Alpha spiral cation polypeptide molecule and preparation method and application thereof

A cationic polypeptide and helical technology, which is applied to alpha helical cationic polypeptide molecules and their preparation methods and application fields, to achieve the effects of high charge density, high gene expression efficiency and mild synthesis conditions

Inactive Publication Date: 2011-04-13
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the research on gene carrier materials has received much attention and some progress has been made, there are still many problems to be solved

Method used

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  • Alpha spiral cation polypeptide molecule and preparation method and application thereof
  • Alpha spiral cation polypeptide molecule and preparation method and application thereof
  • Alpha spiral cation polypeptide molecule and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The general molecular formula of the polypeptide in claim 1 is taken as n1=3, n2=1, x=1, y=1, z=0, B1 is Ala, and B2 is Lys. The structure of the polypeptide molecule is:

[0055]

[0056] Abbreviated as RC16. This example illustrates the synthesis of RC16 and its use in gene delivery. RC16 was synthesized by a peptide synthesizer and purified by HPLC.

[0057] Dissolve 10mg of RC16 in 20mM PBS buffer (pH 5.0), mix the RC16 solution with DNA according to a certain nitrogen-phosphorus ratio (N / P), and the N / P value is 0, 1, 5, 10, 20 , 30, 40 and 50. The mixture was left at room temperature for 30 minutes to fully combine RC16 with DNA to form a stable RC16 / DNA complex. The complex had an average particle diameter of 540 nm and a zeta potential of 22 mV.

[0058] HepG2 cells were seeded in 24-well plates (8×10 4 cells / well), put into an incubator, and when the confluence of the cells reaches 70%, remove the medium, rinse the cells twice with PBS buffer, and then...

Embodiment 2

[0060] The molecular general formula of the polypeptide in claim 1 is set as n1=5, n2=4, x=7, y=12, z=5, B1 is His, and B2 is Arg. n2=4 means that the molecule is a tetra-crosslinked polypeptide, referred to as tetra-RC49, and the corresponding monomer molecule is referred to as RC49.

[0061] Its structural formula is:

[0062]

[0063] This example illustrates the synthesis of tetra-RC49 and its use in gene delivery. The structural formula of tetra-RC49 is:

[0064]

[0065] Firstly, RC49 was synthesized by a peptide synthesizer, and purified by preparative liquid chromatography to obtain a monomeric polypeptide molecule.

[0066] Dissolve 10 mg of purified RC49 in deionized water to a concentration of 10 mg / ml; dissolve 400 mg of azodicarbonamide (N, N, N', N'-tetramethylazodicarboxamide) in DMSO to a concentration of 0.1 mg / μL. Then the two solutions were mixed so that the molar ratio of RC49 and azodicarbonamide was about 1:100. After stirring at room temperatu...

Embodiment 3

[0070] In claim 1, the molecular general formula of the polypeptide takes n1=4, n2=2, x=2, y=4, z=3, B1 is His, and B2 is Lys. n2=2 means that the molecule is a di-crosslinked polypeptide, referred to as di-RC30, and the corresponding monomer molecule is referred to as RC30, and its structural formula is:

[0071]

[0072] This example illustrates the synthesis of di-RC30 and its use in gene delivery. The structural formula of di-RC30 is:

[0073]

[0074] Firstly, RC30 was synthesized by a peptide synthesizer and purified by HPLC.

[0075] 10 mg of purified RC30 was dissolved in deionized water at a concentration of 50 mg / ml; 24 mg of azodicarbonamide (N, N, N', N'-tetramethylazodicarboxamide) was dissolved in DMSO at a concentration of 2 mg / μL. Then the two solutions were mixed so that the molar ratio of RC30 to azodicarbonamide was about 1:52. After stirring at room temperature for 3 days, the by-products were removed by dialysis with deionized water (MWCO=500Da). A...

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Abstract

The invention relates to an alpha spiral cation polypeptide molecule and a preparation method and application thereof. The polypeptide molecule consists of an alpha spiral main chain and a side chain. The main chain consists of lysine, arginine, leucine and histidine; and the side chain mainly comprises histidine, lysine, arginine and a targeted group. Histidine contributes to releasing a gene by an endosome and enhances the expression effect of the gene; the lysine or arginine can provide positive charges which are combined with DNA to form a stable compound; and the targeted group is favorable for improving the expression efficiency of the gene. The sizes of polypeptide/ DNA compound particles prepared from the alpha spiral cation polypeptide molecule are between 100 and 500 nm, and positive charges are carried on the surfaces of the particles. The invention has the characteristics of narrow molecular weight distribution of polypeptide, simple method for compounding the polypeptide and the gene, high stability and expression efficiency of the gene, low toxicity and the like.

Description

technical field [0001] The present invention mainly relates to the field of biomedical macromolecular material and its application, in particular to an alpha helical cationic polypeptide molecule and its preparation method and application. Background technique [0002] Gene therapy has entered clinical trials for the first time in 1989, and it has been more than 20 years since it provided a new treatment method for many diseases (especially cancer). Gene therapy must have a carrier system that can introduce the target gene into the cells of the body. Whether gene therapy is successfully applied clinically, the technical bottleneck is whether a safe and efficient gene drug delivery system can be developed (International Journal of Pharmaceutics, 2001, 229: 1-21; Nature Biotechnology, 2000, 18: 33-21). 37; Gene Therapy, 2005, 12:1734-1751). Currently, there are two types of vector systems commonly used for gene delivery: viral vectors and non-viral vectors. The former has h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K14/00C12N15/63
Inventor 郭新东钱宇章莉娟
Owner SOUTH CHINA UNIV OF TECH
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