阅读说明：本技术 一种新型的高分子纳米输送体系及其治疗神经损伤的制备方法 (Novel polymer nano delivery system and preparation method thereof for treating nerve injury ) 是由 张鲁中 姚珂 杨宇民 李贵才 于 2019-09-26 设计创作，主要内容包括：本发明公开了一种新型的高分子纳米输送体系,所述高分子纳米输送体系是由具有树枝状结构的聚赖氨酸-聚乙二醇嵌段共聚物组成。其治疗神经损伤的制备方法如下：(1)细胞因子模拟肽的合成；(2)Lys-G4为核的树枝状炔基化聚β-氨基酯的合成；(3)细胞因子模拟肽偶联到树枝状炔基化聚β-氨基酯。本发明通过点击化学反应将细胞因子模拟肽与树枝状聚合物结合开发一种新型的高分子纳米输送体系来治疗神经损伤。可以实现较高的载药量,可控释放且具有靶向性,具有很高的临床应用价值。(The invention discloses a novel macromolecule nano conveying system which is composed of polylysine-polyethylene glycol block copolymer with a dendritic structure. The preparation method for treating nerve injury comprises the following steps: (1) synthesis of cytokine mimetic peptides; (2) synthesizing dendritic alkynyl poly-beta-amino ester taking Lys-G4 as core; (3) cytokine mimetic peptides were coupled to dendritically alkynylated poly- β -amino esters. The invention combines cytokine analogue peptide with dendritic polymer by click chemical reaction to develop a novel macromolecule nano-delivery system for treating nerve injury. Can realize higher drug loading, controllable release and targeting property, and has very high clinical application value.)

1. A novel polymer nanometer conveying system is characterized in that: the macromolecule nanometer conveying system is composed of polylysine-polyethylene glycol block copolymer with a dendritic structure.

2. A novel polymeric nanosupport system according to claim 1, wherein: the dendritic polymer carrying the cytokine mimic peptide is prepared by connecting the cytokine mimic peptide to azide, connecting polyethylene glycol to alkynyl, and connecting the cytokine mimic peptide to the azide through a click chemical reaction between the azide and the alkynyl under the catalysis of copper ions, wherein the ratio of the cytokine mimic peptide to the polyethylene glycol is 1: 4.

3. A method for preparing the novel macromolecule nano delivery system for treating nerve injury according to claim 1 or 2, which is characterized in that: the method comprises the following steps:

(1) synthesis of cytokine mimetic peptides

The cytokine mimic peptide is prepared by Fmoc chemical strategy through solid phase synthesis technology;

(2) synthesis of dendritically alkynylated Poly-beta-amino ester with Lys-G4 as core

The alkynyl poly-beta-amino ester containing polyethylene glycol is prepared by carrying out Michael addition reaction on polyethylene glycol acrylate and propargylamine; 1, 2-bis (2-aminoethoxy) ethane and Boc protected lysine are used as starting materials, and a dendritic molecule Lys-G4 of the lysine is constructed through amidation reaction of a divergent synthesis method; the amino at the end of Lys-G4 and the acrylate at the end group of poly-beta-amino ester are subjected to Michael addition reaction to obtain the dendritic alkynyl poly-beta-amino ester taking Lys-G4 as a core;

(3) coupling of cytokine mimetic peptides to dendritic alkynylated poly-beta-amino esters

Cytokine mimetic peptides were coupled to dendritically alkynylated poly-beta-amino esters by click chemistry.

4. The method for preparing the novel macromolecule nano delivery system for treating nerve injury according to claim 3, is characterized in that: the specific operation of the synthesis of the cytokine mimetic peptide is as follows: step 1: (1) swelling the 2-chloro-trityl chloride resin in a solid phase reaction tube by dichloromethane; (2) amino acid condensation, adding Fmoc-D-Pro-OH and a DMF solution of DIEA, and reacting for 2 hours at room temperature; (3) methanol blocking of unreacted resin; (4) removing Fmoc protection, and adding 20% DMF solution for reaction for 30 min; (5) amino acid condensation, adding DMF solution of Fmoc-Arg (Pbf) -OH, HBTU and DIEA, and reacting for 2h at room temperature; (6) repeating the step (4-5), and sequentially carrying out Fmoc-Lys (Boc) -OH and Fmoc-azidolysine amino acid condensation; (7) cutting off the amino acid sequence from the resin by using trifluoroethanol lysate; (8) cyclizing amino acid, adding DMF solution of HATU and DIEA, and reacting at room temperature for 2 h; (9) removing the protecting group with TFA/TIPS/H₂Removing the protecting group; (10) and separating and purifying the crude product by reversed-phase high performance liquid chromatography to obtain the azide-containing brain-derived neurotrophic factor (BDNF) mimic peptide.

5. The method for preparing the novel macromolecule nano delivery system for treating nerve injury according to claim 4, is characterized in that: the dosage of DIEA and HBTU is twice of the molar weight of amino acid, TFA/TIPS/H₂The ratio of O is 38:1:1, the reverse phase high performance liquid chromatography column is an 18C column, and the mobile phase is acetonitrile and water.

6. The method for preparing the novel macromolecule nano delivery system for treating nerve injury according to claim 3, is characterized in that: the specific operation of the synthesis of the dendriform alkynyl poly beta-amino ester taking Lys-G4 as a core is as follows:

a. polyethylene glycol acrylate (2.124g,3.54mmol) and propargylamine (0.165g,3.54mmol) are dissolved in 10mL of chloroform with stirring under the protection of nitrogen; after reacting for several days, precipitating by using anhydrous ether to obtain alkynyl poly beta-amino ester;

b. carrying out amidation reaction on 1, 2-bis (2-aminoethoxy) ethane and (S) -2, 6-di-tert-butoxycarbonylamino caproic acid under the condition that HBTU and HOBt are condensing agents to obtain Lys-G1Boc 4; removing the Boc protecting group under the condition of trifluoroacetic acid, carrying out amidation reaction step by step to obtain Lys-G4Boc32, and finally removing the protecting group by using trifluoroacetic acid to obtain Lys-G4;

dissolving Lys-G4 in dimethyl sulfoxide solution, and slowly adding into dimethyl sulfoxide solution of alkynyl poly beta-amino ester; stirring and reacting for 2 days at the temperature of 50 ℃ to obtain the dendritic alkynyl poly-beta-amino ester taking Lys-G4 as a core.

7. The method for preparing the novel macromolecule nano delivery system for treating nerve injury according to claim 6, is characterized in that: in the reaction for obtaining the alkynyl poly beta-amino ester, the reaction is carried out for 6 hours at 50 ℃, 12 hours at 80 ℃ and 24 hours at 90 ℃.

8. The method for preparing the novel macromolecule nano delivery system for treating nerve injury according to claim 6, is characterized in that: and b, in the amidation reaction, the reaction temperature is room temperature, and the reaction time is 2h each time.

9. The method for preparing the novel macromolecule nano delivery system for treating nerve injury according to claim 3, is characterized in that: the specific operation of coupling the cytokine mimic peptide to the dendritically alkynylated poly-beta-amino ester is as follows: performing Michaels addition reaction on the terminal group double bond of the dendritic alkynyl poly beta-amino ester and sulfhydrylated rhodamine (Rho-SH) to obtain dendritic alkynyl poly beta-amino ester coupled with Rho; under the catalysis of copper ions, the polypeptide-coupled poly beta-amino ester is obtained by a Click chemical reaction with BDNF mimic peptide cyclo (DPRKK-N3) with azide.

10. The method for preparing the novel macromolecule nano delivery system for treating nerve injury according to claim 9, is characterized in that: the concentration of the copper ions is 3.4mg/ml, the reaction time is 2 hours, and the collected product is dialyzed and freeze-dried.

Technical Field

The invention belongs to the field of polymer nano, and particularly relates to a novel polymer nano delivery system carrying cytokine mimic peptide for treating nerve injury through click chemistry reaction and a preparation method thereof.

Background

The nervous system is part of the most important system of the human body. It is the pathway of brain signal transmission and plays an important role. But the ability to regenerate and recover after nerve damage in higher mammals is very weak. How to effectively and accurately repair the damaged nerve becomes a hot point of research.

The nerve growth factor is a protein and polypeptide which is produced by tissues and glial cells innervated by nerves, is in addition to essential nutrients necessary for maintaining survival and has special nutritional effects on nerve cells, but the clinical application of the nerve growth factor is limited by the defects of instability in vivo, short half-life, easy hydrolysis by proteolytic enzyme, poor tissue infiltration capability, immunogenicity and the like, and the amino acid sequence of the cytokine mimic peptide is mainly derived from cytokines, has a clear structure, a clear action mechanism and close quality control to small molecular chemical drugs. They can combine with the target of receptor to have the biological activity of cell factor, the activity is high, compare with cell factor easier to synthesize and structure transformation, have no or low antigenicity, it is not easy to cause immune reaction, etc. the advantages are widely used in research, but the polypeptide drug also has the disadvantages of easy degradation, short half-life period, poor bioavailability, etc. Therefore, the polypeptide modified by the water-soluble polymer represented by polyethylene glycol can effectively prolong the biological half-life period, enhance the stability, improve the curative effect and has no immunogenicity.

In a high-molecular nano delivery system, the dendritic polymer can be widely applied to a nano drug delivery system because the molecular size of the dendritic polymer can be accurately controlled, the pharmacokinetic and pharmacological property reproducibility is good, and the high functional group density on the surface is favorable for functional modification such as polymerization, intelligence, targeting and the like. The click chemistry reaction aims to quickly and reliably complete the chemical composition of various color molecules through the splicing of small units, and molecular diversity can be simply and efficiently obtained.

The research of the macromolecule nano-drug delivery system provides a new idea for the prevention, diagnosis and treatment of various diseases. However, the existing polymer nano-carriers still have many problems to be solved, for example, the linear polymer assembly is difficult to realize effective control on the size, morphology, stability and surface properties, which causes great difficulty in vivo and in vitro biological behavior research of nano-drug delivery systems. The clinical application of the cell trophic factor is limited by the defects of instability in vivo, short half-life, easy hydrolysis by proteolytic enzyme, poor tissue infiltration capacity, immunogenicity and the like of the cell trophic factor serving as an important component part for disease recovery.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the defects of the prior art, the invention provides a novel macromolecule nano delivery system for treating nerve injury by connecting a cytokine mimic peptide with a dendrimer through a click chemistry reaction.

The technical scheme is as follows: the invention provides a novel macromolecule nano conveying system which is composed of polylysine-polyethylene glycol block copolymer with a dendritic structure.

Furthermore, the dendritic polymer carrying the cytokine mimic peptide is used for connecting the cytokine mimic peptide to azide, connecting polyethylene glycol to alkynyl, and connecting the cytokine mimic peptide to the alkyne through a click chemical reaction between the azide and the alkynyl under the catalysis of copper ions, wherein the ratio of the cytokine mimic peptide to the polyethylene glycol is 1: 4.

The preparation method of the novel macromolecule nano delivery system for treating nerve injury comprises the following steps:

(1) synthesis of cytokine mimetic peptides

The cytokine mimic peptide is prepared by Fmoc chemical strategy through solid phase synthesis technology;

(2) synthesis of dendritically alkynylated Poly-beta-amino ester with Lys-G4 as core

The alkynyl poly-beta-amino ester containing polyethylene glycol is prepared by carrying out Michael addition reaction on polyethylene glycol acrylate and propargylamine; 1, 2-bis (2-aminoethoxy) ethane and Boc protected lysine are used as starting materials, and a dendritic molecule Lys-G4 of the lysine is constructed through amidation reaction of a divergent synthesis method; the amino at the end of Lys-G4 and the acrylate at the end group of poly-beta-amino ester are subjected to Michael addition reaction to obtain the dendritic alkynyl poly-beta-amino ester taking Lys-G4 as a core;

(3) coupling of cytokine mimetic peptides to dendritic alkynylated poly-beta-amino esters

Cytokine mimetic peptides were coupled to dendritically alkynylated poly-beta-amino esters by click chemistry.

Further, the specific operation of the cytokine mimetic peptide synthesis is as follows: step 1: (1) swelling the 2-chloro-trityl chloride resin in a solid phase reaction tube by dichloromethane; (2) amino acid condensation, adding Fmoc-D-Pro-OH and a DMF solution of DIEA, and reacting for 2 hours at room temperature; (3) methanol blocking of unreacted resin; (4) removing Fmoc protection, and adding 20% DMF solution for reaction for 30 min; (5) amino acid condensation, adding DMF solution of Fmoc-Arg (Pbf) -OH, HBTU and DIEA, and reacting for 2h at room temperature; (6) repeating the step (4-5), and sequentially carrying out Fmoc-Lys (Boc) -OH and Fmoc-azidolysine amino acid condensation; (7) cutting off the amino acid sequence from the resin by using trifluoroethanol lysate; (8) cyclizing amino acid, adding DMF solution of HATU and DIEA, and reacting at room temperature for 2 h; (9) removing the protecting group with TFA/TIPS/H₂Removing the protecting group; (10) and separating and purifying the crude product by reversed-phase high performance liquid chromatography to obtain the azide-containing brain-derived neurotrophic factor (BDNF) mimic peptide.

Furthermore, the amount of DIEA and HBTU is twice of the molar amount of amino acids, TFA/TIPS/H₂The ratio of O is 38:1:1, the reverse phase high performance liquid chromatography column is an 18C column, and the mobile phase is acetonitrile and water.

Further, the specific operation of synthesizing the dendritically alkynylated poly-beta-amino ester with Lys-G4 as the core is as follows:

a. polyethylene glycol acrylate (2.124g,3.54mmol) and propargylamine (0.165g,3.54mmol) are dissolved in 10mL of chloroform with stirring under the protection of nitrogen; after reacting for several days, precipitating by using anhydrous ether to obtain alkynyl poly beta-amino ester;

b. carrying out amidation reaction on 1, 2-bis (2-aminoethoxy) ethane and (S) -2, 6-di-tert-butoxycarbonylamino caproic acid under the condition that HBTU and HOBt are condensing agents to obtain Lys-G1Boc 4; removing the Boc protecting group under the condition of trifluoroacetic acid, carrying out amidation reaction step by step to obtain Lys-G4Boc32, and finally removing the protecting group by using trifluoroacetic acid to obtain Lys-G4;

dissolving Lys-G4 in dimethyl sulfoxide solution, and slowly adding into dimethyl sulfoxide solution of alkynyl poly beta-amino ester; stirring and reacting for 2 days at the temperature of 50 ℃ to obtain the dendritic alkynyl poly-beta-amino ester taking Lys-G4 as a core.

Further, in the reaction for obtaining the alkynyl poly beta-amino ester, the reaction is carried out for 6 hours at 50 ℃, 12 hours at 80 ℃ and 24 hours at 90 ℃.

Further, in the step b, the reaction temperature in the amidation reaction is room temperature, and the reaction time is 2 hours each time.

Further, the specific operation of coupling the cytokine mimetic peptide to the dendritically alkynylated poly β -amino ester is as follows: performing Michaels addition reaction on the terminal group double bond of the dendritic alkynyl poly beta-amino ester and sulfhydrylated rhodamine (Rho-SH) to obtain dendritic alkynyl poly beta-amino ester coupled with Rho; under the catalysis of copper ions, the polypeptide-coupled poly beta-amino ester is obtained by a Click chemical reaction with BDNF mimic peptide cyclo (DPRKK-N3) with azide.

Further, the concentration of the copper ions is 3.4mg/ml, the reaction time is 2 hours, and the collected product is dialyzed and freeze-dried.

Has the advantages that: the invention provides a novel macromolecule nano delivery system for treating nerve injury by connecting polypeptide and polyethylene glycol through click chemical reaction, wherein the polyethylene glycol in poly beta-amino ester can prolong the half-life period of mimic peptide, and the dendritic poly beta-amino ester has good biocompatibility, good degradability, good structure controllability and good pharmacokinetics and pharmacology reproducibility.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below so that those skilled in the art can better understand the advantages and features of the present invention, and thus the scope of the present invention will be more clearly defined. The embodiments described herein are only a few embodiments of the present invention, rather than all embodiments, and all other embodiments that can be derived by one of ordinary skill in the art without inventive faculty based on the embodiments described herein are intended to fall within the scope of the present invention.