CN110639023A - Novel polymer nano delivery system and preparation method thereof for treating nerve injury - Google Patents
Novel polymer nano delivery system and preparation method thereof for treating nerve injury Download PDFInfo
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- CN110639023A CN110639023A CN201910914300.3A CN201910914300A CN110639023A CN 110639023 A CN110639023 A CN 110639023A CN 201910914300 A CN201910914300 A CN 201910914300A CN 110639023 A CN110639023 A CN 110639023A
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Classifications
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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- C—CHEMISTRY; METALLURGY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
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.
Description
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.
Examples
The invention relates to a preparation method for treating nerve injury by connecting polypeptide with azide group, connecting polyethylene glycol with propargylamine and connecting cytokine analogue peptide with polylysine-polyethylene glycol block copolymer with dendritic structure as a novel macromolecule nano-delivery system through click chemical reaction between azide and alkynyl, which comprises the following steps:
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, and removing the protecting group by TFA/TIPS/H2O; (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.
Wherein, the dosage of DIEA and HBTU is twice of the molar weight of amino acid, the ratio of TFA/TIPS/H2O is 38:1:1, the reverse-phase high performance liquid chromatography column is 18C column, and the mobile phase is acetonitrile and water.
Step 2: polyethylene glycol acrylate (2.124g,3.54mmol), propargylamine (0.165g,3.54mmol) were dissolved in 10mL of chloroform with stirring under nitrogen. After reacting for several days, precipitating by absolute ethyl ether to obtain the alkynylated poly-beta-amino ester. Wherein the reaction is carried out for 6h at 50 ℃, 12h at 80 ℃ and 24h at 90 ℃.
And step 3: and 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. And 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. Wherein the reaction temperature is room temperature, and the reaction time is 2h each time.
And 4, step 4: Lys-G4 was dissolved in dimethyl sulfoxide solution and slowly added to the solution of alkynylated poly β -amino ester in dimethyl sulfoxide. 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.
And 5: and (3) carrying out Michaels addition reaction on the terminal group double bond of the dendritic alkynyl poly beta-amino ester and sulfhydrylated rhodamine (Rho-SH) to obtain the 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. Wherein, the concentration of the added copper ions is 3.4mg/ml, the reaction time is 2h, and the collected product is dialyzed and lyophilized.
Specific example 1:
step 1: (1) taking 1.0g of 2-chloro-trityl chloride resin for swelling; (2) amino acid condensation, adding DMF solution of Fmoc-Arg-OH (2eq) and DIEA (4eq) and reacting for 2h at room temperature; (3) methanol blocking of unreacted resin; (4) removing Fmoc protection, and adding 20% piperidine DMF solution for reaction for 30 min; (5) amino acid condensation, adding Fmoc-Trp-OH, HBTU (4eq) and DIEA DMF solution, reacting for 2h at room temperature; (6) repeating the steps (4) to (5) and sequentially condensing Fmoc-Ala-OH, Fmoc-Gln-OH, Fmoc-Lys-OH, Fmoc-Gly-OH, Fmoc-Asp-OH, Fmoc-Met-OH, Fmoc-Thr-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Lys-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Thr-OH and Fmoc-azidolysine amino acids; (9) removing the protecting group, and removing the protecting group by TFA/TIPS/H2O; (10) and separating and purifying the crude product by using reverse phase high performance liquid chromatography to obtain the Nerve Growth Factor (NGF) mimic peptide with azide. The product was obtained in an amount of about 500 mg.
Step 2: polyethylene glycol acrylate (2.124g,3.54mmol), propargylamine (0.165g,3.54mmol) were dissolved in 10mL of water with stirring under nitrogen. Reacting at 50 ℃ for 6h, and reacting at 90 ℃ for 12h, and then precipitating by anhydrous ether to obtain the alkynyl poly beta-amino ester.
And step 3: the dendritic alkynyl poly beta-amino ester end group double bond (0.696mmol) and sulfhydrylated rhodamine (Rho-SH) are subjected to Michaels addition reaction to obtain the dendritic alkynyl poly beta-amino ester coupled with Rho. Under the catalysis of copper ions (0.0087mmol), the NGF mimic peptide (N3-TFVKALTMDGKQAAWR) with azide (0.174mmol) undergoes Click chemical reaction to obtain poly beta-amino ester coupled with polypeptide.
And 4, step 4: dissolving poly beta-amino ester linked with polypeptide in sterilized water, adding into dorsal root ganglion at different concentrations, and observing growth of neurofibrillary filaments by confocal observation.
Tests prove that the poly beta-amino ester of the coupled NGF mimetic peptide has obvious effect of promoting the growth of dorsal heel ganglion fibers.
Specific example 2:
step 1: (1) taking 1.0g of 2-chloro-trityl chloride resin for swelling; (2) amino acid condensation, adding DMF solution of Fmoc-Arg-OH (2eq) and DIEA (4eq) and reacting for 2h at room temperature; (3) methanol blocking of unreacted resin; (4) removing Fmoc protection, and adding 20% piperidine DMF solution for reaction for 30 min; (5) amino acid condensation, adding Fmoc-Ser-OH, HBTU (4eq) and DIEA DMF solution, reacting for 2h at room temperature; (6) repeating the steps (4) to (5), and sequentially carrying out Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Tyr-OH and Fmoc-azidolysine amino acid condensation; (9) removing the protecting group, and removing the protecting group by TFA/TIPS/H2O; (10) the crude product is separated and purified by reversed phase high performance liquid chromatography to obtain retinoic acid (YIGSR) mimic peptide with azide, and the obtained product is about 500 mg.
Step 2: polyethylene glycol acrylate (2.124g,3.54mmol), propargylamine (0.165g,3.54mmol) were dissolved in 10mL of water with stirring under nitrogen. Reacting at 50 ℃ for 6h, and reacting at 90 ℃ for 12h, and then precipitating by anhydrous ether to obtain the alkynyl poly beta-amino ester.
And step 3: the dendritic alkynyl poly beta-amino ester end group double bond (0.696mmol) and sulfhydrylated rhodamine (Rho-SH) are subjected to Michaels addition reaction to obtain the dendritic alkynyl poly beta-amino ester coupled with Rho. Under the catalysis of copper ions (0.0087mmol), the poly-beta-amino ester coupled with the polypeptide is obtained by the Click chemical reaction with retinoic acid mimic peptide (YIGSR) with azide (0.174 mmol).
And 4, step 4: dissolving poly beta-amino ester linked with polypeptide in sterilized water, adding into dorsal root ganglion at different concentrations, observing growth of nerve fiber silk by confocal method, and testing proliferation.
Tests prove that the poly beta-amino ester of the coupling retinoic acid mimic peptide has obvious effects of promoting the growth of dorsal root ganglion fibers and proliferating Schwann cells.
Specific example 3:
step 1: (1) taking 1.0g of 2-chloro-trityl chloride resin for swelling; (2) amino acid condensation, adding Fmoc-Phe-OH (2eq) and DIEA (4eq) in DMF, and reacting at room temperature for 2 h; (3) methanol blocking of unreacted resin; (4) removing Fmoc protection, and adding 20% piperidine DMF solution for reaction for 30 min; (5) amino acid condensation, adding Fmoc-Phe-OH, HBTU (4eq) and DIEA solution in DMF, and reacting at room temperature for 2 h; (6) repeating the steps (4) to (5), and sequentially condensing ibuprofen and Fmoc-azidolysine amino acid; (9) removing the protecting group, and removing the protecting group by TFA/TIPS/H2O; (10) the crude product was purified by reverse phase high performance liquid chromatography to obtain ibuprofen with azide, yielding about 500mg of product.
Step 2: polyethylene glycol acrylate (2.124g,3.54mmol), propargylamine (0.165g,3.54mmol) were dissolved in 10mL of water with stirring under nitrogen. Reacting at 50 ℃ for 6h, and reacting at 90 ℃ for 12h, and then precipitating by anhydrous ether to obtain the alkynyl poly beta-amino ester.
And step 3: the dendritic alkynyl poly beta-amino ester end group double bond (0.696mmol) and sulfhydrylated rhodamine (Rho-SH) are subjected to Michaels addition reaction to obtain the dendritic alkynyl poly beta-amino ester coupled with Rho. Under the catalysis of copper ions (0.0087mmol), the poly-beta-amino ester coupled with ibuprofen is obtained by a Click chemical reaction with ibuprofen (0.174mmol) with azide.
And 4, step 4: dissolving poly beta-amino ester linked with ibuprofen in sterilized water, adding into fibroblasts at different concentrations, and detecting the biological toxicity.
Tests prove that the toxic effect of the poly beta-amino ester coupled with the ibuprofen on fibroblasts is obviously reduced.
The invention uses the polypeptide connected with azide group, the polyethylene glycol connected with propargylamine, and uses the cell factor analog peptide connected with the polylysine-polyethylene glycol block copolymer with dendritic structure as a novel macromolecule nano-conveying system through the click chemical reaction between azide and alkynyl, thus having higher application value clinically.
The above-described embodiments are intended only to illustrate the present invention further, and it should be noted that modifications and improvements made on the concept of the present invention by those skilled in the art are also considered to be within the scope of the present invention.
Claims (10)
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.
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