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Nano gene drug for non-alcoholic fatty liver disease and preparation method thereof

A gene drug and non-alcoholic technology, applied in the field of nano-gene drug and its preparation for non-alcoholic fatty liver disease, can solve the problem of not being able to compress DNA well, and achieve high therapeutic effect, good stability, and high transformation The effect of dyeing efficiency

Active Publication Date: 2020-04-28
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Penetrin is a positively charged short peptide under physiological conditions. It has a strong ability to penetrate the cell membrane and can effectively achieve efficient cell endocytosis and lysosome escape. Its disadvantage is that it cannot compress DNA well; Studies have shown that the combination of penetrating peptides and other gene delivery vectors can effectively overcome their shortcomings and improve gene transfection efficiency [Liu C, Jiang K, TaiL, Liu Y, Wei G, Lu W, et al. Facile Noninvasive Retinal Gene Delivery Enabled by Penetratin.ACS Appl Mater Interfaces.2016.8:19256-67]

Method used

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  • Nano gene drug for non-alcoholic fatty liver disease and preparation method thereof
  • Nano gene drug for non-alcoholic fatty liver disease and preparation method thereof
  • Nano gene drug for non-alcoholic fatty liver disease and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation and characterization of embodiment 1 nano gene drug (CDPIA)

[0043] The composition and structure of the nano gene drug of the present invention are as follows: figure 1 Shown in A; First, the therapeutic gene (pIA plasmid) was dissolved in ultrapure water with a mass percentage of 5, and the Chitosan-Metformin cationic polymers of different mass ratios were added rapidly; the mixture was vortexed for 60 seconds, and the Incubate for one hour under high temperature conditions; Sephadex gel electrophoresis of cationic polymers containing different components such as figure 1 As shown in B and C, it shows that when the Chitosan-Metformin: pIA mass ratio is 1, the therapeutic gene can be fully packaged;

[0044] Then, positively charged Chitosan-Metformin, penetratingin, and DSPE-PEG2000 were dissolved in five percent by mass ultrapure water, and the negatively charged therapeutic gene was added dropwise to the mixed solution; the mixture was vortexed for 60 ...

Embodiment 2

[0046] Example 2 The uptake of hepatocytes to CDPIA

[0047] Use laser confocal microscope to observe the uptake of nano-gene drug (CDPIA) by hepatocytes; first, use fluorescent dye TOTO-3 to label therapeutic gene pIA and FAM to label penetranin, and prepare fluorescent probe-labeled CDPIA by self-assembly method Nanogene drug (CDPIA); then 1×10 5 The HepG2 cells and Huh7 cells were inoculated in confocal small dishes, and when the cells grew to 70%, fluorescently labeled pIA, CMIA, and CDPIA were added to the cells for 2 hours; after washing with pre-cooled PBS for 3 times, the laser confocal Observe the uptake of hepatocytes to nano-gene drugs (CDPIA) under a focusing microscope; figure 2 As shown, the results show that the uptake rate of hepatocytes to the nanogene drug (CDPIA) modified by penetranin and self-assembled is significantly increased, while the naked plasmid (pIA) and the nanogene drug (CMIA) without penetratingin The uptake rate is low; through fluorescence...

Embodiment 3

[0048] Example 3 CDPIA can escape the clearance of hepatic lysosomes and has low cytotoxicity

[0049] According to literature reports, lysosome escape is the prerequisite for the good transfection efficiency of nanogene drug after ingestion; this example further investigates the subcellular localization and lysosome escape ability of nanogene drug (CDPIA) in liver cells. CDPIA was labeled with TOTO-3 dye, lysosome was labeled with Lysotracker Green dye, and nucleus was labeled with Hoechst33342 to observe the localization of CDPIA, lysosome and nucleus; 1×10 5 HepG2 cells and Huh7 cells were inoculated in confocal small dishes; when the cells grew to a fusion rate of 70%, CDPIA was added, and then the localization of CDPIA, lysosomes and nuclei was observed at different time points; image 3 As shown in A and B, the experimental results show that CDPIA is taken up by hepatocytes within 1 hour and some nanoparticles and lysosomes co-localize (yellow); after 4 hours, the green ...

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Abstract

The invention belongs to the technical field of biological pharmacy, and relates to a novel nano gene drug for non-alcoholic fatty liver disease and a preparation method thereof. The nano gene drug for non-alcoholic fatty liver disease is formed by taking metformin grafted chitosan, a cell-penetrating agent and DSPE-PEG2000 as carriers and carrying out self-assembly on the carriers and an anion treatment gene. The metformin grafted chitosan in the nano gene drug can have high transfection efficiency and protect the intrinsic pharmacological activity of the non-alcoholic fatty liver disease atthe same time. The nano gene drug can effectively target the liver part, and a treatment effect is achieved by overexpression or interference of synthesis of certain key proteins. The nano gene drug prepared by the invention has the characteristics of high safety, good stability and controllable quality. The nano gene drug can effectively treat non-alcoholic fatty liver diseases and obesity-related metabolic syndrome, and is expected to become a drug for protecting liver injury and regulating metabolic-related diseases.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a nano-gene drug for non-alcoholic fatty liver disease and a preparation method thereof. The prepared nano-gene drug can be used to treat non-alcoholic fatty liver disease, drug-induced liver injury, and autoimmunity liver disease, liver cancer, viral hepatitis, alcoholic liver and other liver diseases, diabetes, obesity-related metabolic syndrome, and cardiovascular and cerebrovascular diseases. Background technique [0002] The report disclosed that with the rapid development of the world economy and the continuous improvement of human living standards, non-alcoholic fatty liver disease has gradually become one of the most common chronic liver diseases, affecting nearly one-third of the world's population [Issa D, Patel V, Sanyal AJ. Future therapy for non-alcoholic fatty liver disease. LiverInt. Suppl. 2018.1:56-63]. Surveys show that among obese people, the incide...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K47/69A61K47/64A61K47/61A61K31/155A61P1/16A61P35/00A61P31/14A61P31/20A61P3/04A61P3/10A61P9/00
CPCA61K31/155A61K47/61A61K48/0041A61K48/005A61K47/6935A61K47/64A61P1/16A61P35/00A61P31/14A61P31/20A61P3/04A61P3/10A61P9/00
Inventor 鞠佃文宰文静刘洪瑞陈伟
Owner FUDAN UNIV
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