACA TCATCACGTC GCATTCAAGA CGTGCGACGT GATGATGTTG CTTTTTTG, and the antisense strand is: AGCTCAAAAA AGCAACATCA TC...

Embodiment 2

[0037] Example 2 RNAi expression plasmid vector construction

[0038] Firstly, the upstream and downstream primers of the DNA template encoding the shRNA corresponding to the target sequence were designed and synthesized; the upstream and downstream fragments (1 OD) of the primers were dissolved with 30 μL of annealing buffer respectively. Take 2 μL of the above solution + 16 μL of annealing buffer, mix well, heat to 94°C, then anneal naturally and cool to room temperature. Take 1 μL of the annealed product + 99 μL of water to make a 100-fold dilution. The shRNA plasmid expression vector pGenesil-2.1 or pGenesil2.4 was digested with Eco31I restriction endonuclease, the digested product was subjected to 1% agarose gel electrophoresis, and the large plasmid fragment was recovered from the gel. The annealed fragment of CLDN3 was linearized with pGenisil-2.1. The ligation product was transformed into competent DH5α Escherichia coli, spread on the LB agarose medium plate containi...

Embodiment 3～8

[0039] Examples 3-8 Preparation of folic acid-targeted lipids using α-carboxy-ω-amino polyethylene glycol or aminocaproic acid as a spacer

[0040] The specific feeding is as follows:

[0041]

[0042] The specific operation is: take 0.5mmol of α-carboxy-ω-aminopolyethylene glycol or aminocaproic acid (amino-terminal protection), 0.75mmol of lipid material (cholesterol or phospholipid), 0.75mmol of 4-dimethylaminopyridine and 1- Dissolve 0.75mmol of ethyl-3–(3-dimethylaminopropyl)-carbodiimide in 100ml of dichloromethane, and react for about 72~96h. The organic solvent was removed by rotary evaporation under reduced pressure, and the crude product of aminopolyethylene glycol cholesterol was obtained by vacuum drying. Weigh 1mmol of folic acid, 2.5mmol of N-hydroxysuccinimide, 2.5mmol of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 10mmol of triethylamine in 50ml of anhydrous DMSO Add about 0.5mmol aminopolyethylene glycol crude product of cholesterol, react at 25~30...

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Abstract

The invention relates to a targeting liposome containing a Claudin3 gene interference sequence. The targeting liposome takes the Claudin3 gene interference sequence as an active ingredient, and phospholipid and folic acid coupling lipid as carrier materials. The composition can be used for delivering the Claudin3 gene interference sequence to tumor cells in a targeting manner, and can give play to relatively good antitumor activity by efficiently interfering the expression of a Claudin3 gene.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a folic acid receptor-targeted targeted liposome for interfering with Claudin3 gene expression, a preparation method and application thereof. Background technique [0002] Cancer is a major public health problem facing countries all over the world today. In developed countries, cancer is the leading cause of death; in developing countries, cancer is also the second cause of death. According to the latest cancer statistics, there are 12.66 million new cancer cases and 7.56 million deaths in the world every year. In developed countries such as the United States, there are about 1.66 million new cases of cancer each year, and about 580,000 deaths due to cancer, and 1 out of every 4 people dies of cancer. According to the "2012 China Cancer Registration Annual Report", there are about 3.5 million new cancer cases in my country every year, and about 2.5 million cancer deaths. Every 6 minutes...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K9/127A61K47/24A61K47/28A61P35/00
Inventor 魏于全何治尧魏霞蔚宋相容
Owner SICHUAN UNIV
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