Active targeted gene delivery nanoparticle, and preparation method and application thereof

A gene delivery and active targeting technology, applied in the field of nanobiotechnology gene therapy, can solve the problems of short circulation time in vivo, non-specific adhesion, cytotoxicity, etc., achieve active targeted transfection efficiency and improve safety and stability, broad clinical application prospects

Active Publication Date: 2019-04-02
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Usually, after the cationic polymer is complexed with nucleic acid, the surface of the complex will have excess positive charge, so that the adhesion between the complex and the negatively charged cell membrane is not specific, and when the amount of positive charge is too large, it will cause cell damage. Toxic effects, in addition, excess positive charges are easy to interact with proteins in the blood and are easily removed by the reticuloendothelial system (RES), and the circulation time in the body is short

Method used

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  • Active targeted gene delivery nanoparticle, and preparation method and application thereof
  • Active targeted gene delivery nanoparticle, and preparation method and application thereof
  • Active targeted gene delivery nanoparticle, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preparation of cationic polymer / gene complex

[0034]The PAsp(DET) and sFlt-1 plasmids were dissolved in HEPES (10mM, pH7.4), respectively, the concentration of the sFlt-1 plasmid was 0.05μg / μl, and the concentration of the PAsp(DET) solution was adjusted according to different N / P ratio requirements. Mix the PAsp(DET) solution and 2 times the volume of sFlt-1 plasmid solution and vortex for 30 seconds to prepare a PAsp(DET) / pDNA complex solution with a final concentration of sFlt-1 plasmid of 33.3 μg / mL. Using the same method and procedure, PAsp(EDA), PAsp(TET) and PAsp(TEP) or their derivatives can be used to construct cationic polymer / pDNA complexes.

[0035] Also, according to the same steps and methods as above, PAsp(EDA), PAsp(DET), PAsp(TET) and PAsp(TEP) or their derivatives and mRNA, siRNA, oligonucleotides, etc. can be used to form cationic polymers / mRNA complexes, cationic polymer / siRNA complexes, cationic polymer / oligonucleotide complexes, etc. ...

Embodiment 2

[0036] Embodiment 2: Preparation of PAsp(DET) / DNA / HA nanoparticles

[0037] In this example, PAsp(DET) is poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartic acid}, HA is hyaluronic acid, and HA is made form a chemical bond with the core of the complex. EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and NHS is N-hydroxysuccinimide.

[0038] PAsp(DET) and EGFP plasmids were prepared into solutions with concentrations of 2.0 μg / μl and 0.05 μg / μl with Tris-HCl (10 mM, pH 7.4), respectively. A mixed solution of HA, EDC and NHS was prepared with water, and the concentrations of HA, EDC and NHS were 1.0 µg / µl, 0.15 µg / µl and 0.25 µg / µl, respectively. Use Tris-HCl to adjust the PAsp(DET) and EGFP plasmid solution to the desired concentration, mix the PAsp(DET) and EGFP plasmid solution in an appropriate ratio and vortex for 30 seconds to prepare the PAsp(DET) / DNA complex substance solution. After the complex solution is left at room temperature for 30 minutes, add t...

Embodiment 3

[0042] Example 3: Comparison of Cytotoxicity

[0043] Using the above preparation method, prepare PAsp(DET) / DNA / HA nanoparticles, and use CCK-8 experiment to investigate different N / P / COO - The ratio of cytotoxicity of the gene-delivered nanoparticles. After HUVEC / B16F10 cells in the logarithmic growth phase were digested with trypsin, 5×10 3 Cells / well were seeded in 96-well plate, 50 μl cell suspension per well, at 37°C, 5% CO 2 After culturing in the incubator for 24 hours, add the sample solution adjusted to 50 μl with the medium, and prepare four kinds of each sample solution with N / P ratio of 5:1, 10:1, 20:1 and 40:1. For solutions with different N / P ratios, the sample solution added to each well contained 1.0 μg of EGFP plasmid, and PEG-PAsp (DET) with low toxicity and 25K PEI with high toxicity were used as controls. After culturing for 24 hours, add 10 μl of CCK-8 solution to each well, continue culturing for 2 hours, and measure the absorbance value (A) at 450 nm ...

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Abstract

The invention discloses an active targeted gene delivery nanoparticle, and a preparation method and application thereof. The active targeted gene delivery nanoparticle consists of a cationic polymer,hyaluronic acid and a negatively charged gene. The cationic polymer and the negatively charged gene form a composite core; the hyaluronic acid coats on the surface of the composite core by charge action and chemical bond bonding; and the cationic polymer is one of PAsp (EDA), PAsp (DET), PAsp (TET) and PAsp (TEP), or one of chemically modified derivatives of the PAsp (EDA), PAsp (DET), PAsp (TET)and PAsp (TEP). The hyaluronic acid used in the invention has excellent biocompatibility, not only reduces the cytotoxicity caused by the cationic polymer, but also actively targets a hyaluronic acid-specific receptor which is highly expressed on surfaces of tumor cells, theeby more efficiently delivering exogenous source genes into the tumor cells, and increasing cellular uptake and transfectionefficiency.

Description

technical field [0001] The invention belongs to the field of nano-biotechnology gene therapy, and relates to nano-biomaterials and gene delivery systems, in particular to a novel active-targeting gene delivery nanoparticle with hyaluronic acid as a target molecule and its preparation method and application. Background technique [0002] Gene therapy refers to the introduction of exogenous target genes into target cells of patients to correct or compensate diseases caused by gene defects and abnormalities, so as to achieve therapeutic purposes. After nearly ten or twenty years of development, the research of gene therapy has made a lot of progress, and its development trend is encouraging, especially as a new treatment method for cancer, a major human disease, it has become a research topic at home and abroad. hotspot. Gene delivery vectors are the most critical part of gene therapy, including viral vectors and non-viral vectors. Although viral vectors have the advantage of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/61A61K47/64A61P35/00
CPCA61K48/005A61K47/61A61K47/64A61P35/00
Inventor 米鹏魏于全张华萍
Owner SICHUAN UNIV
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