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cRGD-quaternized chitosan oligosaccharide modified ES2 peptide-methotrexate conjugate as well as a preparation method and application thereof

A technology for converting chitosan oligosaccharide and methotrexate, applied in the field of biomedicine, can solve the problems of poor stability, low cell affinity, short half-life, etc., and achieve the effects of high stability and strong biological activity

Pending Publication Date: 2021-10-15
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ES2 also has shortcomings such as poor stability, low cell affinity, unstable activity, and short half-life. Studies have shown that chemical modification is expected to improve these shortcomings.

Method used

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  • cRGD-quaternized chitosan oligosaccharide modified ES2 peptide-methotrexate conjugate as well as a preparation method and application thereof
  • cRGD-quaternized chitosan oligosaccharide modified ES2 peptide-methotrexate conjugate as well as a preparation method and application thereof
  • cRGD-quaternized chitosan oligosaccharide modified ES2 peptide-methotrexate conjugate as well as a preparation method and application thereof

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Experimental program
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preparation example Construction

[0041] The second aspect of the present invention provides the preparation method of the cRGD-quaternized chitosan-modified ES2 peptide-methotrexate conjugate described in the first aspect, the preparation method comprising the following steps: activating the carboxyl part of cRGD, weakly Add quaternized chitosan oligosaccharide (QCOS) to react under alkaline conditions to obtain CR conjugates; couple the activated ES2 peptide with CR conjugates to obtain CRE conjugates; dissolve methotrexate in alkaline solution, Coupling with the CRE conjugate under catalysis to obtain the above quaternized chitosan modified ES2 peptide-methotrexate conjugate (CREM).

[0042] Preferably, in the above preparation method, the specific preparation method of the CR conjugate is as follows: add 1-ethyl-3(3-dimethylpropylamine) carbodiimide (EDCI) and N-hydroxysuccinimide to the cRGD aqueous solution Amine (NHS) was used as a catalyst to activate the carboxyl group in cRGD. After the activation, t...

Embodiment 1

[0077] 1. Preparation of cRGD-quaternized chitooligosaccharification modification (CRE) of ES2 peptide

[0078](1) Dissolve an appropriate amount of cRGD in double-distilled water to obtain a cRGD solution, then add EDCI and NHS catalyst in a ratio of 2:1, mix well, stir slowly at room temperature, and adjust the pH value to 7.40 after stirring. Dissolve QCOS in double-distilled water, add the QCOS solution dropwise to the cRGD solution, and slowly stir for 24 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a MWCO of 1000 Da and dialyzed with double distilled water for two days to remove impurities. After the dialysis is completed, the reaction solution is collected and placed in a freeze dryer to freeze-dry to obtain the CR conjugate.

[0079] (2) Dissolving ES2 short peptide (synthesized by solid-phase synthesis method) in double distilled water to obtain ES2 solution. Add EDCI and NHS to it respectively, stir slowly an...

Embodiment 2

[0083] Example 2 Inhibitory effect of ES2 peptide, CRE and CREM conjugates on endothelial cell proliferation

[0084] (1) Experimental drug: ES2 peptide, CRE conjugate and CREM conjugate prepared in Example 1.

[0085] (2) Experimental method: Collect EAhy926 cells grown in logarithmic phase, adjust the cell suspension to an appropriate concentration, and add 5×10 3 Cells were seeded into 96-well plates, placed in a carbon dioxide incubator, and cultured overnight at 37°C until the cells adhered to the wall. Then add ES2, CRE and CREM three kinds of drugs respectively, the drug concentration is respectively 5μg / mL, 25μg / mL, 50μg / mL, 75μg / mL, 100μg / mL, 200μg / mL, 500μg / mL (the concentration is based on ES2 concentration ), and 8 replicate wells were set up for each drug. The wells containing only DMEM medium were set as the blank control group, and the wells with cells and no drug-containing medium were set as the negative control group. Place the 96-well plate in a carbon di...

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Abstract

The invention relates to a cRGD-quaternized chitosan oligosaccharide modified ES2 peptide-methotrexate conjugate as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The CREM peptide conjugate can be prepared by controlling the conditions such as the supply quantity of the cRGD, the ES2 peptide and the MTX, the pH of a reaction system, the reaction time and the like. Compared with the ES2 peptide, the cRGD-quaternized chitosan oligosaccharide modified and methotrexate combined conjugate of the ES2 peptide keeps the anti-angiogenesis and anti-tumor activity of the ES2 peptide, integrates the tumor targeting property of cRGD and the anti-tumor activity of methotrexate and the like, has the characteristics of higher stability, hydrophilicity, targeting property and the like; therefore, the oligosaccharide has better use effect and application value.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a cRGD-quaternized ammonium chitosan modified ES2 peptide-methotrexate conjugate and a preparation method of the conjugate, and also provides a preparation method comprising the cRGD-quaternary ammonium Composition of chitooligosaccharide-modified ES2 peptide-methotrexate conjugate and application of the conjugate and composition. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Endostatin (Endostatin, ES) is an endogenous angiogenesis inhibitor isolated and purified from the supernatant of mouse endothelial cell tumors. Metabolic pathway, thereby playing an ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/64A61K47/61A61K47/55A61K38/39A61K31/519A61K31/722A61K45/06A61P35/00A61P9/10A61P27/02A61P37/04A61P39/06A61P29/00A61P31/04A61P31/10A61P9/00A61P25/00A61P1/00A61P1/16A61P3/10A61P3/06A61P3/04A61P11/06A61P19/02A61P17/06A23L33/10A23L33/18
CPCA61K47/64A61K47/61A61K47/55A61K38/39A61K31/519A61K31/722A61K45/06A61P35/00A61P9/10A61P27/02A61P37/04A61P39/06A61P29/00A61P31/04A61P31/10A61P9/00A61P25/00A61P1/00A61P1/16A61P3/10A61P3/06A61P3/04A61P11/06A61P19/02A61P17/06A23L33/10A23L33/18A23V2200/00A61K2300/00A23V2200/30A23V2200/308A23V2200/314A23V2200/32A23V2200/324A23V2200/326A23V2200/3262A23V2200/328A23V2200/332A23V2250/55Y02A50/30
Inventor 谭海宁孙凤唐雯李妍侯慧文王洁符家爱卢鲁
Owner SHANDONG UNIV
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