Polyamide amine, part degradation product or complex of polyamide amine-Math1 gene nano microparticles and application of gene nano microparticles in treating deafness

A technology of polyamidoamine and degradation products, which is applied in the field of nanomaterials, can solve unseen problems and achieve the effects of adjustable particle size, low cytotoxicity, and uniform size

Inactive Publication Date: 2012-07-18
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, there have been no reports on the preparation of PAMAM and its derivatives-Math1 gene nanoparticles and the use of PAMAM-Math1 gene nanoparticles to transfect in vitro cultured cells or transfect cochlea in vivo and express Math1 gene

Method used

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  • Polyamide amine, part degradation product or complex of polyamide amine-Math1 gene nano microparticles and application of gene nano microparticles in treating deafness
  • Polyamide amine, part degradation product or complex of polyamide amine-Math1 gene nano microparticles and application of gene nano microparticles in treating deafness
  • Polyamide amine, part degradation product or complex of polyamide amine-Math1 gene nano microparticles and application of gene nano microparticles in treating deafness

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Embodiment 1: Construction of PRK5-Math1 plasmid

[0050] The total RNA of the 16-day brain tissue of embryonic mice was extracted by the Trizol method, cDNA was synthesized by reverse transcription, and the Math1 gene containing the E box was synthesized by the PCR method, and ECOR1 and BamH1 restriction sites were added to the 5' and 3' ends. The PCR amplified product was digested with ECOR1 and BamH1, and after purification, it was ligated with the PRK5 plasmid (Clontech Company) digested with ECOR1 and BamH1 to construct the PRK5-Math1 plasmid. The Math1 gene has Figure 5 sequence shown.

[0051] Amplification primers are as follows:

[0052] F: 5'-GGAATTAAAATAGTTGGGGGACC-3',

[0053] R: 5'-TGGACAGCTTCTTGTTGGCTT-3',

[0054] The amplification conditions were: 94°C for 5min; 94°C for 1min; 58°C for 40sec; 72°C for 40sec; 35 cycles, 72°C for 5min.

Embodiment 2

[0055] Embodiment 2: Construction of PRK5-Math1-EGFP plasmid

[0056] The plasmid pEGFP-C2 (Invitrogen Company) containing the EGFP gene and the PRK5-Math1 plasmid of Example 1 were double-digested with Hpa1 and Xbal1 enzymes, respectively, purified and recovered, and connected by T4 ligase to construct the PRK5-Math1-EGFP plasmid.

Embodiment 3

[0057] Example 3: Amplification and purification of PRK5-Math1-EGFP

[0058] Take 5 μl of plasmid PRK5-Math1-EGFP, add 100 μl of competent Escherichia coli DH5α bacteria, mix well, ice-bath for 30 minutes, heat shock at 42°C for 1 minute, ice-bath for 2 minutes, add 800 μl of LB medium, and incubate at 37°C for 1 hour. Take 100 μl of the bacterial solution and spread it on a plate containing ampicillin, and incubate it upside down at 37°C for 16 hours. Pick a single colony from the plate, inoculate it in 5 ml of LB liquid medium containing ampicillin, shake at a constant temperature of 37°C overnight, and grow the bacteria to late logarithmic phase. Plasmids were extracted according to the instructions of the plasmid extraction kit (QIAGEN).

[0059] Take 0.5-1 μg of the plasmid, add 5U of endonuclease (no more than 1 / 10 of the total reaction volume), the reaction volume is 20 μl, bathe in a warm water for 2 hours, and take a small amount of samples for agarose gel electropho...

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Abstract

The invention relates to polyamide amine and / or polyamide amine complex-Math1 gene nano microparticles as well as a preparation method and application thereof. The gene nano microparticles are prepared by performing complex coacervation on polyamide amine or polyamide amine complex and plasmids containing Math1 gene. The gene nano particulates are controllable in particle size and uniform in size, are beneficial to surface modification, can improve the expression and delivery capability of the Math1 gene, and can be used for treating sensorineural hearing loss caused by hair cell deficiency due to noise, drug poisoning and other causes.

Description

technical field [0001] The invention belongs to the technical field of nanomaterials, and specifically relates to polyamidoamine (PAMAM), its partial degradation product or its complex-Math1 gene nanoparticle, its preparation method and its application in the treatment of deafness. Background technique [0002] In recent years, gene therapy research using non-viral materials as gene carriers has attracted widespread attention, among which the use of cationic polymers and genes to form nanoparticles to simulate virus-like structures as gene carriers is an important aspect. Nanoparticle (NP) gene carrier is a solid colloidal nanoscale particle carrier synthesized by polymer materials, which can transfer DNA, RNA, PNA (peptide nucleotide), dsRNA (double-stranded RNA) and other gene therapy molecules Wrapped in nanoparticles or adsorbed on the surface of nanoparticles, the nanoparticles enter the cells through cell endocytosis, and the gene therapy molecules are gradually releas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63A61K48/00A61P27/16
CPCA61K48/005C12N15/85A61K9/5138A61K9/146B82Y5/00A61K38/1709A61K48/0041A61K9/5146A61K9/5161A61P27/16
Inventor 杨仕明吴南吴雁韩东郭维维赵立东翟所强高维强杨伟炎
Owner GENERAL HOSPITAL OF PLA
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