Preparation and anti-tumor effect of RGD/KLA integrated lipopeptide

A lipopeptide and resin technology, applied in the field of anticancer lipopeptide preparation, can solve the problems of easy hydrolysis by protease, restricted development, low cell membrane permeability and the like

Pending Publication Date: 2022-07-29
BINZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although ACPs have good anticancer activity, they still have disadvantages such as low cell membrane permeability, poor stability, and easy hydrolysis by proteases, which limit their further development.

Method used

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  • Preparation and anti-tumor effect of RGD/KLA integrated lipopeptide
  • Preparation and anti-tumor effect of RGD/KLA integrated lipopeptide
  • Preparation and anti-tumor effect of RGD/KLA integrated lipopeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Preparation method of anticancer lipopeptide

[0022] The synthesis of the anticancer lipopeptide of the present invention is carried out based on the Fmoc-protected solid-phase peptide synthesis method. The sequence of anticancer lipopeptide is C 8 H 15 O-Lys-Ala-Leu-Lys-Ala-Leu-Lys-Lys-Ala-Leu-Lys-Ala-Leu-Lys-Asp-Gly-Arg, the specific synthesis steps are as follows:

[0023] (1) Fmoc-Lys(Boc)-Ala-Leu-Lys(Boc)-Ala-Leu-Lys(Boc)-Lys(Boc)-Ala-Leu-Lys(Boc)-Ala-Leu-Lys(Boc) - Synthesis of Asp(OtBu)-Gly-Arg(Pbf)-Wang Resin;

[0024] Fmoc-Lys(Boc)-Ala-Leu-Lys(Boc)-Ala-Leu-Lys(Boc)-Lys(Boc)-Ala-Leu-Lys(Boc)-Ala-Leu- Lys(Boc)-Asp(OtBu)-Gly-Arg(Pbf)-Wang Resin sample; use DMF to swell Fmoc-Arg(Pbf)-Wang Resin for 30min; take a small amount of resin for ninhydrin detection, if no If the color changes, add a DMF solution containing 20% ​​piperidine to react for 30 minutes to remove the Fmoc protecting group; rinse the resin with DMF, DCM, and DMF in turn, and then ...

Embodiment 2

[0027]Example 2: In vitro anticancer activity assay of anticancer lipopeptides

[0028] The in vitro anticancer activity of anticancer lipopeptides was tested by MTT assay. Select H22 cells in logarithmic growth phase, inoculate H22 cells into 96-well plates, and then put them in 5% CO. 2 , 37 ℃ incubator for cultivation, use medium to prepare lipopeptide into different concentrations, the concentration is set to 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL. 100 μL of drug-containing medium was added to each well, and 5 replicate wells were set for each concentration to reduce the influence of errors caused by experimental chance. After co-culturing the cells and the drug for 24 h, add another 20 μL (5 mg·mL) to each well. -1 ) MTT solution, placed in a cell incubator for 4 h. After 4 h, the supernatant was discarded, 150 μL of DMSO solution was added to each well, and the 96-well plate was protected from light and...

Embodiment 3

[0029] Example 3: Secondary structure determination of anticancer lipopeptides

[0030] The secondary structure of lipopeptide in water and SDS solution was detected by circular dichroism (CD). The lipopeptide was dissolved in pure water and a 30 mM SDS solution to form a 150 μM lipopeptide solution, respectively. The wavelength setting range is 180-260nm, and the optical path selection is 0.5mm. The SDS solution environment can simulate the surface environment of the cancer cell membrane, so that the secondary structure changes when the lipopeptide binds to the cancer cell membrane can be observed. The data is transformed by the following formula:

[0031] θ M =θobs×1000 / cln

[0032] θ M is the average residue ellipticity (deg cm 2 ·dmol -1 ), θobs is the ellipticity (mdeg) observed at a given wavelength, c is the sample concentration (mM), l is the optical path (mm), and n is the number of peptide residues.

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Abstract

The invention relates to preparation of anti-cancer lipopeptide C8H15O-Lys-Ala-Lu-Lys-Ala-Lu-Lys-Ala-Lu-Lys-Ala-Lu-Lys-Asp-Gly-Arg and application of the anti-cancer lipopeptide C8H15O-Lys-Ala-Lu-Lys-Asp-Gly-Arg in anti-tumor treatment, and belongs to the field of biological medicine. The anti-cancer lipopeptides are prepared based on key biological characteristics (positive charges, alpha-helical structure and amphipathy) of membrane lysis peptides. The invention provides a solid-phase synthesis method of the anticancer lipopeptide. The anti-cancer lipopeptide has the characteristics of low hemolytic activity and good serum stability, can effectively overcome the defect of high hemolytic toxicity of most anti-cancer peptides, and solves the problem of poor in-vivo stability of peptide drugs. An in-vitro anti-cancer experiment and an in-vivo anti-tumor experiment prove that the anti-cancer lipopeptide has a good anti-tumor effect and a good application prospect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the preparation of an artificially synthesized anticancer lipopeptide with good anticancer activity and its application in tumor treatment. Background technique [0002] Cancer, also known as malignant tumor, is a disease caused by abnormal cell division and proliferation mechanism. It has become one of the most serious diseases threatening human health worldwide. Currently, surgery, radiation therapy (radiotherapy), and chemotherapy (chemotherapy) are the main treatments for cancer. However, although traditional anticancer drugs have high anticancer efficiency, they also have some disadvantages, such as low selectivity, obvious side effects, immunosuppression, nerve and gastrointestinal damage, etc. More importantly, the combined use of these anticancer drugs It is very easy to cause multidrug resistance (MDR) of tumors. Therefore, new breakthroughs are expected in the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/04C07K1/06A61K38/10A61P35/00
CPCC07K7/08A61P35/00A61K38/00
Inventor 闫苗苗马林浩张琳靖
Owner BINZHOU MEDICAL COLLEGE
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