CN116036370B

CN116036370B - Preparation method of pH fluorescent response polypeptide self-assembled 3D printing ink

Info

Publication number: CN116036370B
Application number: CN202310294163.4A
Authority: CN
Inventors: 张磊; 李勇
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2023-03-24
Filing date: 2023-03-24
Publication date: 2023-06-13
Anticipated expiration: 2043-03-24
Also published as: CN116036370A

Abstract

The invention discloses a preparation method of pH fluorescent response polypeptide self-assembled 3D printing ink, and belongs to the field of biological material preparation. The composition comprises water-soluble synthetic polymer with crosslinking function, water-soluble natural polymer with crosslinking function, bioactive component capable of spontaneously forming special ultrastructure, crosslinking initiator and solvent, and further comprises bioactive component; the invention overcomes the defects that the traditional 3D printing ink solution has single component structure, does not have good biological activity, needs to use an organic solvent, is difficult to consider cell compatibility, biological activity, mechanical property and the like, and has physical crosslinking before printing for maintaining a certain shape, biological activity and rapid curing function after printing, and can form biological ink with a certain microstructure, so that hydrogel obtained by curing and molding the ink has controllable mechanical property, good structural stability, fidelity effect, good biological compatibility and biological activity.

Description

Preparation method of pH fluorescent response polypeptide self-assembled 3D printing ink

Technical Field

The invention relates to a preparation method of pH fluorescent response polypeptide self-assembled 3D printing ink, and belongs to the technical field of biological materials.

Technical Field

The 3D biological printing is a tissue engineering technology combining the development of multiple fields such as cell biology, computer aided design, biological material science and the like on the basis of the development of a rapid prototyping technology, and the final aim is to realize organ printing. The 3D biological printing technology can overcome the limitation of the traditional tissue engineering technology, and can realize the three-dimensional accurate positioning of seed cells with different densities in different stent materials by accurately positioning biological materials, biochemical molecules and living cell interlayer positions, and the three-dimensional accurate positioning is used for manufacturing a three-dimensional structure by controlling the spatial positions of functional components. Although 3D printing technology attracts enough attention in biomedical fields such as disease models, drug sustained release, tissue engineering and regenerative medicine, so far, the manufacture of organs or tissues truly suitable for in vivo implantation using 3D printing technology faces a great challenge, wherein the development of biomaterials suitable for 3D printing is critical. The current biological materials suitable for 3D printing mainly stay in the scientific research stage, the 3D printing technology applied to biological medical treatment widely adopts synthetic materials including thermoplastic degradable absorbing polyester plastics (PLA, PLGA, PCL, PEG and copolymers thereof) and some natural high polymer materials (alginate, fibrin, collagen, gelatin, chitosan, hyaluronic acid and other materials) which are mostly subjected to 3D printing by adopting organic solvents or high temperature, and the printed bracket has stable structure but limited precision and can not be mixed with cells for printing.

In the 3D bio-printing process, the ideal bio-ink needs to meet the requirements of highly controlling the printing speed, resolution, cell concentration and droplet volume in the printing process, and in addition, has good biocompatibility and degradability, and also needs to maintain proper mechanical strength in the printing process and after molding. Therefore, few materials are currently available for 3D bioprinting, with hydrogel-like materials being considered the most potential soft tissue-making materials because of their better biomaterial properties and similarity to natural tissue. The hydrogel mainly comprises two major types of natural materials and synthetic materials, wherein the natural materials mainly comprise: gelatin, cellulose, alginic acid, hyaluronic acid, chitosan and the like; the synthetic material mainly comprises: alcohols, acrylic acid and, polyacrylic acid, polymethacrylic acid, polyacrylamide, and the like. The main guest chemistry is a simple and effective strategy for constructing a 'plug and play' multiple nano-delivery system by utilizing intermolecular non-covalent acting force. The cyclodextrin compound has good biocompatibility and is easy to modify, and the cyclodextrin compound is very beneficial to introducing a functional module by combining a guest molecule, so that complex synthesis steps are avoided.

The gel is based on the design concept from bottom to top, and the micro-nano colloid particles are taken as basic units to form a novel hydrogel material with a fine microstructure and a stable macroscopic structure through physical or chemical cross-linking. The micro-nano colloid particles are assembled through physical interactions such as hydrogen bonds, static electricity, hydrophile and hydrophobic property to form a colloid network. The physical interaction between the particles enables the colloidal gel to have excellent mechanical properties of injectability and self-repairing, and can be used as an injectable filling material for minimally invasive surgery and 3D biological printing ink for biological manufacture. Meanwhile, the colloid gel is based on a micro-nano size structure and can be used as a carrier for loading and releasing medicines/proteins, so that the colloid gel has potential application in the medicine slow release field. However, the traditional gel is assembled only through physical interaction, so that macroscopic mechanical strength is not high, the repair of bearing tissues or organs cannot be satisfied in tissue repair, and meanwhile, the mechanical property of a bracket obtained as the biological printing ink is weak, so that further application of the gel material in the biomedical field is limited. The design strategy of the double-network hydrogel provides a new means for solving the problem of poor mechanical strength of the traditional gel. Double-network hydrogels are a class of hydrogel materials with high mechanical strength and toughness. It achieves excellent mechanical properties by compounding two polymers that can independently form a hydrogel network in the same hydrogel. The double-network hydrogels reported at present are often based on high molecular composition and do not have injectability, which does not meet the requirements of tissue filling materials and bio-printing inks on materials. Therefore, designing a colloid gel material with high mechanical strength and excellent biological performance, which can be printed and injected, is still a technical blank today.

Disclosure of Invention

In order to solve the problem of lack of the biological ink for 3D printing at present, the invention provides the 3D printing ink which has the functions of biological activity, fluorescence intensity pH response, physical pre-crosslinking forming and rapid photo-curing, can form a certain microstructure and can be degraded and absorbed, and has good biocompatibility.

A preparation method of pH fluorescence response polypeptide self-assembled 3D printing ink specifically comprises the following steps:

(1) A solution of N-t-butoxycarbonyl-L-tyrosine was prepared at a concentration of 4.0-8.0g/L using a boric acid buffer solution with a solvent of 0.1-0.25M, pH =8.0-10.0.

(2) Adding 3% hydrogen peroxide solution according to the volume ratio of the hydrogen peroxide solution to the boric acid buffer solution of 0.02-0.05:1, adding horseradish peroxidase with the mass of 0.01-0.02 times of the mass of the N-t-butoxycarbonyl-L-tyrosine until the N-t-butoxycarbonyl-L-tyrosine and the horseradish peroxidase are completely dissolved, and stirring and reacting for 6-24h at the temperature of 32-37 ℃.

(3) Beta-mercaptoethanol is added according to the volume ratio of the beta-mercaptoethanol to the boric acid buffer solution of 0.005-0.01:1 to stop the reaction, and the volume of the solvent is concentrated to 2.0-6.0% of the reaction system.

(4) Taking the concentrated solution obtained in the step (3) for column chromatography, and n-propanol: the volume ratio of ammonia water is 8:1-9:1, and the mixture is dried after elution.

(5) Dissolving the object dried in the step (4) in ethanol, precipitating with acetone to obtain N, N-diboron dityrosine (DBDY), and storing for later use, wherein the synthesis route of the DBDY is as follows:

(6) And (3) weighing dihydroxyl PEG, dissolving in dichloromethane to prepare PEG solution with the concentration of 60-70g/L, sequentially adding silver iodide and p-toluenesulfonyl chloride, wherein the mass ratio of the silver iodide to the dihydroxyl PEG is 0.1-0.3:1, the mass ratio of the p-toluenesulfonyl chloride to the dihydroxyl PEG is 0.1-0.2, reacting at room temperature for 4-5h, filtering the reaction solution by using diatomite, washing three times by using Dichloromethane (DCM), and drying to obtain a TsO-PEG-OH crude product, wherein the volume of the reaction solution is preferably 10-25% in order to save the consumption of raw material dichloromethane.

(7) Dissolving the obtained crude TsO-PEG-OH product in ammonia water with the mass percentage concentration of 25% (the addition of the ammonia water can lead the crude TsO-PEG-OH product to be completely dissolved), adding ammonium chloride, stirring and reacting for 8-24h, wherein the mass of the ammonium chloride is 1.0-1.5 times that of dihydroxyPEG; then extracted with Dichloromethane (DCM), the organic layer was collected and dried to give NH ₂ -PEG-OH product retentionUsing; NH (NH) ₂ The synthetic route for PEG-OH is as follows:

(8) Polyethylene glycol (PEG) is weighed and dissolved in methylene dichloride to prepare PEG solution with the concentration of 80-100g/L, maleic anhydride is added according to the proportion of 10-11 g/L, and nitrogen is introduced and stirred to dissolve the solid.

(9) Pyridine is added for reaction for 1.0-6.0h at 40-100 ℃, wherein the mass volume ratio of polyethylene glycol to pyridine is 1 (0.5-0.8), g is mL.

(10) After the reaction is finished, concentrating the organic solvent under reduced pressure, dripping ice petroleum ether into the concentrated solution to precipitate reactants, filtering, washing the solid by petroleum ether to obtain white solid powder HO-PEG-COOH, wherein the synthetic route of HO-PEG-COOH is as follows:

(11) Dissolving white solid powder HO-PEG-COOH obtained in the step (10) in dichloromethane to prepare HO-PEG-COOH solution with the concentration of 60-100g/L, introducing nitrogen and stirring to dissolve the solid, and adding triethylamine according to the volume ratio of dichloromethane to triethylamine of 1:0.25-0.5; according to the mass ratio of HO-PEG-COOH to the acryloyl chloride of 1:0.15-0.25, dissolving the acryloyl chloride in Dichloromethane (DCM) to prepare an acryloyl chloride solution with the concentration of 5-10%, and then dropwise adding the acryloyl chloride solution into the HO-PEG-COOH solution.

(12) After the reaction is finished, diatomite is adsorbed and filtered, the organic solvent is concentrated under reduced pressure at 30-45 ℃, ice petroleum ether is dripped into the concentrated solution, the mixture is kept stand for 2-6 hours at the temperature of 20 ℃ below zero, the solid is washed by petroleum ether after the filtration, and MA-PEG-COOH is obtained for standby, and the synthetic route of MA-PEG-COOH is as follows:

(13) Dissolving the N, N-diboron dityrosine obtained in the step (5) into N, N-diboron dityrosine solution prepared in the 1, 4-dioxane according to the proportion of 2.0-3.0g/L, and adding N-hydroxysuccinimide (NHS) with the mass of 0.3-0.6 times of that of the N, N-diboron dityrosine; n, N-dicyclohexyl carbodiimide (DCC) with the mass of 0.6-0.8 times of that of N, N-diboron dityrosine is weighed and dissolved in 1, 4-dioxane to prepare 18.0-20.0g/LN, N-dicyclohexyl carbodiimide (DCC) solution, the N, N-dicyclohexyl carbodiimide (DCC) solution is dripped into the reaction solution, and the organic solvent is concentrated under reduced pressure at 35-60 ℃ after the reaction is finished.

(14) The concentrate was washed with acetonitrile and deionized water in sequence and the resulting DBDY-SCM was stored at-20 ℃.

(15) Weighing the NH obtained in the step (7) ₂ PEG-OH, weighing DBDY-SCM obtained in the step (14), dissolving in dichloromethane, adding N, N-Dimethylformamide (DMF) with the volume of 4.0-5.0 times of that of the dichloromethane, and Triethylamine (TEA) with the volume of 50.0-60.0 times of that of the dichloromethane, and reacting for 18-36h at the temperature of 32.0-40.0 ℃.

(16) Adding excessive petroleum ether to precipitate the reactant to obtain 2-ARM-PEG-DBDY crude product, wherein the synthetic route of the 2-ARM-PEG-DBDY is as follows:

(17) Weighing the white solid powder MA-PEG-COOH obtained in the step (12), dissolving the white solid powder MA-PEG-COOH in 1, 4-dioxane to prepare a solution with the concentration of 8.0-10.0%, and adding N-hydroxysuccinimide (NHS) with the mass of 0.2-0.5 times that of MA-PEG-COOH; weighing N, N-Dicyclohexylcarbodiimide (DCC) with the mass of 0.5-0.6 times of that of MA-PEG-COOH, dissolving in 1, 4-dioxane to prepare a solution with the concentration of 25-30g/L, dripping the N, N-Dicyclohexylcarbodiimide (DCC) solution into the reaction solution, and concentrating the organic solvent under reduced pressure after the reaction is finished.

(18) The concentrate of step (17) was dissolved in Dichloromethane (DCM), filtered to remove N, N-dicyclohexylcarbodiimide, and the filtrate was concentrated in vacuo to give MA-PEG-SCM.

(19) Dissolving the 2-arm-PEG-DBDY crude product obtained in the step (16) in Dichloromethane (DCM), adding EDT, and reacting for 15-30min; the reaction was precipitated by adding excess petroleum ether to give 2-arm-PEG-DBDY.

(20) Weighing 2-arm-PEG-DBDY in the step (19) according to the proportion of 10g/L, weighing MA-PEG-SCM obtained in the step (18) according to the proportion of 50-80 g/L, dissolving in Dichloromethane (DCM), adding N, N-Dimethylformamide (DMF) (the volume ratio of N, N-Dimethylformamide (DMF) to Dichloromethane (DCM) is 1:2-5), triethylamine (TEA) (the volume ratio of Triethylamine (TEA) to Dichloromethane (DCM) is 1:60), and reacting for 15-36h at 35-45 ℃; the reaction was precipitated by adding excess petroleum ether to give crude 4-arm-PEG-DBDY.

(21) Dissolving the light yellow powder 4-ARM-PEG-DBDY crude product obtained in the step (20) in deionized water, dialyzing with 2500Da dialysis bag, and freeze-drying, wherein the synthetic route of the 4-ARM-PEG-DBDY is as follows:

(22) Under the protection of inert gas, the beta-cyclodextrin and 18-crown ether-6 are weighed and dissolved in anhydrous DMF, the concentration of the beta-cyclodextrin is 20-25g/L, the mass ratio of the 18-crown ether-6 to the beta-cyclodextrin is 1.0:1.5-2.0, and then potassium hydride (KH) which is 0.2-0.5 times of the mass of the beta-cyclodextrin is added and continuously stirred to fully react with the hydroxyl of the cyclodextrin.

(23) Dissolving glycidol in anhydrous DMF, slowly dropwise adding the mixture into the reaction solution in the step (22), wherein the volume ratio of the glycidol to the anhydrous DMF is 1.0:1.3-1.5, the volume mass ratio of glycidol to beta-cyclodextrin is 16.0-18.0; after the reaction is finished, a small amount of water is added to terminate the reaction, the reaction is dialyzed and freeze-dried, and light yellow beta-CD-HPG solid is obtained, and the synthetic route of the beta-CD-HPG is as follows:

(24) Weighing the beta-CD-HPG obtained in the step (23) according to the proportion of 180-200g/L, weighing succinic anhydride with the mass of 0.5-0.6 times of that of the beta-CD-HPG, and dissolving the beta-CD-HPG and the succinic anhydride in DMF simultaneously.

(25) The beta-CD-HPG mass is weighed to be 0.6-0.75 times of 4-Dimethylaminopyridine (DMAP), and Triethylamine (TEA) is weighed according to the proportion of 10 times of the mass volume of the 4-Dimethylaminopyridine (DMAP), and both are dissolved in DMF.

(26) The mixture was added to the reaction solution of step (25) (the volume ratio of the reaction was 1:2.5), and stirred at room temperature under nitrogen for 3-5d to give β -CD-HPG-COOH.

(27) The beta-CD-HPG-COOH (mwco=1000) obtained after the reaction of step (26) was dialyzed against distilled water for 3-7 days, and then the solution was freeze-dried.

(28) beta-CD-HPG-COOH was weighed and dissolved in deionized water to prepare a beta-CD-HPG-COOH solution at a concentration of 50-100g/L, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and an aqueous N-hydroxysuccinimide solution (NHS) (the mass ratio of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) to aqueous N-hydroxysuccinimide solution (NHS) was 1:1, wherein the mass ratio of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) to beta-CD-HPG-COOH was 1:18-20), and the pH of the solution was adjusted to 4.0-5.0 by adding PBS buffer and stirring.

(29) Measuring a certain volume of RGD peptide aqueous solution, adding and stirring to react for 18-36h, and obtaining beta-CD-

HPG-RGD was dialyzed against distilled water for 3-5 days and then lyophilized, and the synthesis route of beta-CD-HPG-RGD was as follows:

(30) Under the condition of avoiding light, a certain amount of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) is weighed and dissolved in deionized water to prepare 5-10g/L LAP solution;

(31) And (3) weighing the beta-CD-HPG-RGD prepared in the step (29), dissolving the beta-CD-HPG-RGD in LAP solution at 35-80 ℃, adding a certain mass of 4-arm-PEG-DBDY after the beta-CD-HPG-RGD is completely dissolved, and stirring and reacting to obtain the 3D printing ink.

All concentrations in the present invention are mass percent concentrations unless specifically stated otherwise.

The invention has the beneficial effects that:

(1) Based on the limitations of the 3D biological printing technology, most of materials need to be subjected to 3D printing by adopting an organic solvent or high temperature, and the printed bracket structure is unstable, has limited precision, can not be mixed with cells for printing, and has poor mechanical property, simulation and stability. According to the invention, the physical pre-crosslinking is carried out by combining the main body with the ligand, the hydrogel is endowed with a certain structure maintaining function before 3D printing, the stability of a printing structure can be maintained during printing, and the printed bracket structure is stable, high in precision, good in mechanical property, good in simulation and stability through photo-crosslinking after printing; meanwhile, polypeptide is utilized to be covalently combined with the material, so that the hydrogel material has good biological compatibility and biological activity.

(2) The 4-arm-PEG-DBDY provided by the invention has the pH fluorescence response function, when the pH of the environment is different and the fluorescence intensity is different, the larger the pH is, the larger the fluorescence intensity is, and the hydrogel material has the fluorescence indication function; the main body beta-CD-HPG-RGD has good drug loading capacity after glycidol modification, and the loaded micromolecular drugs can be flexibly adjusted according to the use requirements.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of a compound BY-DBDY;

FIG. 2 Compound NH ₂ -PEG-OH nuclear magnetic hydrogen spectrometry;

FIG. 3D print ink compression experiment;

FIG. 4 3D print ink rheological behavior experiments;

FIG. 5 3D print ink collapse experiments;

FIG. 6 fluorescence intensity detection;

FIG. 7 3D print ink compression cycle experiment;

FIG. 8 is a schematic diagram showing a 3D printing process using the 3D printing ink obtained in example 3;

FIG. 9 cell adhesion assay.

Detailed Description

The invention will be described in further detail with reference to specific embodiments, but the scope of the invention is not limited to the description.

Example 1

(1) 4.0g of N-t-butoxycarbonyl-L-tyrosine was weighed out and dissolved in 1000mL of boric acid buffer, 0.25M, pH =10.0.

(2) Then 20mL hydrogen peroxide solution with the concentration of 3% and 40mg horseradish peroxidase are added, and the mixture is stirred and reacted for 15 hours at the temperature of 35 ℃ to completely dissolve the N-tert-butoxycarbonyl-L-tyrosine and the horseradish peroxidase.

(3) After the reaction, 5mL of beta-mercaptoethanol was added to terminate the reaction, and the solvent was concentrated to 20mL.

(4) Taking the concentrated solution obtained in the step (3) for column chromatography, and n-propanol: the volume ratio of ammonia water is 8:1, and the solution is dried after elution.

(5) Dissolving the dried object obtained in the step (4) in 200mL of ethanol, precipitating with acetone to obtain N, N-diboron dityrosine (DBDY), and storing for later use, wherein the nuclear magnetic hydrogen spectrum is shown in figure 1; as can be seen from FIG. 1, the nuclear magnetic hydrogen spectrum of the compound DBDY shows BY-DBDY ¹ H-NMR mass spectrometry; by means of DBDY ¹ Comparison of the H-NMR spectrum with the peak positions of 18 methyl protons (indicated by chemical shift of about 1.6 in the nuclear magnetic hydrogen spectrum of the compound DBDY of FIG. 1) confirmed that pure DBDY was obtained.

(6) 60.0g of dihydroxyl PEG is weighed and dissolved in 1000mL of dichloromethane, then 6.0g of silver iodide and 6.0g of p-toluenesulfonyl chloride are sequentially added for reaction at room temperature for 4 hours, the reaction solution is filtered by diatomite, and the Dichloromethane (DCM) is washed for three times, and the crude TsO-PEG-OH product is obtained after drying.

(7) The obtained crude TsO-PEG-OH product is dissolved in 600mL of concentration: 60.0g of ammonium chloride was added to 25% aqueous ammonia, the mixture was stirred and reacted for 8 hours, then extracted with Dichloromethane (DCM), and the organic layer was collected and dried to give NH ₂ -the PEG-OH product is left for later use; detecting the obtained compound NH ₂ The nuclear magnetic hydrogen spectrum of PEG-OH is shown in FIG. 2; from FIG. 2, it can be seen that the nuclear magnetic hydrogen spectrum of the compound NH2-PEG-OH shows the 1H-NMR spectrum of PEG and PEG-NH2, and the chemical shift of the figure is about 7.8, and the modified amino functional group is shown; comparison of proton peaks in the figures confirms that PEG-NH2 has been successfully prepared.

(8) 80.0g polyethylene glycol (PEG) and 10.3g maleic anhydride were weighed out and dissolved in 1000mL Dichloromethane (DCM), and the solid was dissolved by stirring with nitrogen.

(9) 40mL of pyridine was added and the reaction was carried out at 40℃for 1.0h.

(10) After the reaction, the organic solvent was concentrated under reduced pressure, and 500mL of ice petroleum ether was added dropwise to the concentrate to precipitate the reaction product, and after filtration, the solid was washed with petroleum ether to obtain white solid powder HO-PEG-COOH.

(11) Taking 60.0g of white solid powder HO-PEG-COOH obtained in the step (10), dissolving in 1000mL of Dichloromethane (DCM), introducing nitrogen, stirring to dissolve the solid, and adding 400mL of triethylamine; 9.0g of acryloyl chloride was dissolved in 90.0mL of Dichloromethane (DCM) and then added dropwise to the HO-PEG-COOH solution.

(12) After the reaction is finished, diatomite is adsorbed and filtered, the organic solvent is concentrated under reduced pressure at 30 ℃, ice petroleum ether is dripped into the concentrated solution, the mixture is stood for 2 hours at the temperature of 20 ℃ below zero, and the solid is washed by petroleum ether after the filtration, so that MA-PEG-COOH is obtained for standby.

(13) Weighing 2.0g of the N, N-diboron dityrosine obtained in the step (5), dissolving in 1000mL of 1, 4-dioxane, and adding 0.6g of N-hydroxysuccinimide (NHS); 1.2g of N, N-Dicyclohexylcarbodiimide (DCC) was weighed and dissolved in 66.7mL of 1, 4-dioxane, the N, N-Dicyclohexylcarbodiimide (DCC) solution was added dropwise to the reaction solution, and after the completion of the reaction, the organic solvent was concentrated under reduced pressure at 35 ℃.

(14) The concentrate was washed with 200mL acetonitrile and 200mL deionized water, and the resulting DBDY-SCM was stored at-20 ℃.

(15) Weighing 0.50g of NH obtained in step (7) ₂ PEG-OH, 0.18g of DBDY-SCM obtained in step (14) was dissolved in 10.0mL, 40.0mL of LN, N-Dimethylformamide (DMF), 500.0mL of Triethylamine (TEA) was added, and the mixture was reacted at 32℃for 36 hours.

(16) Adding excessive petroleum ether to precipitate the reactant to obtain 2-arm-PEG-DBDY crude product.

(17) Weighing 8.0g of the white solid powder MA-PEG-COOH obtained in the step (12), dissolving in 100mL of 1, 4-dioxane, and adding 1.6g of N-hydroxysuccinimide (NHS); 4.0g of N, N-Dicyclohexylcarbodiimide (DCC) was weighed and dissolved in 160mL of 1, 4-dioxane, and the N, N-Dicyclohexylcarbodiimide (DCC) solution was added dropwise to the reaction solution, followed by concentrating the organic solvent under reduced pressure after the completion of the reaction.

(18) The concentrate of step (17) was dissolved in 200mL of Dichloromethane (DCM), filtered to remove N, N-dicyclohexylcarbodiimide, and the filtrate was concentrated in vacuo to give MA-PEG-SCM.

(19) The crude 2-arm-PEG-DBDY obtained in step (16) was dissolved in 400mL of Dichloromethane (DCM) and 3mLEDT was added for 30min.

(20) The reaction was precipitated by adding excess petroleum ether to give 2-arm-PEG-DBDY.

(21) 0.30g of 2-arm-PEG-DBDY and 1.1g of MA-PEG-SCM obtained in the step (18) were weighed out, dissolved in 20mL of Dichloromethane (DCM), 60mLN, N-Dimethylformamide (DMF) and 1000mL of Triethylamine (TEA) were added and reacted at 35℃for 15 hours.

(22) The reaction was precipitated by adding excess petroleum ether to give crude 4-arm-PEG-DBDY.

(23) Dissolving the light yellow powder 4-arm-PEG-DBDY crude product obtained in the step (22) in deionized water, dialyzing with 2500Da dialysis bag, and freeze-drying.

(24) 2.0g of beta-cyclodextrin and 3.2g of 18-crown-6 are dissolved in 100.0mL of anhydrous DMF under inert gas protection, and then 0.4g of KH is added and stirring is continued to allow them to react fully with the hydroxyl groups of the cyclodextrin.

(25) 35.2mL of glycidol is dissolved in 50mL of anhydrous DMF and slowly added dropwise to the reaction solution in step (24); after the reaction is finished, a small amount of water is added to terminate the reaction, and the mixture is dialyzed and freeze-dried to obtain a pale yellow beta-CD-HPG solid.

(26) 1.5g of the beta-CD-HPG obtained in step (25) and 0.8g of succinic anhydride were weighed out and dissolved in 8 mM LDMF.

(27) 1.0g of 4-Dimethylaminopyridine (DMAP) and 10.0mL of Triethylamine (TEA) were weighed into 20mL of LDMF, and the mixture was added to the reaction system of step (26) and stirred at room temperature under nitrogen for 3d to give β

-CD-HPG-COOH。

(28) The beta-CD-HPG-COOH (mwco=1000) obtained after the reaction was dialyzed against distilled water for 3 days, and then the solution was freeze-dried.

(29) 3.0g of beta-CD-HPG-COOH obtained in the step (28) was weighed and dissolved in 60mL of deionized water, 0.15g of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and 0.15g of an aqueous N-hydroxysuccinimide solution (NHS) were added, and the pH of the solution was adjusted to 4.0 by adding PBS buffer and stirring.

(30) 15mLRGD peptide aqueous solution was weighed and added and stirred for reaction for 18h, and the obtained beta-CD-HPG-RGD was dialyzed against distilled water for 3 days and then freeze-dried.

Example 2

(1) 8.0g of N-t-butoxycarbonyl-L-tyrosine was weighed out and dissolved in 1000mL of boric acid buffer, 0. M, pH =8.0.

(2) 50mL of 3% hydrogen peroxide solution, 40mg of horseradish peroxidase, and horseradish peroxidase were added until N-t-butoxycarbonyl-L-tyrosine and horseradish peroxidase were completely dissolved, and the reaction was stirred at 32℃for 6 hours.

(3) After the reaction, 10mL of beta-mercaptoethanol was added to terminate the reaction, and the solvent was concentrated to 60mL.

(5) And (3) dissolving the dried object obtained in the step (4) in 200mL of ethanol, precipitating with acetone to obtain N, N-diboron dityrosine (DBDY), and storing for later use.

(6) 65.0g of dihydroxyPEG is weighed and dissolved in 1000mL of dichloromethane, 13g of silver iodide and 9.7g of p-toluenesulfonyl chloride are sequentially added for reaction at room temperature for 5h, the reaction solution is filtered by diatomite, washed three times with Dichloromethane (DCM), and the crude product of TsO-PEG-OH is obtained after drying.

(7) The crude TsO-PEG-OH product was dissolved in 600.0mL of 25% ammonia, 78.0g of ammonium chloride was added, and after stirring for 15h, the reaction was extracted with Dichloromethane (DCM), the organic layer was collected and dried to give the NH2-PEG-OH product, which was retained for use.

(8) 90.0g polyethylene glycol (PEG) and 10g maleic anhydride were weighed out and dissolved in 1000mL Dichloromethane (DCM), and the solid was dissolved by stirring with nitrogen.

(9) 56mL of pyridine was added and the reaction was carried out at 100℃for 3.0h.

(11) Taking 100.0g of white solid powder HO-PEG-COOH obtained in the step (10), dissolving in 1000mL of Dichloromethane (DCM), introducing nitrogen, stirring to dissolve the solid, and adding 500.0mL of triethylamine; 25.0g of acryloyl chloride was dissolved in 500.0mL of Dichloromethane (DCM) and then added dropwise to the HO-PEG-COOH solution;

(12) After the reaction is finished, diatomite is adsorbed and filtered, the organic solvent is concentrated under reduced pressure at 45 ℃, ice petroleum ether is dripped into the concentrated solution, the mixture is stood for 6 hours at the temperature of-20 ℃, and the solid is washed by petroleum ether after the filtration, so that MA-PEG-COOH is obtained for standby.

(13) Weighing 3.0g of the N, N-diboron dityrosine obtained in the step (5), dissolving in 1000mL of 1, 4-dioxane, and adding 1.8g of N-hydroxysuccinimide (NHS); 2.4g of N, N-Dicyclohexylcarbodiimide (DCC) was weighed and dissolved in 120.5mL of 1, 4-dioxane, the N, N-Dicyclohexylcarbodiimide (DCC) solution was added dropwise to the reaction solution, and the organic solvent was concentrated under reduced pressure at 60℃after the completion of the reaction.

(14) The concentrate was washed with 300mL of acetonitrile and 300mL of deionized water, and the resulting DBDY-SCM was stored at-20 ℃.

(15) Weighing 0.50g of NH obtained in step (8) ₂ PEG-OH, 0.18g of DBDY-SCM obtained in step (14) was dissolved in 10.0mL, 50.0mL of LN, N-Dimethylformamide (DMF), 600.0mL of Triethylamine (TEA) was added, and reacted at 40℃for 18 hours.

(17) 9.0g of the white solid powder MA-PEG-COOH obtained in the step (12) is weighed and dissolved in 100mL of 1, 4-dioxane, and 4.0g of N-hydroxysuccinimide (NHS) is added; 4.5g of N, N-Dicyclohexylcarbodiimide (DCC) was weighed and dissolved in 160mL of 1, 4-dioxane, the N, N-Dicyclohexylcarbodiimide (DCC) solution was added dropwise to the reaction solution, and after the completion of the reaction, the organic solvent was concentrated under reduced pressure.

(18) The concentrate of step (17) was dissolved in 300mL of Dichloromethane (DCM), filtered to remove N, N-dicyclohexylcarbodiimide, and the filtrate was concentrated in vacuo to give MA-PEG-SCM.

(21) 0.32g of 2-arm-PEG-DBDY and 1.3g of MA-PEG-SCM obtained in the step (18) were weighed out, dissolved in 20mL of Dichloromethane (DCM), 90mLN, N-Dimethylformamide (DMF) and 1200mL of Triethylamine (TEA) were added and reacted at 45℃for 36 hours.

(24) 2.5g of beta-cyclodextrin and 4.8g of 18-crown-6 are dissolved in 100.0mL of anhydrous DMF under inert gas protection, and then 1.25g of KH is added and stirring is continued to allow them to react well with the hydroxyl groups of the cyclodextrin.

(25) 34.3mL of glycidol is dissolved in 50mL of anhydrous DMF and slowly added dropwise to the reaction solution in step (24); after the reaction is finished, a small amount of water is added to terminate the reaction, and the mixture is dialyzed and freeze-dried to obtain a pale yellow beta-CD-HPG solid.

(26) 1.5g of the β -CD-HPG obtained in step (25) and 1.0g of succinic anhydride were weighed out and dissolved in 10 mM LDMF.

(27) 1.5g of 4-Dimethylaminopyridine (DMAP) and 15.0mL of Triethylamine (TEA) were weighed into 30mL of LDMF, and the mixture was added to the reaction system of step (26) and stirred at room temperature under nitrogen for 5d to give β

-CD-HPG-COOH。

(28) The beta-CD-HPG-COOH (mwco=1000) obtained after the reaction was dialyzed against distilled water for 7 days, and then the solution was freeze-dried.

(29) 0.6g of beta-CD-HPG-COOH obtained in the step (28) was weighed and dissolved in 60mL of deionized water, and 0.15g of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and 0.18g of an aqueous N-hydroxysuccinimide solution (NHS) were added, and the pH of the solution was adjusted to 5.0 by adding PBS buffer and stirring.

(30) 30mLRGD peptide aqueous solution was measured and added and stirred for reaction for 36 hours, and the obtained beta-CD-HPG-RGD was dialyzed against distilled water for 5 days and then freeze-dried.

Example 3

(1) 5.2g of N-t-butoxycarbonyl-L-tyrosine was weighed out and dissolved in 1000mL of boric acid buffer, 0. M, pH =9.5.

(2) 30mL of 3% hydrogen peroxide solution, 78mg of horseradish peroxidase, and N-t-butoxycarbonyl-L-tyrosine were added and the mixture was stirred at 37℃for 24 hours.

(3) After the reaction, 8mL of beta-mercaptoethanol was added to terminate the reaction, and the solvent was concentrated to a volume of 40mL.

(4) Taking the concentrated solution obtained in the step (3) for column chromatography, and n-propanol: the volume ratio of ammonia water is 9:1, and the mixture is dried after elution.

(5) Dissolving the object dried in the step (4) in ethanol, precipitating with acetone to obtain N, N-diboron dityrosine (DBDY), and storing for later use.

(6) 70g of dihydroxyPEG is weighed and dissolved in 1000mL of dichloromethane, 21g of silver iodide and 14g of p-toluenesulfonyl chloride are sequentially added for reaction for 5 hours at room temperature, the reaction solution is filtered BY diatomite, dichloromethane (DCM) is used for washing three times, and a crude TsO-PEG-OH product (BY-DBDY for short) is obtained after drying.

(7) The obtained crude TsO-PEG-OH product is dissolved in 1200mL concentration: 105g of ammonium chloride was added to 25% aqueous ammonia, stirred for 24 hours, extracted with Dichloromethane (DCM), and the organic layer was collected and dried to give NH ₂ The PEG-OH product is left ready for use.

(8) 100.0g polyethylene glycol (PEG) and 11g maleic anhydride were weighed out and dissolved in 1000mL Dichloromethane (DCM), and the solid was dissolved by stirring with nitrogen.

(9) 80mL of pyridine was added and the reaction was carried out at 60℃for 2 hours.

(11) Taking 80.0g of white solid powder HO-PEG-COOH obtained in the step (10), dissolving in 1000mL of Dichloromethane (DCM), introducing nitrogen, stirring to dissolve the solid, and adding 400mL of triethylamine; 16g of acryloyl chloride was dissolved in 200mL of Dichloromethane (DCM) and added dropwise to the HO-PEG-COOH solution.

(12) After the reaction is finished, diatomite is adsorbed and filtered, the organic solvent is concentrated under reduced pressure at 35 ℃, ice petroleum ether is dripped into the concentrated solution, the mixture is stood for 4 hours at the temperature of-20 ℃, and the solid is washed by petroleum ether after the filtration, so that MA-PEG-COOH is obtained for standby.

(13) Weighing 2.5g of the N, N-diboron dityrosine obtained in the step (5), dissolving in 1000mL of 1, 4-dioxane, and adding 1.25g of N-hydroxysuccinimide (NHS); 1.75g of N, N-Dicyclohexylcarbodiimide (DCC) was weighed and dissolved in 100mL of 1, 4-dioxane, and the N, N-Dicyclohexylcarbodiimide (DCC) solution was added dropwise to the reaction solution, followed by concentration of the organic solvent at 55℃under reduced pressure.

(14) The concentrate was washed with 500mL acetonitrile and 500mL deionized water, and the resulting DBDY-SCM was stored at-20 ℃.

(15) Weighing 1.2g of NH obtained in step (7) ₂ PEG-OH, 0.35g of DBDY-SCM obtained in step (14) was dissolved in 20mL, 90mLN, N-Dimethylformamide (DMF), 1120mL Triethylamine (TEA) were added, and reacted at 38℃for 24 hours.

(17) Weighing 10.0g of the white solid powder MA-PEG-COOH obtained in the step (12), dissolving in 100mL of 1, 4-dioxane, and adding 2.9g of N-hydroxysuccinimide (NHS); 5.2g of N, N-Dicyclohexylcarbodiimide (DCC) was weighed and dissolved in 200mL of 1, 4-dioxane, the N, N-Dicyclohexylcarbodiimide (DCC) solution was added dropwise to the reaction solution, and after the completion of the reaction, the organic solvent was concentrated under reduced pressure.

(18) The concentrate of step (17) was dissolved in 500mL of Dichloromethane (DCM), filtered to remove N, N-dicyclohexylcarbodiimide, and the filtrate was concentrated in vacuo to give MA-PEG-SCM.

(21) 0.35g of 2-arm-PEG-DBDY and 1.2g of MA-PEG-SCM obtained in the step (18) were weighed out, dissolved in 20mL of Dichloromethane (DCM), 80mLN, N-Dimethylformamide (DMF) and 1120mL of Triethylamine (TEA) were added and reacted at 38℃for 24 hours.

(24) 2.4g of beta-cyclodextrin and 4g of 18-crown-6 are dissolved in 100mL of anhydrous DMF under inert gas protection, and then 2.0g of KH is added and stirring is continued to allow them to react fully with the hydroxyl groups of the cyclodextrin.

(25) Dissolving 108.6mL of glycidol in 100mL of anhydrous DMF, and slowly dropwise adding the solution into the reaction system obtained in the step (24); after the reaction is finished, a small amount of water is added to terminate the reaction, and the mixture is dialyzed and freeze-dried to obtain a pale yellow beta-CD-HPG solid.

(26) 2.0g of the β -CD-HPG obtained in step (25) and 1.0g of succinic anhydride were weighed out and dissolved in 10 mM LDMF.

(27) 1.2g of 4-Dimethylaminopyridine (DMAP) and 10.0mL of Triethylamine (TEA) were weighed into 20mL of LDMF, and the mixture was added to the reaction system of step (26) and stirred at room temperature under nitrogen for 3d to give β

-CD-HPG-COOH。

(28) The beta-CD-HPG-COOH (mwco=1000) obtained after the reaction was dialyzed against distilled water for 3-7 days, and then the solution was freeze-dried.

(29) 0.6g of the β -CD-HPG-COOH obtained in step (28) was weighed and dissolved in 60mL of deionized water, 0.15g of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and 0.15g of an aqueous N-hydroxysuccinimide solution (NHS) were added, and the pH of the solution was adjusted to 4.30 by adding PBS buffer and stirring.

(30) 15mLRGD peptide aqueous solution was weighed and added and stirred for reaction for 20 hours, and the obtained beta-CD-HPG-RGD was dialyzed against distilled water for 5 days and then freeze-dried.

Analysis of experimental results:

the following 3D printing ink was prepared by taking 4-arm-PEG-DBDY, beta-CD-HPG-RGD prepared in example 3 as an example, and the specific steps are as follows:

experiment one: 1.0g of lithium phenyl-2, 4, 6-trimethylbenzoyl phosphonate (LAP) was weighed and dissolved in 200mL of deionized water under dark conditions; 5g, 15g and 30g of beta-CD-HPG-RGD are respectively weighed, 50mL of the beta-CD-HPG-RGD is dissolved in phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) solution, and 5g of 4-arm-PEG-DBDY is added into the solutions with different concentrations in 3, so that 3D printing ink with different hardness degrees is obtained.

Compression experiments are shown in fig. 3, and the compression experiments of the 3D printing ink show the compression modulus of the inks with different proportions after crosslinking; the higher the beta-CD-HPG-RGD concentration, the higher the compression modulus.

Rheological experiments are shown in fig. 4, and the rheological behavior experiments of the 3D printing ink show the rheological properties of the inks with different proportions; the higher the beta-CD-HPG-RGD concentration, the higher the viscosity, and the shear thinning occurs along with the increase of the shear rate; from the viscosity-shear rate of the graph, it can be seen that the 3D printing ink has extrusion properties while the β -CD-HPG-RGD is first physically crosslinked with 4-arm-PEG-DBDY to maintain the printed force performance; the time-modulus spectrum shows that the storage modulus and the loss modulus after photocrosslinking are rapidly increased, the storage modulus and the loss modulus after photocrosslinking are stable after about 15s, the 3D printed shape can be well maintained, a certain mechanical supporting behavior exists before photocrosslinking, and the method is a very important factor for extrusion type 3D, and has remarkable effects on shape maintenance and printing fidelity. And can print multi-layer structure or print complex structure; from the angular frequency-modulus, it can be seen that 3D printing inks have a certain shape-sustaining capability; from the strain-modulus it can be seen that 3D printing inks have the ability to deform.

Collapse experiments are shown in fig. 5, and the 3D printing ink collapse experiments show the shape maintenance capability of line center control after the 3D printing ink with different proportions is subjected to photo-curing; the offset angles of the three lines with different intensities are measured by changing different pitches, and the lines tend to be more straight when the material tends to stabilize the structure and the shape maintenance performance is stronger.

Experiment II: 6g of beta-CD-HPG-RGD was weighed and dissolved in 10mL of a solution of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) at 55℃and 1g of 4-arm-PEG-DBDY was added to the solution and stirred until completely dissolved. After dividing the solution into 12 equal parts and placing the solution in a 3.5cm dish under light with a wavelength of 405nm for 30s, respectively placing the solution in pH=3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14, and taking a photograph of a Nikon fluorescence microscope after balancing for 30min, as shown in FIG. 6.

FIG. 6 fluorescence intensity measurements show that the fluorescence intensity of 3D printing ink in different pH environments increases with increasing pH; the fluorescence intensity tends to be maximum when the pH is close to 10, at which point the fluorescence is brightest to the maximum; as the pH increases, the fluorescence intensity decreases; the experiment proves that the 3D printing ink prepared by the invention has a pH response function, and the fluorescence intensity can be changed along with the change of pH.

Experiment III: separately, 30g of beta-CD-HPG-RGD was dissolved in 50mL of deionized water at 55deg.C, and 5g of 4-arm was added

PEG-DBDY, and the obtained 3D printing ink is subjected to a cyclic compression experiment, as shown in FIG. 7; as can be seen from fig. 7: the 3D printing ink compression cycle experiment shows that the 3D printing ink is suitable for the compression modulus of 100 cycles compression of the 3D printing ink with the optimal combination ratio of 60% concentration of beta-CD-HPG-RGD and 10% concentration of 4-arm-PEG-DBDY under the condition that the strain is 10%; the material has certain strain capacity and the ink has good mechanical property.

Experiment IV: 30g of beta-CD-HPG-RGD is weighed, 50mL of beta-CD-HPG-RGD is dissolved in deionized water at 55 ℃, 5g of 4-arm-PEG-DBDY is added, stirring is carried out for 5min, 3D printing is carried out, as shown in FIG. 8, 3D printing shows a physical image after 3D printing, a printed object has blue fluorescence, printing details are obvious, ink printing precision is high, and good 3D printing capability is achieved.

Experiment five: sterilizing the grid structure obtained by printing in the step (3) with 75% ethanol, co-culturing Human Umbilical Vein Endothelial Cells (HUVEC), wherein the cell adhesion experiment in FIG. 9 is that the grid bracket after 3D printing is sterilized and then cultured with vascular endothelial cells (red fluorescent markers) on a low-adsorption culture dish as shown in FIG. 9; the figure shows that the vascular endothelial cells have good adhesion and growth conditions on the 3D printing grid bracket and have a certain proliferation effect; it shows that the 3D printing ink has good biocompatibility and bioactivity.

The 3D printing inks prepared using the methods described in examples 2 and 3 perform similarly to example 1.

Claims

1. The preparation method of the pH fluorescence response polypeptide self-assembled 3D printing ink is characterized by comprising the following steps of:

(1) Preparing N-t-butoxycarbonyl-L-tyrosine solution with the concentration of 4.0-8.0g/L, wherein the solvent is boric acid buffer solution;

(2) Adding hydrogen peroxide solution according to the volume ratio of the hydrogen peroxide solution to the boric acid buffer solution of 0.02-0.05:1, adding horseradish peroxidase with the mass of 0.01-0.02 times of that of N-t-butoxycarbonyl-L-tyrosine until the N-t-butoxycarbonyl-L-tyrosine and the horseradish peroxidase are completely dissolved, and stirring and reacting at room temperature;

(3) Adding beta-mercaptoethanol according to the volume ratio of the beta-mercaptoethanol to the boric acid buffer solution of 0.005-0.01:1 to terminate the reaction, and concentrating the volume of the solvent to 2.0-6.0% of the reaction system;

(4) Taking the concentrated solution obtained in the step (3) for column chromatography, and spin-drying after elution; dissolving the dried object in ethanol, precipitating with acetone to obtain N, N-diboron dityrosine (DBDY) for later use:

(5) Dissolving dihydroxyl PEG in dichloromethane to prepare PEG solution with the concentration of 60-70g/L, sequentially adding silver iodide and p-toluenesulfonyl chloride, wherein the mass ratio of the silver iodide to the dihydroxyl PEG is 0.1-0.3:1, the mass ratio of the p-toluenesulfonyl chloride to the dihydroxyl PEG is 0.1-0.2, reacting at room temperature for 4-5 hours, filtering the reaction solution with diatomite, washing with dichloromethane, and drying to obtain a TsO-PEG-OH crude product;

(6) Dissolving the obtained TsO-PEG-OH crude product in ammonia water with the mass percentage concentration of 25%, and adding ammonium chloride Stirring and reacting for 8-24h, wherein the mass of ammonium chloride is 1.0-1.5 times that of dihydroxyl PEG; then extracting with dichloromethane, collecting the organic layer, and drying to obtain NH ₂ -the PEG-OH product is left for later use;

(7) Weighing polyethylene glycol, dissolving in dichloromethane, preparing PEG solution with concentration of 80-100g/L, adding maleic anhydride according to the proportion of 10-11 g/L, introducing nitrogen and stirring to dissolve the solid;

(8) Adding pyridine, and reacting for 1.0-6.0h at 40-100 ℃, wherein the mass volume ratio of polyethylene glycol to pyridine is 1 (0.5-0.8), and g is mL;

(9) After the reaction is finished, concentrating the organic solvent under reduced pressure, dripping ice petroleum ether into the concentrated solution to precipitate reactants, filtering, and washing the solid with petroleum ether to obtain white solid powder HO-PEG-COOH;

(10) Dissolving white solid powder HO-PEG-COOH obtained in the step (9) in dichloromethane to prepare HO-PEG-COOH solution with the concentration of 60-100g/L, introducing nitrogen and stirring to dissolve the solid, and adding triethylamine according to the volume ratio of dichloromethane to triethylamine of 1:0.25-0.5; dissolving the acryloyl chloride in dichloromethane to prepare an acryloyl chloride solution with the concentration of 5% -10%, and then dropwise adding the acryloyl chloride solution into the HO-PEG-COOH solution, wherein the mass ratio of the HO-PEG-COOH to the acryloyl chloride is 1:0.15-0.25;

(11) After the reaction is finished, diatomite is subjected to adsorption filtration, an organic solvent is concentrated, ice petroleum ether is dripped into the concentrated solution, the concentrated solution is kept stand, and the solid is washed by petroleum ether after filtration to obtain MA-PEG-COOH for standby;

(12) Dissolving the N, N-diboron dityrosine obtained in the step (4) into 1, 4-dioxane according to the proportion of 2.0-3.0g/L to prepare N, N-diboron dityrosine solution, and adding N-hydroxysuccinimide with the mass of 0.3-0.6 times of that of the N, N-diboron dityrosine; weighing N, N-dicyclohexyl carbodiimide with the mass of 0.6-0.8 times of that of N, N-diboron dityrosine, dissolving the N, N-dicyclohexyl carbodiimide in 1, 4-dioxane to prepare 18.0-20.0g/LN, dripping the N, N-dicyclohexyl carbodiimide solution into a reaction solution, and concentrating an organic solvent under reduced pressure at 35-60 ℃ after the reaction is finished;

(13) Sequentially adding acetonitrile and deionized water into the concentrate for washing, and preserving the obtained DBDY-SCM at the temperature of minus 20 ℃;

(14) Weighing the NH obtained in the step (6) ₂ -PEG-OH, weighing DBDY-SCM obtained in the step (13), dissolving in dichloromethane, adding N, N-dimethylformamide with the volume of 4.0-5.0 times of that of the dichloromethane, and triethylamine with the volume of 50.0-60.0 times of that of the dichloromethane, and reacting for 18-36h at the temperature of 32.0-40.0 ℃;

(15) Adding excessive petroleum ether to precipitate the reactant to obtain a crude product of 2-arm-PEG-DBDY;

(16) Weighing the white solid powder MA-PEG-COOH obtained in the step (11), dissolving the white solid powder MA-PEG-COOH in 1, 4-dioxane to prepare a solution with the concentration of 8.0-10.0%, and adding N-hydroxysuccinimide with the mass of 0.2-0.5 times that of MA-PEG-COOH; weighing N, N-dicyclohexylcarbodiimide with mass of 0.5-0.6 times of that of MA-PEG-COOH, dissolving in 1, 4-dioxane to prepare a solution with concentration of 25-30g/L, dripping the N, N-dicyclohexylcarbodiimide solution into a reaction solution, and concentrating an organic solvent under reduced pressure after the reaction is finished;

(17) Dissolving the concentrate in the step (16) in dichloromethane, filtering to remove N, N-dicyclohexylcarbodiimide, and concentrating the filtrate under reduced pressure to obtain MA-PEG-SCM;

(18) Dissolving the 2-arm-PEG-DBDY crude product obtained in the step (15) in dichloromethane, adding EDT, and reacting for 15-30min;

(19) Adding excessive petroleum ether to precipitate the reactant to obtain 2-arm-PEG-DBDY;

(20) Weighing 2-arm-PEG-DBDY in the step (19) according to the proportion of 10g/L, weighing MA-PEG-SCM obtained in the step (17) according to the proportion of 50-80 g/L, dissolving in dichloromethane, adding N, N-dimethylformamide and triethylamine, wherein the volume ratio of N, N-dimethylformamide to dichloromethane is 1:2-5, the volume ratio of triethylamine to dichloromethane is 1:60, and reacting for 15-36h at the temperature of 35-45 ℃;

(21) Adding excessive petroleum ether to precipitate the reactant to obtain a crude product of 4-arm-PEG-DBDY;

(22) Dissolving the light yellow powder 4-arm-PEG-DBDY crude product obtained in the step (21) in deionized water, dialyzing with 2500Da dialysis bag, and freeze-drying;

(23) Under the protection of inert gas, beta-cyclodextrin and 18-crown ether-6 are weighed and dissolved in anhydrous DMF, the concentration of the beta-cyclodextrin is 20-25g/L, the mass ratio of the 18-crown ether-6 to the beta-cyclodextrin is 1.0:1.5-2.0, and then potassium hydride which is 0.2-0.5 times of the mass of the beta-cyclodextrin is added and continuously stirred to fully react with the hydroxyl of the cyclodextrin;

(24) Dissolving glycidol in anhydrous N, N-dimethylformamide, slowly dropwise adding the anhydrous N, N-dimethylformamide into the reaction solution in the step (23), wherein the volume ratio of the glycidol to the anhydrous DMF is 1.0:1.3-1.5, and the volume mass ratio of the glycidol to beta-cyclodextrin is 16.0-18.0; adding a small amount of water to terminate the reaction after the reaction is finished, dialyzing, and freeze-drying to obtain a pale yellow beta-CD-HPG solid;

(25) Weighing beta-CD-HPG obtained in the step (24) according to the proportion of 180-200g/L, weighing succinic anhydride with the mass of 0.5-0.6 times of that of the beta-CD-HPG, and dissolving the beta-CD-HPG and the succinic anhydride into N, N-dimethylformamide simultaneously;

(26) Weighing 4-dimethylaminopyridine with the mass of 0.6-0.75 times of that of the beta-CD-HPG, weighing triethylamine according to the proportion of 10 times of that of the 4-dimethylaminopyridine, dissolving both in DMF, adding the mixed solution into the reaction solution in the step (25), wherein the volume ratio of the mixed solution to the reaction solution is 1:2.5, and stirring for 3-5d at room temperature under nitrogen to obtain beta-CD-HPG-COOH;

(27) Dialyzing the beta-CD-HPG-COOH obtained after the reaction in the step (26) with distilled water for 3-7 days, and then freeze-drying the solution;

(28) Weighing beta-CD-HPG-COOH obtained in the step (27), dissolving in deionized water, preparing a beta-CD-HPG-COOH solution with the concentration of 50-100g/L, adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride and an N-hydroxysuccinimide aqueous solution, wherein the mass ratio of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride to the N-hydroxysuccinimide aqueous solution is 1:1, and the mass ratio of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride to the beta-CD-HPG-COOH is 1:18-20; adjusting the pH of the solution to 4.0-5.0 by adding PBS buffer and stirring;

(29) Measuring a certain volume of RGD peptide aqueous solution, adding and stirring to react for 18-36h, dialyzing the obtained beta-CD-HPG-RGD with distilled water for 3-5 days, and freeze-drying;

(30) Under the condition of avoiding light, weighing phenyl-2, 4, 6-trimethyl benzoyl lithium phosphonate, dissolving in deionized water, and preparing 5-10g/L LAP solution;

(31) And (3) weighing the beta-CD-HPG-RGD prepared in the step (29), dissolving the beta-CD-HPG-RGD in LAP solution at 35-80 ℃, adding 4-arm-PEG-DBDY after the beta-CD-HPG-RGD is completely dissolved, and stirring and reacting to obtain the 3D printing ink.

2. The method for preparing the pH fluorescence response polypeptide self-assembled 3D printing ink according to claim 1, wherein the method comprises the following steps: the concentration of the boric acid buffer solution in the step (1) is 0.1-0.25M, pH =8.0-10.0.

3. The method for preparing the pH fluorescence response polypeptide self-assembled 3D printing ink according to claim 1, wherein the method comprises the following steps: in the step (2), the mass percentage concentration of the hydrogen peroxide solution is 3%, and the reaction conditions are as follows: stirring and reacting for 6-24h at 32-37 ℃.

4. The method for preparing the pH fluorescence response polypeptide self-assembled 3D printing ink according to claim 1, wherein the method comprises the following steps: eluting in the step (4) by using a mixed solution of n-propanol and ammonia water, wherein n-propanol: the volume ratio of the ammonia water is 8:1-9:1.

5. The method for preparing the pH fluorescence response polypeptide self-assembled 3D printing ink according to claim 1, wherein the method comprises the following steps: the specific process of the step (11) is as follows: concentrating the organic solvent under reduced pressure at 30-45deg.C, dripping ice petroleum ether into the concentrated solution, standing at-20deg.C for 2-6 hr, filtering, and washing the solid with petroleum ether.