Hydroxyl-enriched nano gene vector as well as preparation method and application thereof

A gene carrier and nanotechnology, which is applied in the field of hydroxy-rich nanogene carrier and its preparation, mediates the efficient delivery of miRNA in esophageal cancer cells, and can solve the problems of unstable intake and deficiency of miRNA. , to achieve good miRNA delivery efficiency, induce cell proliferation inhibition, and inhibit invasion and metastasis effects

Inactive Publication Date: 2017-05-31
SHANDONG RES INST OF TUMOUR PREVENTION TREATMENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, intracellular instability and insufficient uptake of miRNAs in targeted tissues

Method used

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  • Hydroxyl-enriched nano gene vector as well as preparation method and application thereof
  • Hydroxyl-enriched nano gene vector as well as preparation method and application thereof
  • Hydroxyl-enriched nano gene vector as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A method for preparing a hydroxyl-rich nano gene carrier, comprising the steps of:

[0041] (1) Add pentaerythritol and 2-bromoisobutyryl bromide to dimethylformamide respectively, the molar ratio of pentaerythritol and 2-bromoisobutyryl bromide is 1:6, and react at 50°C for 24 hours to obtain PER- Br;

[0042] (2) Add glycidyl methacrylate (GMA) to the PER-Br prepared in step (1), then add CuBr and N,N,N',N",N"-hexamethyldivinyl Tetramine (PMDETA), the molar ratio of PER-Br, glycidyl methacrylate, CuBr and N,N,N',N",N"-hexamethyldivinyltetramine is 1:350:1: 2. The star-shaped s-PGMA was prepared by atom transfer radical polymerization at room temperature for 3 hours, and then s-PGMA and ethanolamine were added to dimethyl sulfoxide (DMSO) respectively, and the s-PGMA and ethanolamine The molar ratio is 1:3500, and the reaction is carried out at 80°C for 40 minutes to obtain s-PGEA;

[0043] (3) Dissolve the s-PGMA prepared in step (2) in dimethyl sulfoxide, then add...

experiment example 1

[0052] Molecular weight was determined by gel permeation chromatography (GPC). The GPC instrument was equipped with a Wsters 2414 Refractive Index Detector and a Waters 2487 Dual Wavelength UV Detector. The chemical structure of the polymer to be detected utilizes a 1 H NMR ( 1 H NMR) spectrometer detection, the instrument is equipped with a Bruker ARX 400MHz spectrometer.

[0053] Polycation / RNA complexes under different N / P ratios (expressed as the ratio of nitrogen in the polycation to phosphorus in the RNA) were obtained according to the following steps:

[0054] After mixing equal amounts of the polycation to be detected and the RNA solution (dissolved in DEPC water), vibrate with a vortex shaker, and let stand at room temperature for 30 minutes. The particle size and potential of polycation / RNA complexes were measured using a Zetasizer Nano ZS (Malvern Instruments, Southborough, MA). Images of the topography of the polycation / RNA complexes (with a resolution of 512 p...

experiment example 2

[0058] Experimental Example 2 Cell Culture and Transfection Analysis

[0059] Inoculate KYSE30, KYSE450 and KYSE150 cells into 1640 medium containing 10% fetal bovine serum (Hyclone) at 37°C with 5% CO 2 for 3 days in an incubator. Transfer the cultured cells to a 6-well plate, 2×10 per well 5 After 12 hours, the nanomaterial PEI was used as the material control to compare the transfection efficiency of the two nanocomplexes of s-PGEA / miRNA and s-PGEA-FA / miRNA. At the same time, using NC as a negative control and MALAT1-6108 (siR-M) as a positive control, the effects of miR-101, miR-217 and MALAT1-6108 (siR-M) in three esophageal cancer cells were compared by fluorescent quantitative PCR experiments. the transfection efficiency.

[0060] Trizol (purchased from Invitrogen) was used to extract RNA from cells or tissue samples, genomic DNA was removed with RNase-free DNase, and reverse-transcribed into cDNA (purchased from TOYOBO, Osaka, Japan). Using U6 as an internal refere...

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Abstract

The invention relates to a hydroxyl-enriched nano gene vector as well as a preparation method and application thereof. The vector adopts pentaerythritol as a core, has four arms and is of a star-shaped structure, and the arms have equal molecular weights. The invention discloses the nano gene vector based on a star-shaped polycation derivative (s-PGEA-FA) for a first time, lateral group of the nano gene vector comprises FA ligand and/or rich hydrophilic hydroxyl and secondary amine groups, and the vector is synthesized through atom transfer radical polymerization, ring-opening reaction and amidation reaction, is capable of mediating efficient delivery of miR-101 and miR-217 in different esophagus cancer cell systems, is capable of efficiently silencing the expression of lncRNA MALAT1 and furthermore killing esophagus cancer cells and inhibiting invasion and metastasis of the esophagus cancer cells, and has wide application prospects in treatment on esophagus cancer.

Description

technical field [0001] The present invention relates to a hydroxyl-rich nano-gene carrier and its preparation method and application, in particular to a hydroxyl-rich nano-gene carrier and its preparation method and its application in mediating efficient delivery of miRNA in esophageal cancer cells The medicine belongs to the technical field of medicine carrier. Background technique [0002] As a malignant tumor with a very high mortality rate, the incidence of esophageal cancer is increasing year by year in Asian countries including China. A large amount of epidemiological evidence shows that many environmental risk factors, including excessive drinking, excessive smoking, micronutrient deficiency, and intake of carcinogens in daily diet, can lead to the occurrence of esophageal cancer. However, only some of the individuals exposed to these risk factors eventually develop esophageal cancer, suggesting that human personal genetic factors may also affect the occurrence of es...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C08F120/32C08F8/32
Inventor 杨明徐福建任艳利李瑞全
Owner SHANDONG RES INST OF TUMOUR PREVENTION TREATMENT
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