siRNA for inhibiting HMGB1 gene, stable nucleic acid lipid nanoparticle containing siRNA, and application of siRNA and stable nucleic acid lipid nanoparticle

A lipid nanoparticle, stable technology, applied in the field of medicine, to achieve the effects of improving utilization rate, inflammation and remission of bullous steatosis, and stable process

Active Publication Date: 2020-07-03
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The following technical problems exist in the prior art: there is a lack of an siRNA that can efficiently target macrophages to silence HMGB1 protein and can more effectively treat NASH

Method used

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  • siRNA for inhibiting HMGB1 gene, stable nucleic acid lipid nanoparticle containing siRNA, and application of siRNA and stable nucleic acid lipid nanoparticle
  • siRNA for inhibiting HMGB1 gene, stable nucleic acid lipid nanoparticle containing siRNA, and application of siRNA and stable nucleic acid lipid nanoparticle
  • siRNA for inhibiting HMGB1 gene, stable nucleic acid lipid nanoparticle containing siRNA, and application of siRNA and stable nucleic acid lipid nanoparticle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Screening of siRNA Effective Sequence and Preparation and Characterization of Stable Nucleic Acid Lipid Nanoparticles

[0058] Specific steps are as follows:

[0059] 1.1 Design and synthesis of siRNA

[0060] According to the cDNA sequence of mouse HMGB1 gene provided by GenBank, the inventor firstly designed 3 pairs of siRNA sequences targeting HMGB1 with a length of 21 nucleotides using the online design siRNA software, which were respectively denoted as HMGB1 siRNA-1, HMGB1 siRNA-2 and HMGB1 siRNA-3.

[0061] The sequences of HMGB1 siRNA-1, HMGB1 siRNA-2, and HMGB1 siRNA-3 are as follows:

[0062]

[0063] 1.2 Screening of effective siRNA sequences

[0064] RAW246.7 cells were cultured with RPMI-1640 medium, and the cells were collected and counted after trypsinization, and the cells were divided into 6×10 5 Each well was inoculated in a six-well plate with 2 mL of culture medium per well. Transfection was started when the cell density reached 30%-...

Embodiment 2

[0084] Example 2 Mannose receptor-mediated targeting of macrophages and investigation of targeting effects in vivo

[0085] Specific steps are as follows:

[0086] 2.1 Receptor blocking experiment: co-incubate cells with different concentrations of dextran or mannose, then add HMGB1-siRNA-cy5@SNALP-Man, and then use confocal laser to observe the amount of fluorescence in the cells to investigate the glucan Fluorescence differences in cells of the sugar and mannose groups. The preparation method of HMGB1-siRNA-cy5@SNALP-Man is the same as the preparation method of HMGB1-siRNA@SNALP-Mannose in Example 1, the difference is that the cy5 fluorescent dye is modified at the end of the siRNA.

[0087] The result is as figure 2 As shown, the dextran group ( figure 2 A) Cell fluorescence uptake did not change significantly with the increase of dextran concentration, but the mannose group ( figure 2 B) As the concentration of mannose increases, the intracellular fluorescence gradu...

Embodiment 3

[0089] Example 3 Investigation of HMGB1 silencing effect in vivo

[0090] In order to investigate the effect of silencing the expression of HMGB1 protein and controlling diet on NASH disease, and whether modifying mannose on the surface of the carrier will improve the drug utilization and enhance the therapeutic effect.

[0091] The experimental animals were divided into five groups, namely PBS group, Control-siRNA@SNALP, HMGB1-siRNA@SNALP, HMGB1-siRNA@SNALP-Mannose and HMGB1-siRNA@SNALP-Mannose+NFD group, (NFD is NORMALfeeding diets, that is, the normal feed group).

[0092] The mice in each group were injected into the tail vein twice a week. At the same time, they were fed with high-fat diet and normal diet according to the experimental design. After 8 times of administration, the mice were dissected and their livers were taken for QPCR experiments and Western Blot. Experiments, HE staining and immunohistochemical experiments. The differences in the expression of HMGB1 am...

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Abstract

The invention provides siRNA for inhibiting an HMGB1 gene, a stable nucleic acid lipid nanoparticle containing the siRNA, and an application of the siRNA and the stable nucleic acid lipid nanoparticle. The positive-sense strand of an siRNA molecule is SEQ ID NO:1, and the antisense chain is SEQ ID NO:2. The technical effects are as follows that the siRNA aiming at the HMGB1 is encapsulated in thestable nucleic acid lipid nanoparticle, so that the siRNA is prevented from being degraded by in-vivo enzymes; the prepared nanoparticle has a significant anti-inflammatory effect; and the siRNA aiming at the HMGB1 can be targeted and delivered to liver macrophages for release to enhance the anti-inflammatory effect, so that non-alcoholic steatohepatitis (NASH) can be better treated.

Description

technical field [0001] The invention relates to siRNA capable of effectively silencing the expression of HMGB1 in a NASH model, stable nucleic acid lipid nanoparticles loaded with siRNA and applications thereof, belonging to the technical field of medicine. Background technique [0002] In Western countries, nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. NAFLD includes simple fatty liver, nonalcoholic steatohepatitis (NASH) and its related fibrosis and cirrhosis. Even liver cancer. NASH is a more aggressive form of fatty liver disease that can progress to cirrhosis and complications of cirrhosis, including liver failure and hepatocellular carcinoma, and is expected to be the most common indication for liver transplantation over the next decade. Unlike alcoholic hepatitis, patients with NASH have no history of past alcohol use and are histologically characterized by steatosis, inflammation, and hepatocellular injury or associated liver fib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61K9/51A61K47/24A61K47/14A61K31/713A61P1/16
CPCC12N15/113A61K31/713A61P1/16A61K9/5146A61K9/5123C12N2310/141
Inventor 俞磊刘丽韩宇桥方小燕贾裕杰周靖娥王依婷闫志强王镜
Owner EAST CHINA NORMAL UNIV
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