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Nano gene medicine for liver-related diseases as well as preparation method and application thereof

A gene medicine and nanotechnology, which is applied in the field of nanogene medicine and its preparation, can solve the problems of off-target effect monocyte-macrophage system clearance, reduce the transmembrane penetration and transfection efficiency, etc., and achieve good liver parenchymal cell targeting. Distribution effect, high penetration efficiency of hepatocyte membrane, and effect of protecting hepatocyte damage

Active Publication Date: 2020-01-24
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This concept has promoted the development of non-viral carrier nano-gene drug technology, and a large amount of capital has been invested in the development of more effective nano-gene drug [Kim, B.Y.; Rutka, J.T.; Chan, W.C.Nanomedicine.N.Engl.J .Med.2010, 363, 2434-2443.]; However, nano-gene drugs have not been widely used clinically so far, including off-target effects, mononuclear macrophage system clearance, difficult to penetrate the cell membrane, and lysosome clearance The delivery problem is one of the most critical obstacles [Wilhelm, S.; Tavares, A.J.; Dai, Q.; Ohta, S.; Audet, J. Analysis of nanoparticle delivery to tumors. Nat. Rev. Mater. 2016, 1, 16014]
[0004] Penetranins are a class of polypeptides that can penetrate cell membranes efficiently. In recent years, they have been widely used in nanotechnology to help nanoparticles penetrate target cell membranes and escape from lysosome clearance; however, studies have shown that penetranins lack sufficient positive charge to compress the negatively charged therapeutic gene; not only that, the negatively charged therapeutic gene can neutralize the positive charge of penetranin, thereby reducing the transfection and transfection efficiency of penetrin [Meade, B.R.; Dowdy, S.F. Exogenous siRNA Delivery Using Peptide Transduction Domains / Cell Penetrating Peptides. Adv. Drug Delivery Rev. 2007, 59, 134-140]

Method used

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  • Nano gene medicine for liver-related diseases as well as preparation method and application thereof
  • Nano gene medicine for liver-related diseases as well as preparation method and application thereof
  • Nano gene medicine for liver-related diseases as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]Embodiment 1, preparation and characterization of nano-gene drug (PDPIA)

[0038] First, the therapeutic gene (pIA plasmid) was dissolved in a glucose solution with a mass percentage of 5, and PAMAM3.0 cationic polymers of different mass ratios were quickly added, the mixture was vortexed for 60 seconds, and incubated at 37 degrees for one Hours, cationic polymer sephadex gel electrophoresis with different components such as figure 1 As shown in A and B, it shows that PAMAM3.0: pIA mass ratio is 2 can fully wrap the therapeutic gene;

[0039] Then, positively charged penetratingin, PAMAM3.0, and DSPE-PEG2000 were dissolved in a glucose solution with a mass percentage of 5, and the negatively charged therapeutic gene was added dropwise to the mixed solution, and the mixture was vortexed for 60 seconds. And incubate at 37 degrees for one hour, keep the ratio of PAMAM3.0: DSPE-PEG2000 at 10, adjust the mass ratio of penetratingin: pIA to form nanocomposites with different ...

Embodiment 2

[0041] Embodiment 2, the uptake experiment of hepatic cell to PDPIA

[0042] Using flow cytometry and fluorescent confocal microscopy to observe the uptake of hepatocytes to nano-gene drugs (PDPIA): first, the therapeutic gene pIA was labeled with fluorescent dye TOTO-3 and the penetratingin was labeled with FAM, and prepared by self-assembly method. Fluorescent probe-labeled nanogene drug (PDPIA), and then 1×10 5 HepG 2 cells and Huh 7 cells were seeded in a six-well plate, with the cells growing to 70%, adding fluorescently labeled pIA, DPIA, and PDPIA to the cells for 4 hours, collecting the cells with a cell scraper and washing them twice with PBS Finally, the uptake of hepatocytes to nanogene drug (PDPIA) was detected under flow cytometry, and the results showed (such as figure 2 As shown in A), the uptake rate of hepatocytes to the nano-gene drug (PDPIA) modified by penetranin and self-assembly is as high as about 80%, while the uptake rate of the nano-gene drug withou...

Embodiment 3

[0044] Example 3, PDPIA can escape the clearance of liver cell lysosomes and has a lower cytotoxicity test

[0045] Based on the fact that nanogene drugs with good lysosome escape ability have the ability to resist ribozyme decomposition, so this characteristic is the premise that nanogene drugs have excellent transfection efficiency. Literature reports point out that both phospholipidated PEG and PAMAM have Excellent lysosome escape ability, so this application speculates that the nano-gene drug composed of penetratingin, DSPE-PEG2000, PAMAM 3.0, and therapeutic gene also has excellent lysosome escape ability: this application uses YOYO dye to label PDPIA, and uses LysotrackerRed dye labeled lysosomes, observed the localization of PDPIA and lysosomes, 1×10 5 HepG 2 cells and Huh 7 cells were inoculated into culture dishes, and PDPIA was added when the cells grew to a confluence rate of 70%, and then the localization of PDPIA and lysosomes was observed at different time points...

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Abstract

The invention belongs to the field of biological pharmacy, and relates to a nano medicine drug for liver-related diseases as well as a preparation method and an application of the nano gene drug, andthe nano gene medicine is formed by taking polyamide-amine dendrimer (PAMAM 3.0), DSPE-PEG2000 and penetratin as carriers and performing self-assembly on the carriers and anion therapy genes. The nano-gene medicine can target liver parenchyma cells and generate a therapeutic effect by overexpression or interference of synthesis of certain proteins. The invention also provides a preparation methodof the nano gene medicine. The nano gene medicine has the characteristics of high stability, controllable quality and relative safety. The nano gene medicine can remarkably protect liver cells from being damaged, and is expected to become a medicine for protecting liver cells from being damaged.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a nano-gene drug for liver-related diseases and a preparation method thereof, and is used in the treatment of drug-induced liver injury, non-alcoholic fatty liver disease, autoimmune liver disease, liver cancer, viral hepatitis, Alcoholic liver and other liver diseases and diabetes applications. Background technique [0002] The prior art discloses that hepatitis seriously threatens human life and health, yet the drugs for this class of diseases are very limited [Saito, C.; Zwingmann, C.; Jaeschke, H.Novel mechanisms of protection against acetaminophen hepatitis in mice by glutathione and N-acetylcysteine. Hepatology 2010, 51, 246-254]. Studies have shown that risk factors for hepatitis include infection, drug abuse, chronic alcoholism, high-fat diet, etc., which will aggravate liver cell damage. Studies have shown that the injury of hepatic parenchymal cells is the most importa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/713A61K47/42A61K47/34A61K47/24A61P1/16A61P35/00A61P31/12A61P3/10
CPCA61K48/005A61K31/713A61K47/42A61K47/34A61K47/24A61P1/16A61P35/00A61P31/12A61P3/10
Inventor 鞠佃文陈伟宰文静范佳君章旭耀栾静韵
Owner FUDAN UNIV
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