Ph-sensitive polypeptide, polypeptide-polymer conjugate, preparation method and application

A technology of polymers and conjugates, which is applied in the field of biomedicine, can solve the problems of high production cost of natural antimicrobial peptides, limit the application of natural antimicrobial peptides, and prevent large-scale production of antimicrobial peptides, and achieve the effect of sensitive conformational changes

Active Publication Date: 2022-03-29
HENAN UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the high production cost of natural antimicrobial peptides cannot be applied to large-scale production of antimicrobial peptides. At the same time, its non-specific toxicity to mammalian normal cells limits the application of natural antimicrobial peptides

Method used

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  • Ph-sensitive polypeptide, polypeptide-polymer conjugate, preparation method and application
  • Ph-sensitive polypeptide, polypeptide-polymer conjugate, preparation method and application
  • Ph-sensitive polypeptide, polypeptide-polymer conjugate, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 has the synthesis of the polypeptide Pep of pH sensitivity

[0065] The amino acid sequence of the polypeptide Pep is COOH-(XXXLXXV) n -C-NH 2 , wherein n: 3-6; V: valine; L: leucine; C: cysteine; X: any amino acid.

[0066] The polypeptide Pep sequence in the present invention is designed according to the α-helical structure of the polypeptide, with 7 amino acids as a repeating unit, and the amino acid position number in each repeating unit XXXLXXV is gfedcba, where the a and d positions are usually hydrophobic valine Acid and leucine, forming a hydrophobic core; and e, f positions are usually oppositely charged polar amino acids, such as lysine, glutamic acid, e, f positions are oppositely charged, forming an electrostatic attraction; each repeat The specific position in the unit is set as leucine or valine, so the polypeptide Pep in the present invention has an α-helical structure; finally, the so-called leucine zipper structure is formed by using elec...

Embodiment 2

[0071] The synthesis of embodiment 2 polymer PMAA

[0072] Step 1, Synthesis of Initiator

[0073] (1) Dissolve 200mg of hydroxyethylmaleimide (N-(2-hydroxyethyl)maleimide) in 5mL of dichloromethane solution, add 250μL of triethylamine, and mix well to obtain hydroxyethylmaleimide basemaleimide solution;

[0074] (2) Dissolve 210 μL of dibromoisobutyl bromide in 2 mL of dichloromethane to obtain a dibromoisobutyl bromide solution;

[0075] (3) In an ice-water bath, slowly drop the dibromoisobutyryl bromide solution into the hydroxyethylmaleimide solution, and control the dropping time for a total of 30 minutes;

[0076] (4) After the dropwise addition is completed, place the above-mentioned mixed solution at room temperature to react overnight, and the reaction time is more than or equal to 12h;

[0077] (5) After the reaction is finished, use ultrapure water to extract three times, collect the organic phase, and pass the organic phase through anhydrous MgSO 4 After drying...

Embodiment 3

[0093] The synthesis of embodiment 3 polypeptide-polymer conjugate Pep-PMAA

[0094] (1) Prepare 15 mL of an aqueous solution containing 10 mg of polypeptide Pep, and the specific sequence of polypeptide Pep is NH 2 -C-VAQLEVKVAQLESK-VSKLESK-VSSLESK-COOH;

[0095] (2) Add 6 mg of polymer PMAA to the above aqueous solution, and stir and react at room temperature for 12 hours;

[0096] (3) The above reaction system was placed in a dialysis bag with a molecular cut-off of 3500, and the dialysate was replaced every 6 hours for a total of 48 hours of dialysis;

[0097] (4) Freeze-dry the dialyzed aqueous solution in the bag to a solid state to obtain the conjugate Pep-PMAA.

[0098] The chemical reaction between the polypeptide Pep and PMAA relies on the Michael addition reaction between the sulfhydryl group at one end of the polypeptide molecule and the N-methylmaleimide at one end of the polymer molecule to realize the construction of the conjugate.

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Abstract

The invention belongs to the technical field of biomedicine, and specifically relates to a pH-sensitive polypeptide, a polypeptide-polymer conjugate, a preparation method and an application. The amino acid sequence of the polypeptide Pep is COOH‑(XXXLXXV) n ‑C‑NH 2 , where n: 3‑6; V: valine; L: leucine; C: cysteine, X: any amino acid; the amino acid position number in XXXLXXV is gfedcba, where the a and d positions are hydrophobic of amino acids. The polypeptide-polymer conjugate is obtained by Michael addition reaction coupling between the thiol at one end of the polypeptide molecule and the N-methylmaleimide at one end of the polymer PMAA molecule; its molecular structure is COOH-(XXXLXXV ) n The molecular weights of ‑C‑PMAA, PMAA and polypeptide‑polymer conjugates are all 1000‑10000. By controlling the molecular weight of the polymer, a series of conjugates of polypeptide-polymers with different pH sensitive points can be obtained, and the polypeptides or conjugates can destroy the membrane structure of tumor cells in an acidic environment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a pH-sensitive polypeptide, a polypeptide-polymer conjugate, a preparation method and an application. Background technique [0002] Cancer is a major cause of morbidity and mortality in today's society. In 2019, the National Cancer Center released the latest national cancer statistics. In 2015, the incidence of malignant tumors in the country was about 4 million, a growth rate of 3.2% compared with 2014, which means that more than 10,000 people are diagnosed with cancer every day, and 7.5 people are diagnosed with cancer every minute. Cancer remains a huge challenge to human health around the world. Radiotherapy and chemotherapy are traditional cancer treatment methods. However, while killing tumor cells, they will also cause damage to normal cells and immune cells, and the resulting drug resistance will greatly reduce the efficacy of treatment. Therefore, the ne...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C07K14/00C07K1/10A61K38/16A61K47/58A61P35/00C08F8/12C08F120/16G01N21/64
CPCC07K14/00A61K47/58A61P35/00C08F8/12G01N21/6428A61K38/00G01N2021/6439C08F120/16
Inventor王斯佳李圆圆李朋伟吕品孙月
OwnerHENAN UNIV OF CHINESE MEDICINE