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Gene/drug composite lipid preparation and preparation method and application thereof

A technology for compounding lipids and preparations, which can be used in drug combinations, gene therapy, pharmaceutical formulations, etc., and can solve problems such as unsatisfactory results.

Active Publication Date: 2021-05-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, no satisfactory results have been found so far whether targeting TGF-β secretion, blocking its interaction with receptors, or targeting downstream intracellular targets.

Method used

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  • Gene/drug composite lipid preparation and preparation method and application thereof
  • Gene/drug composite lipid preparation and preparation method and application thereof
  • Gene/drug composite lipid preparation and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Preparation and Characterization of Example 1 Gene / Drug Composite Lipid Carrier

[0076] (1) Reagent and lipid stock solution: DOTAP / Chol / DPPC stock solution: 25mg / mL, weigh 100mg DOTAP / Chol / DPPC dissolved in 4mL chloroform; DMG-PEG2000 stock solution: 5mg / mL, weigh 20mgDMG - PEG2000 dissolved in 4mL chloroform; ammonium sulfate solution: 200mM, prepared by weighing 1.057g solid ammonium sulfate powder and dissolving it in 40mL ultrapure water;

[0077] (2) Pipette an appropriate amount of DOTAP / Chol / DPPC / DMG-PEG2000 stock solution into a round-bottomed flask with a micro-syringe needle, the molar ratio is 50:40:9:1, add an appropriate amount of chloroform, and place in a 50°C water bath Rotary evaporation to remove organic solvent;

[0078] (3) drying up the organic solvent remaining in the round-bottomed flask of step (2) with nitrogen to make lipid film;

[0079] (4) adding an appropriate amount of 200mM ammonium sulfate solution preheated to 50°C into the lipid fi...

Embodiment 2

[0084] The luciferase gene transfection effect evaluation of embodiment 2 gene / drug composite lipid carrier

[0085] (1) 24 hours in advance, mouse hepatic stellate cells (JS1) were treated with 1*10 4 Seed into a 96-well plate at the density of each well, add 100uL DMEM complete medium containing 10% FBS to each well, and enter the next step after the cell confluency reaches 80%;

[0086] (2) Blank cationic liposomes were co-incubated with pGL3 plasmid at a nitrogen-to-phosphorus ratio of 4:1. Add about 250ng of plasmid to each well, add DMEM medium to a total volume of 100uL per well, and place in a 37°C incubator without serum transfer. Change the medium after 4-6 hours of staining, add complete medium and continue to culture for 24 hours;

[0087] (3) 24 hours after transfection, wash with pre-cooled PBS to remove residual medium, add 50uL cell lysate to each well, lyse at room temperature for 10 minutes, and centrifuge at 4,000rpm in an orifice centrifuge for 2 minutes; ...

Embodiment 3

[0090] Example 3 Efficiency Evaluation of Gene / Drug Composite Lipid Carrier Importing Cy5-siRNA into Hepatic Stellate Cells

[0091] (1) 24 hours in advance of JS1 cells with 1*10 4 Seed into a 96-well plate at the density of each well, add 100uL DMEM complete medium containing 10% FBS to each well, and enter the next step after the cell confluency reaches 80%;

[0092] (2) Blank cationic liposomes were incubated with cy5-siRNA at a nitrogen-to-phosphorus ratio of 4:1 in the dark, and three concentrations of siRNA were set, namely 50nM, 100nM, and 200nM, and DMEM medium was added to make the total volume of each well 100uL. Serum-free transfection in a 37°C incubator;

[0093] (3) Change the medium after 4-6 hours of transfection, wash with pre-warmed PBS three times, and remove unbound free siRNA;

[0094] (4) The transfection efficiency was observed under a fluorescence microscope, and the images were converted into grayscale images using ImageJ software.

[0095] The res...

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Abstract

The invention relates to the technical field of biology, in particular to a gene / small molecule compound complex lipid preparation and a preparation method and application thereof, and particularly relates to a gene / drug composite lipid preparation aiming at a TGF-beta / SMAD signal channel and application thereof. Research finds that a positively charged cationic lipid and a negatively charged nucleic acid drug form a lipid complex through electrostatic interaction, and by means of an active drug loading technology of a small molecule compound, a complex lipid carrier capable of simultaneously delivering a gene drug / small molecule compound into the same target cell can be formed.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene / small molecular compound compound lipid preparation targeting TGF-β / SMAD signaling pathway and its application. Background technique [0002] The TGF-β / SMAD signaling pathway plays a very important role in many pathological and physiological processes. Dysplasia of intrahepatic connective tissue caused by excessive deposition of diffuse extracellular matrix. [0003] The TGF-β / SMAD signaling pathway plays a very important role in the progression of fibrotic diseases, but due to the pleiotropic effects of TGF-β and its role in other physiological processes such as inflammation regulation and immune suppression, direct inhibition of TGF-β The β signaling pathway may have potential systemic adverse effects. TGF-β1 homozygous deletion mice were accompanied by severe immune and inflammatory system dysfunction, and the mice died in utero or died of multi-organ inflammation within ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K31/517A61K9/127A61P1/16
CPCA61K48/005A61K31/517A61K9/1277A61K47/28A61P1/16A61K2300/00Y02A50/30
Inventor 徐宇虹张新健
Owner SHANGHAI JIAO TONG UNIV
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