Targeting nucleic acid drug as well as preparation method and application thereof

A nucleic acid drug and targeting technology, which can be used in drug combinations, pharmaceutical formulations, anti-tumor drugs, etc., and can solve problems such as inability to achieve targeted delivery of drugs

Active Publication Date: 2020-02-07
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese invention patent application CN105199012A discloses a colloidal granule based on hydrophobically modified hyaluronic acid self-assembled colloid, which uses hydrophobically modified hyaluronic acid to make the colloidal granule have good emulsifying properties, biocompatibility and light Responsiveness, however, the colloidal particles cannot achieve targeted drug delivery

Method used

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  • Targeting nucleic acid drug as well as preparation method and application thereof
  • Targeting nucleic acid drug as well as preparation method and application thereof
  • Targeting nucleic acid drug as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Synthesis and characterization of distearoylphosphatidylethanolamine-hyaluronic acid (DSPE-HA) conjugate

[0065] The synthetic route of DSPE-HA conjugate is as follows Figure 1A shown.

[0066]0.2g of sodium hyaluronate was dissolved in 20mL of deionized water, dialyzed in 0.01M HCl for 24h, and then dialyzed in deionized water for 24h to obtain hyaluronic acid in the form of acid groups. Then tetrabutylammonium hydroxide solution was added dropwise to adjust the pH to 9, and stirred at room temperature for 2 h. Finally, it was dialyzed in deionized water for 48 hours to remove excess tetrabutylammonium hydroxide, and freeze-dried to obtain tetrabutylammonium salt of hyaluronic acid (HA-TBA).

[0067] Dissolve 10 μmol HA-TBA in 8 mL of anhydrous dimethyl sulfoxide (DMSO), stir at 60°C until dissolved, dissolve 50 μmol DSPE and 15 μL triethylamine in 2 mL of chloroform, mix them and stir at 60°C for 2 hours. Next, 100 μmol NaBH(OAc) 3 Dissolve in 2mL of ...

Embodiment 2

[0073] Example 2 Preparation and characterization of Gli1 siRNA nanoparticles targeting gastric cancer stem cells

[0074] The preparation process of gastric cancer stem cell-targeted Gli1 siRNA nanoparticles is as follows: figure 2 shown.

[0075] 1. Preparation of targeting Gli1 siRNA nanoparticles

[0076] ① Preparation of cationic liposomes

[0077] Accurately weigh an appropriate amount of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-oleoylphosphatidylethanolamine (DOPE) (1:1, molar ratio), dissolve in chloroform and methanol (3:1, volume ratio) in a mixed solvent, 40 ° C under reduced pressure rotary evaporation to remove the organic reagent, and left overnight to further remove the residual organic solvent. Add 5% glucose solution prepared with DEPC water, ultrasonically hydrate the lipid film, and then further crush it in an ultrasonic cell pulverizer (setting the working time to 10s, the intermittent time to 10s, the whole time to 8min, the protection t...

Embodiment 3

[0097] Example 3 The preparation of targeting Gli1 siRNA nanoparticles against gastric cancer stem cells

[0098] 4.1 Expression of Gli1 in cells

[0099] Inoculate the sorted CD44+ cells and CD44- cell suspensions into 6-well plates with coverslips at 37°C, 5% CO 2 Incubate in the incubator for 24h. Wash three times with 0.1mol / L phosphate PBS, 3 minutes each time; fix with 4% paraformaldehyde at room temperature for 20-30 minutes; wash three times with PBS, 3 minutes each time; 5% BSA for blocking at room temperature for 1 hour, adding primary antibody anti-Gli1 antibody (Abcam Company), overnight at 4°C; washing with PBS three times, each time for 3 minutes; adding FITC-labeled goat anti-rabbit secondary antibody, and incubating for 1 hour at 37°C; PBS Wash three times, each time for 3 minutes; then use 5 μg / mL DAPI for nuclear staining at room temperature for 10 minutes, and rinse three times with PBS. Image analysis was performed with a confocal laser microscope. Ce...

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Abstract

The invention provides a targeting nucleic acid drug as well as a preparation method and an application thereof. The targeting nucleic acid drug comprises an amino lipid-hyaluronic acid conjugate, a cationic liposome and a complex containing protamine, chondroitin sulfate and a nucleic acid drug, the amino lipid part of the amino lipid-hyaluronic acid conjugate is combined with the cationic liposome, and the cationic liposome wraps the complex. The targeting nucleic acid drug inhibits self-renewal and migration invasion of cancer cells by specifically blocking a Hedgehog signal transduction pathway of cell self-proliferation and differentiation, induces cell apoptosis, and can effectively inhibit recurrence of tumors.

Description

technical field [0001] The present invention relates to the field of tumor targeting therapy, in particular to a targeting drug modified with hyaluronic acid and its preparation method and application. Background technique [0002] In recent years, some researchers at home and abroad have used liposomes or nanoparticles to encapsulate anti-tumor stem cell drugs, which increased the accumulation of anti-tumor stem cell drugs in tumor tissues (see Liu Y, Lu WL, Guo J, et al.A potential target Associated with both cancer and cancer stem cells: A combinationtherapy for eradication of breast cancer using vinorelbine stealthy liposomesplus parthenolide stealthy liposomes.J.Control Release, 2008,129(1):18-25; LiRJ, Ying X, Zhang Y, et al .All-trans retinoic acid stealth liposomes prevent therelapse of breast cancer emerging from the cancer stem cells.J.ControlRelease,2011,149:281-291). However, due to the lack of specificity of these liposomes or nanoparticles to tumor cells, espe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/127A61K47/36A61K47/42A61K31/713A61P35/00A61P35/02A61P35/04
CPCA61K9/1271A61K47/36A61K47/42A61K31/713A61P35/00A61P35/02A61P35/04
Inventor 姚红娟李亮宋文凭周晓菲李睿
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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