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Compound polypeptide molecule for targeted killing of cancer cells and preparation method thereof

A cancer cell and molecular technology, which is applied in the field of compound polypeptide molecules that target and kill cancer cells and its preparation, can solve the problem that cracking peptides cannot target and kill cancer cells, achieve targeted killing of cancer cells, prevent damage, simple effects

Pending Publication Date: 2021-04-30
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a compound polypeptide molecule that targets and kills cancer cells and its preparation method, aiming at solving the technical problem that lytic peptides cannot target and kill cancer cells

Method used

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  • Compound polypeptide molecule for targeted killing of cancer cells and preparation method thereof
  • Compound polypeptide molecule for targeted killing of cancer cells and preparation method thereof
  • Compound polypeptide molecule for targeted killing of cancer cells and preparation method thereof

Examples

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preparation example Construction

[0036] The preparation method of the composite polypeptide molecule provided by the embodiment of the present invention has simple process and low cost, and can be realized on a polypeptide solid-phase synthesizer. The finally obtained composite polypeptide molecule includes gallic acid, cleavage peptide, and links between gallic acid and cleavage peptide The MMP-2 specifically recognizes the degraded polypeptide substrate, which can achieve the effect of targeting and killing cancer cells.

[0037] In one embodiment, after synthesizing the complex polypeptide molecule, a step of separating and purifying by high performance liquid chromatography is also included. Specific separation process: Dissolve the peptide sheared from the resin, filter it with a 0.2 μm filter membrane, and separate it through a C18 reverse column. The mobile phase is water and acetonitrile, and the peptide peak is collected (monitored at an ultraviolet wavelength of 225 nm).

Embodiment 1

[0039] Example 1 Research on the effect of splitting peptide

[0040] The lytic peptide can destroy cancer cells and normal cells that are harmful to humans by lysing biofilms. This phenomenon can be verified by CCK-8 activity test.

[0041] (1) 5000-8000 human osteosarcoma cells MG-63 were seeded in a 96-well plate, cultured overnight in DMEM containing 10% FBS, and the culture conditions were 37°C, 5% CO 2 , and then use different concentrations (0, 5 μM, 10 μM, 20 μM) of the cleavage peptide (there are four kinds, respectively: SEQ.ID.No.2, SEQ.ID.No.3, SEQ.ID.No.3, SEQ.ID.No.3, SEQ. .ID.No.4, SEQ.ID.No.5) replace the culture medium, after continuing to culture for 24 hours, the cell survival rate is detected by CCK-8 reagent.

[0042] It was found that the above four lytic peptides can effectively kill half of the cancer cells at a concentration above 5 μM, indicating that the lytic peptides have good anticancer activity. The cleavage peptides SEQ.ID.No.3 and SEQ.ID.No.5...

Embodiment 2

[0045] Example 2 Research on the Molecular Action of Composite Polypeptides

[0046] 5000-8000 human kidney epithelial cells 293-T were seeded in 96-well plates and cultured overnight in DMEM containing 10% FBS at 37°C, 5% CO 2 , and then use the composite polypeptide molecules (wherein, MMP-2 specifically recognizes the degraded polypeptide substrate as SEQ.ID.No.1) of different concentrations (0, 5 μM, 10 μM, 20 μM) of DMEM dissolved in 10% FBS to be cleaved The peptide is SEQ.ID.No.5) to replace the medium, and after continuing to culture for 24 hours, the cell viability was detected by CCK-8 reagent.

[0047] The result is as Figure 5 As shown, since gallic acid neutralizes the positive charge of the cleavage peptide through the negative charge, it effectively inhibits the killing effect of the complex polypeptide molecule on normal cells.

[0048] Based on the above examples, it can be known that the examples of the present invention neutralize the positive charge of t...

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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a compound polypeptide molecule for targeted killing of cancer cells and a preparation method thereof. The compound polypeptide molecule comprises gallic acid, a cracking peptide and a polypeptide substrate which is connected between the gallic acid and the cracking peptide and is used for MMP-2 specific recognition degradation. In the composite polypeptide molecule, the electronegative gallic acid small molecule can effectively neutralize the electropositivity and the split membrane activity of the split peptide, so that the damage of the split peptide to normal cells in the in-vivo circulation process can be effectively prevented, and after the composite polypeptide molecule enters cancer tissues, the cancer cells secrete a large amount of MMP-2 protein, the MMP-2 protein can recognize and degrade a polypeptide substrate connected between gallic acid and lytic peptide, so that free lytic peptide is released, and the effect of killing cancer cells in a targeted manner can be achieved.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a compound polypeptide molecule targeting to kill cancer cells and a preparation method thereof. Background technique [0002] In the treatment of malignant tumors, chemotherapy has always occupied an irreplaceable position, but its clinical therapeutic effect is limited, mainly due to the resistance of tumor cells to chemotherapy drugs. Therefore, how to develop a therapeutic regimen that can overcome drug resistance is an important issue in cancer treatment. [0003] Matrix metalloproteinases (MMPs) are a group of Ca-dependent 2+ , Zn 2+ Extracellular enzymes such as metal ions as cofactors play an important role in the degradation of the extracellular matrix. MMP-2 is one of the members of the MMPs family. MMP-2 belongs to gelatinase. It mainly plays an important role in tumor invasion and metastasis by specifically degrading type IV and type collagen V, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/04C07K1/16
CPCC12N9/6491C12Y304/24024C07K2319/00
Inventor 刘启颂
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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