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Nucleic acid delivery system based on alkylated polypeptide as well as preparation method and application

A delivery system and alkylation technology, applied in the field of biomedicine, to achieve the effects of good biocompatibility, simplified preparation process, and low cost

Inactive Publication Date: 2021-10-29
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the use of cationic polypeptide fragments and hydrophobic fragments shown in SEQ ID NO.1 to form alkylated polypeptides for efficient transfection of nucleic acid drugs into various cancer cells or immune cells

Method used

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  • Nucleic acid delivery system based on alkylated polypeptide as well as preparation method and application
  • Nucleic acid delivery system based on alkylated polypeptide as well as preparation method and application
  • Nucleic acid delivery system based on alkylated polypeptide as well as preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0034] An alkylated polypeptide, the alkylated polypeptide comprises a cationic polypeptide fragment and a hydrophobic fragment, and the cationic polypeptide fragment has an α-helical secondary structure ( image 3 ), and is sensitive to pH; the hydrophobic segment is the lipid part of palmitic acid; the amino acid sequence of the cationic polypeptide segment is shown in SEQ ID NO.1; the N-terminal of the cationic polypeptide segment is connected to the hydrophobic segment.

[0035] The molecular formula of the alkylated polypeptide of this embodiment:

[0036] C 16 -Trp-Glu-Ala-Arg-Leu-Ala-Arg-Ala-Leu-Ala-Arg-His-Leu-Ala-Arg-Ala-Leu-Arg-Ala-Cys-Glu-Ala, (wherein the cationic polypeptide The amino acid sequence of the fragment is shown in SEQ ID NO.1, artificially synthesized).

[0037] SEQ ID NO.1 was entrusted to Jill Biochemical (Shanghai) Co., Ltd. for production and preparation.

[0038] The purity of the alkylated polypeptide was tested using the 1200 liquid chromatog...

Embodiment 2

[0041] A method for preparing an alkylated polypeptide-based nucleic acid delivery system 1, comprising the following steps: dissolving 0.5 mg of an alkylated polypeptide prepared in Example 1 in 1 mL of nuclease-free water (DEPC water), and Head blowing to make it evenly dispersed to obtain a solution of alkylated polypeptide with a concentration of 0.5 mg / mL;

[0042] Add to 1 μg / μL nucleic acid solution 1, so that the molar ratio (N / P) of the protonated nitrogen in the prepared alkylated polypeptide solution to the phosphate group in the nucleic acid solution 1 is equal to 15. The alkylated polypeptide-based nucleic acid delivery system 1 was obtained by electrostatic complex assembly at room temperature for 30 minutes.

[0043] Nucleic acid solution 1: 720-base single-stranded EGFP mRNA (SEQ ID NO.2, enhanced green fluorescent protein messenger RNA), purchased from TriLink BioTechnologies in the United States; EGFP mRNA was purchased as a 1 μg / μL solution, artificially synth...

Embodiment 3

[0052] Application of an alkylated polypeptide-based nucleic acid delivery system 1 prepared in Example 2 in the preparation of anticancer drugs

[0053] (1) Preparation of HEK 293T cell culture medium:

[0054] First add 45mL DMEM high-glucose medium to a 50mL centrifuge tube, then add 5mL Australian fetal bovine serum and 500μL double antibody (penicillin and streptomycin), mix well to obtain HEK 293T cell culture medium;

[0055] (2) Preparation of DC2.4 cell culture medium:

[0056] First add 45mL RPMI-1640 medium to a 50mL centrifuge tube, then add 5mL Australian fetal bovine serum and 500μL double antibody (penicillin and streptomycin), mix well to obtain DC2.4 cell culture medium;

[0057] (3) Place HEK 293T cells and DC2.4 cells in 5% CO 2 , Cultured in a cell culture incubator at 37°C. Resuspend HEK 293T cells and DC2.4 cells in the logarithmic growth phase with HEK 293T cell culture medium and DC2.4 cell culture medium respectively, and press 5×10 4 Cells / well we...

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Abstract

The invention discloses a nucleic acid delivery system based on alkylated polypeptide as well as a preparation method and application. The alkylated polypeptide comprises a cationic polypeptide fragment and a hydrophobic fragment, wherein the cationic polypeptide fragment has an alpha-spiral secondary structure; the hydrophobic fragment is a lipid part of any one of palmitic acid, lauric acid, myristic acid and stearic acid; the amino acid sequence of the cationic polypeptide fragment is as shown in SEQ ID NO. 1; and the N-end of the cationic polypeptide fragment is connected with the hydrophobic fragment. The alkylated polypeptide can form a spherical nucleic acid delivery system based on the alkylated polypeptide with a proper size and a stable structure with nucleic acid molecules in different N / P proportions in a simple mixing manner, and transmembrane delivery of the nucleic acid molecules can be realized. Compared with the existing lipidosome and polymer transfection reagent, the alkylated polypeptide provided by the invention has the advantage that the preparation process for wrapping a nucleic acid medicine is simplified, and has the advantages of low toxicity, good biocompatibility, low cost and the like.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a nucleic acid delivery system using an alkylated polypeptide as a carrier, a preparation method and an application. Background technique [0002] In recent years, with the continuous in-depth research on the mechanism of disease genes, nucleic acid drugs represented by oligonucleotides, plasmid DNA, siRNA or mRNA have been proven to be used for the treatment of various diseases, which has triggered a global Wide attention, some of these nucleic acid drugs have been approved for marketing for clinical treatment. However, a series of obstacles in the transportation of nucleic acids in body fluids and cells, such as rapid degradation in the blood, fast liver and kidney clearance, low cellular uptake efficiency, and difficulty in escaping from endosomes, have greatly hindered the development of nucleic acids. Clinical application of drugs. [0003] At present, most of the gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00A61K47/64A61K47/54A61K31/7088A61P35/00
CPCC07K14/001A61K47/64A61K47/542A61K31/7088A61P35/00
Inventor 王跃飞齐崴胡柳平苏荣欣
Owner TIANJIN UNIV
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