Nano drug delivery system capable of delivering genetic drug towards secondary hepatocyte in targeted mode and application

A technology for gene delivery and drug delivery system, applied in the field of pharmacy, can solve problems such as low targeting efficiency, and achieve the effects of quality control, improved utilization, and long-term stable therapeutic effect

Inactive Publication Date: 2020-09-04
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a long-acting, safe, stable and efficient nano drug delivery system for targeted delivery of gene drugs to secondary liver cells based on the current state of the art and existing defects or deficiencies, so as to better solve the problem of The problem of low targeting efficiency in the targeted delivery of gene drugs to hepatocytes

Method used

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  • Nano drug delivery system capable of delivering genetic drug towards secondary hepatocyte in targeted mode and application
  • Nano drug delivery system capable of delivering genetic drug towards secondary hepatocyte in targeted mode and application
  • Nano drug delivery system capable of delivering genetic drug towards secondary hepatocyte in targeted mode and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Preparation of Interleukin 1 Receptor Antagonist (IL-1Ra) and Apolipoprotein AI (ApoAI) Fusion Gene surface delivery vehicle pVAX1-IL-1Ra-ApoAI (pVAX1-IA)

[0059] The gene sequences of IL-1Ra and ApoAI are derived from GeneBank accession no.NM_173842.2 and GeneBank accession no.X07496 respectively, and the protein sequence added between the gene sequences of IL-1Ra and ApoAI is a flexible short peptide of GGGGS (gene sequence: GGCGGAGGCGGATCC) Connection; the Kozak sequence was added to the upstream sequence of IL-1Ra as an enhancer; the gene sequences of IL-1Ra-GGGGS and ApoAI were synthesized by GenScript Biotechnology Company on the pMD19-Tsimple vector, and the two ends of IL-1Ra-GGGGS The restriction endonuclease cutting sites of HindIII and BamHI are designed, and the restriction endonuclease cutting sites of BamHI and XhoI are designed at both ends of ApoAI;

[0060] Take the pVAX1 expression vector and pMD19-T simple-IL-1Ra-GGGGS encoding gene pla...

Embodiment 2

[0062] Example 2, the expression level of gene drug pVAX1-IA in vivo and in vitro

[0063] HEK-293T cells were planted in a six-well plate. When the cell fusion rate reached 70-80%, 5 μg of pVAX1-IA was transfected with lipofectamine2000, and the cells were cultured for 24 hours. Western blot and ELISA were used to detect intracellular and secreted IL-1Ra content in the supernatant, the results confirmed the successful expression and secretion of IL-1Ra-ApoAI fusion protein ( figure 1 B-C); In order to further verify the expression of pVAX1-IA in mice, C57BL / 6 mice were divided into three groups and injected intramuscularly with PBS, pVAX1-IL-1Ra and pVAX1-IA (the weight of the plasmid was 50 μg) respectively. The specified time detects the content of IL-1Ra in blood and liver of mice ( figure 1 E-G); As shown, pVAX1-IA prolongs the half-life of pVAX1-IL-1Ra in mice, and also increases the content of IL-1Ra in the liver.

Embodiment 3

[0064] Example 3. In vitro activity evaluation of gene drug pVAX1-IA

[0065] HEK-293T cells were planted in a six-well plate, and when the cell fusion rate reached 70-80%, 5 μg of pVAX1-IA was transfected with lipofectamine2000. After continuing to culture for 24 hours, the supernatant of HEK-293T cells transfected with pVAX1-IA was collected and applied to A549 cells. The increase of IL-6 in A549 cells caused by 100pg / ml IL-1β was significantly inhibited, proving that pVAX1-IA The expressed IL-1Ra-ApoAI fusion protein antagonized the activity of IL-1β ( figure 1 D).

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Abstract

The invention belongs to the field of pharmacy, and relates to a nano drug delivery system capable of delivering a genetic drug towards secondary hepatocyte in a targeted mode and application. The nano drug delivery system is the nano drug delivery system capable of delivering the genetic drug towards the secondary hepatocyte in the targeted mode established by adopting cationic liposome preparedby (2,3-dioleacyl-propyl)trimethyl ammoniumchloride and cholesterol as a vector, wrapping a therapeutic plasmid pVAX 1-therapeutic gene-apolipoprotein AI gene for expressing hepatocyte targeted protein apolipoprotein AI, and inserting a hepatocyte-targeted lipid material, distearoyl phosphoethanolamine-polyethylene glycol 34000-galactose. In vitro and in vivo evaluation experiments prove that thenano drug delivery system can target on hepatocyte to achieve efficient and lasting liver protection effect durably, safely, stably and efficiently, and improve the therapeutic index of the drug and reduce the toxic and side effect of the drug; and the nano drug delivery system has good application prospect.

Description

technical field [0001] The invention belongs to the technical field of pharmacy, and relates to a nano-drug delivery system, in particular to a nano-drug delivery system for targeted delivery of gene drugs to secondary liver cells, a preparation method and an application thereof. The nano-drug delivery system can be used to treat non-alcoholic fatty Liver disease, alcoholic fatty liver disease, drug-induced liver injury, autoimmune liver disease, liver cancer, viral hepatitis and other liver diseases. Background technique [0002] Statistics show that the burden of liver disease in my country is extremely serious; according to the latest statistics in 2018, Chinese patients with liver disease account for as high as 30% of the world's liver disease patients; however, chronic viral hepatitis and non-viral liver diseases such as non-alcoholic fatty liver and drugs The treatment of diseases such as chronic liver injury is still a major problem in today's medical science, and no d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/26A61K47/18A61K47/28A61P1/16A61P37/02A61P35/00A61P31/14A61P31/20
CPCA61K48/005A61K48/0025A61K47/26A61K47/186A61K47/28A61P1/16A61P37/02A61P35/00A61P31/14A61P31/20
Inventor 鞠佃文栾静韵陈伟冯桢仪范佳君
Owner FUDAN UNIV
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