Preparation method of electric field response self-assembly based photocuring 3D printing ink and product

Abstract

The invention discloses a preparation method of electric field response self-assembly based photocuring 3D printing ink. The preparation method comprises the following steps: (1) mixing a photoinitiator, photosensitive resin and water to obtain a photosensitive resin aqueous solution; (2) uniformly mixing polypeptide or an amino acid derivative with a photosensitive resin aqueous solution to obtain a mixed solution, and performing self-assembly to obtain the electric field response self-assembly based photocuring 3D printing ink, the structural formulas of the polypeptide are shown as formulas (I)-(IV), and the amino acid derivative is shown as a formula (V); the prepared printing ink can be self-assembled to form a certain microstructure (such as a tube, a fiber, a belt, a ball and the like), and under the action of an electric field, structures which are regularly arranged macroscopically and microscopically and have enhanced mechanical properties can be obtained after 3D printing; the structure obtained by curing and forming the ink has excellent mechanical property, good structural stability, fidelity effect and good biocompatibility. # imgabs0 # imgabs1 #

Description

Technical Field

[0001] The present invention relates to the technical field of biological 3D printing, and in particular to a preparation method and product of electric field responsive self-assembly-based photocurable 3D printing ink. Background Art

[0002] Bio 3D printing technology is a cutting-edge technology that combines 3D printing and biomaterials. It uses cells, biomaterials and bioactive molecules as "ink" and accurately stacks them layer by layer according to a preset three-dimensional model to construct biological structures such as tissues and organs with biological activity. However, to date, the use of 3D printing technology to manufacture organs or tissues that are truly suitable for implantation in the body still faces great challenges, among which the development of biomaterials suitable for 3D printing is the key. However, 3D bioprinting still faces the following challenges: it is difficult for existing materials to balance biocompatibility and mechanical properties whether used alone or in combination. Therefore, bio-inks, especially bio-inks used for soft tissue and organ bionics, enhancing the mechanical properties of bio-inks without changing their biological properties is an urgent problem to be solved in the current field of bio-3D printing.

[0003] After a long evolution, nature has evolved a high-strength, multifunctional spiral array stacked superstructure from simple bio-organic materials, which serves as a framework structure to enhance the mechanical strength of arthropod shells and vertebrate bones. Small molecules such as amino acids and bases, as the basic building blocks of life systems, form self-assembled biomaterials such as peptides, proteins, collagen, and DNA with specific ordered structures or functions. In order to improve the mechanical properties of the structure, it is possible to imitate the array of microstructures in high-strength materials in nature, realize the patterning of supramolecular morphology and the integration of performance, and it is expected to bionic manufacture or design from scratch various supramolecular structures that take into account both biocompatibility and mechanical properties.

[0004] However, the self-assembly-based biomaterials currently suitable for 3D printing mainly stay at the realization of designing and manufacturing diversified and customized array structures on a macro scale. For example, the Chinese patent document with application publication number CN 115887763A discloses a hyaluronic acid-based bio-ink based on peptide dendrimer enhancement and its preparation method, wherein thiol-modified hyaluronic acid is dissolved in a phosphate buffer to obtain a first prepolymer solution; the peptide dendrimer is dissolved in a phosphate buffer to obtain a second prepolymer solution; the first prepolymer solution and the second prepolymer solution are mixed to obtain a first mixed solution, a crosslinking agent and a photoinitiator are added to the first mixed solution and mixed, and a photocrosslinking reaction is performed to obtain a bio-ink. The high mechanical properties of the bio-ink disclosed in this technical solution are based on the structure of peptide dendrimers, but the raw material selection is single and has many restrictions; and the mechanical properties of the printed product are improved compared with the mechanical properties of the product printed by methacrylylated hyaluronic acid as bio-ink, but there is still room for improvement. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention discloses an electric field responsive self-assembly-based photocurable 3D printing ink, which uses 3D printing and electric field to regularly arrange the ink at both macro and micro levels to obtain a printed component with enhanced mechanical properties, high biocompatibility, excellent printing accuracy and shape fidelity, etc., which can be widely used in 3D bioprinting.

[0006] The specific technical solutions are as follows:

[0007] A method for preparing an electric field responsive self-assembly-based photocurable 3D printing ink comprises the following steps:

[0008] (1) mixing a photoinitiator, a photosensitive resin and water to obtain a photosensitive resin aqueous solution;

[0009] (2) uniformly mixing the polypeptide or amino acid derivative with the photosensitive resin aqueous solution to obtain a mixed solution, and preparing an electric field responsive self-assembly-based photocurable 3D printing ink after self-assembly;

[0010] The structural formula of the polypeptide is shown in the following formulas (I) to (IV):

[0011]

[0012] The amino acid derivative is shown in the following formula (V):

[0013]

[0014] The present invention discloses a method for preparing an electric field responsive self-assembly-based photocurable 3D printing ink. Specific polypeptides or amino acid derivatives are screened and obtained through a large number of experiments. The specific polypeptides or amino acid derivatives can not only realize rapid self-assembly in a photosensitive resin system to form self-assembled bodies with different microscopic morphologies, but also have electric field responsiveness. Under the action of the electric field, the self-assembled bodies can be regularly arranged to form a directional structure, thereby significantly improving the mechanical properties.

[0015] In step (1):

[0016] There is no special requirement for the type of photoinitiator in the present invention, and the photoinitiator can be selected from conventional types in the art, such as ethyl 2,4,6-trimethylbenzoylphenylphosphonate, 2,4,6-trimethylbenzoyldiphenylphosphine oxide, Irgacure2022, benzoin dimethyl ether, 1-hydroxycyclohexylphenyl ketone, etc.

[0017] There is no special requirement for the type of photosensitive resin in the present invention, and the photosensitive resin can be selected from conventional types in the art, such as one or more of polyethylene glycol diacrylate, polylactide, and polyglycolide.

[0018] Preferred:

[0019] In the photosensitive resin aqueous solution, the concentration of the photosensitive resin is (3-20) wt%, specifically 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or any value in the range, more preferably (5-10) wt%.

[0020] Calculated by the mass of the photosensitive resin, the mass proportion of the photoinitiator is (0.3-5.0)%, which can be 0.3%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or any value in the range, and more preferably (1-5)%.

[0021] In step (2), the concentration of the polypeptide or amino acid derivative in the mixed solution is 0.1 mmol / L to the maximum saturation concentration of the polypeptide or amino acid derivative in the mixed solution. Preferably, the concentration of the polypeptide or amino acid derivative in the mixed solution is 1 to 10 mmol / L.

[0022] In step (2), the preparation of the polypeptide comprises:

[0023] Step 1, dissolving an amino acid with a protecting group in an organic solvent to obtain an amino acid solution, dissolving an activating agent in an organic solvent to obtain an activating agent solution, dissolving an activating base in an organic solvent to obtain an activating base solution, and dissolving a deprotecting agent in an organic solvent to obtain a deprotecting agent solution; installing each solution on a peptide synthesizer, adding a resin as a carrier, and synthesizing a peptide with the resin;

[0024] Step 2: preparing an acetylation agent. When preparing a polypeptide of formula (I), an acetylation agent is required to perform an acetylation reaction on the polypeptide with resin prepared in step 1. When preparing a polypeptide of formula (II) to (IV), step 2 is not required.

[0025] Step 3: prepare a lysis solution, mix the lysis solution with the polypeptide with resin prepared in step 1, or with the polypeptide with resin after acetylation reaction prepared in step 2, and obtain the polypeptide after lysis.

[0026] In step one:

[0027] The amino acid is selected from one or more of isoleucine, lysine, phenylalanine, aspartic acid, and tyrosine; the amino acid can be selected from different configurations (L-type and D-type).

[0028] Preferably, the concentration of the amino acid solution is 0.04-0.20 mol / L, specifically 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, 0.10 mol / L, 0.11 mol / L, 0.12 mol / L, 0.13 mol / L, 0.14 mol / L, 0.15 mol / L, 0.16 mol / L, 0.17 mol / L, 0.18 mol / L, 0.19 mol / L, 0.20 mol / L or any value within the range; if multiple amino acids are used, the concentrations of different types of amino acid solutions may be the same or different, but the concentration of each prepared amino acid solution is also limited to 0.04-0.20 mol / L.

[0029] The activator is selected from common types in the art, such as N,N′-diisopropylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole, O-benzotriazole-tetramethyluronium hexafluorophosphate, and the like.

[0030] Preferably, the concentration of the activator solution is 0.05-1.00 mol / L, specifically 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, 0.35 mol / L, 0.40 mol / L, 0.45 mol / L, 0.50 mol / L, 0.55 mol / L, 0.60 mol / L, 0.65 mol / L, 0.70 mol / L, 0.75 mol / L, 0.80 mol / L, 0.85 mol / L, 0.90 mol / L, 0.95 mol / L, 1.00 mol / L or any value within the range.

[0031] The activated base is selected from common types in the art, such as ethyl 2-oximecyanoacetate, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, N-methylmorpholine and the like.

[0032] Preferably, the concentration of the activated alkali solution is 0.05-1.00 mol / L, specifically 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, 0.35 mol / L, 0.40 mol / L, 0.45 mol / L, 0.50 mol / L, 0.55 mol / L, 0.60 mol / L, 0.65 mol / L, 0.70 mol / L, 0.75 mol / L, 0.80 mol / L, 0.85 mol / L, 0.90 mol / L, 0.95 mol / L, 1.00 mol / L or any value within the range.

[0033] The deprotecting agent is selected from common types in the art, such as piperidine, piperazine, morpholine and the like.

[0034] Preferably, the concentration of the deprotecting agent solution is (4-20) vol%, specifically 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, 15 vol%, 16 vol%, 17 vol%, 18 vol%, 19 vol%, 20 vol% or any value within the range.

[0035] For ease of preparation, the above raw material solutions use the same solvent, which can be specifically selected from N,N-dimethylformamide, N-methylpyrrolidone, dichloromethane, methanol, acetonitrile:ethanol (3:1) mixed solvent, etc.

[0036] The resin is only used as a carrier, and there is no special selection of specific types. It can be selected from common types in the art, such as Wang-Resin, 4-methylbenzhydrylamine hydrochloride resin (MBHA resin), etc.

[0037] In step 2, the acetylating agent is selected from conventional types in the art, such as acetic anhydride, acetyl chloride, glacial acetic acid, and the like.

[0038] Preferably, a mixed solution consisting of three reagents, acetic anhydride, N,N-diisopropylethylamine and 1-hydroxybenzotriazole, is used. N,N-diisopropylethylamine is used to promote the reaction and help maintain a suitable pH value of the reaction system; 1-hydroxybenzotriazole can improve the reaction activity and reduce the occurrence of side reactions.

[0039] When the acetylation reaction is performed, the relationship between the amount of the acetylation reagent added and the amount of the polypeptide with resin prepared in step 1 is: 4 to 20 mL of the acetylation reagent is added to 1 g of the polypeptide with resin.

[0040] Preferably, the acetylation reaction is carried out by shaking on a shaker at a water bath temperature of 20 to 90° C. for 10 to 60 minutes.

[0041] In step 3, the lysis solution is obtained by mixing trifluoroacetic acid, triisopropylsilane and ultrapure water;

[0042] Preferably, the volume ratio of trifluoroacetic acid:triisopropylsilane:ultrapure water is 95:2.5:2.5.

[0043] The cleavage reaction conditions are continuous shaking on a shaker at room temperature for more than 2 hours. When the polypeptide with the resin contains side chain protecting groups, the cleavage time needs to be appropriately extended.

[0044] The crude product after the cleavage needs to be post-processed;

[0045] The post-treatment includes concentration, precipitation, washing until the pH value is close to neutral, and freeze-drying.

[0046] In step (2), the self-assembly refers to incubation at room temperature for not less than 24 hours.

[0047] The present invention also discloses an electric field responsive self-assembly-based photocurable 3D printing ink prepared according to the above method. The 3D printing ink can self-assemble to form a certain microstructure (such as tubes, fibers, strips, balls, etc.), and under the action of the electric field, a structure with macroscopic and microscopic regular arrangement and enhanced mechanical properties can be obtained after 3D printing, and the structure obtained by curing the ink has excellent mechanical properties, good structural stability, fidelity effect, and good biocompatibility.

[0048] Preferably, the structural formula of the polypeptide is as above formula (I), (III) or (IV); further preferably, the structural formula of the polypeptide is as above formula (III).

[0049] With the continuous optimization of the structural formula of the above polypeptide, the structure obtained by 3D printing of the finally prepared 3D printing ink has a higher storage modulus and loss modulus.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) The electric field responsive self-assembly-based photocurable 3D printing ink of the present invention is photocured 3D printing assisted by an electric field, thereby obtaining a dual arrangement effect at the macroscopic and microscopic levels, thereby effectively enhancing the mechanical properties of the printed components.

[0052] (2) The electric field responsive self-assembly-based photocurable 3D printing ink of the present invention can achieve high printability and shape fidelity after 3D printing.

[0053] (3) The electric field responsive self-assembly-based photocurable 3D printing inks of the present invention are all natural biomaterials with excellent biocompatibility and biodegradability, which are beneficial to 3D bioprinting. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) of the polypeptide prepared in Example 1;

[0055] Figure 2 This is a MALDI-TOF image of the polypeptide prepared in Example 2;

[0056] Figure 3 This is a MALDI-TOF image of the polypeptide prepared in Example 3;

[0057] Figure 4 The electrospray ionization mass spectrum (ESI-MS) of the polypeptide prepared in Example 4;

[0058] Figure 5 This is the MALDI-TOF image of the polypeptide prepared in Comparative Example 1;

[0059] Figure 6 Transmission electron microscopy (TEM) images of the 3D printing bio-inks prepared in Examples 1 to 5 and Comparative Example 1, respectively;

[0060] Figure 7 The microscopic morphology images of the 3D printing bio-inks prepared in Examples 1 to 5 respectively after being arranged in an electric field, wherein Examples 1 to 4 provide atomic force microscope images, and Example 5 provides a TEM image;

[0061] Figure 8 The physical pictures are of the 3D printing bio-ink (ink before curing) and the UV-cured (ink after curing) prepared in Examples 1 to 5 respectively;

[0062] Fig. 9 The 3D printed objects are respectively printed using the 3D printing bio-inks prepared in Examples 1 to 5;

[0063] Fig.10 The rheological characterizations of the 3D printing inks prepared in Examples 1 to 5 after 3D printing are shown, and the rheological characterizations of the 3D printing ink (pure resin) prepared in Comparative Example 2 after 3D printing are given as a comparison. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below in conjunction with examples and comparative examples, but the embodiments of the present invention are not limited thereto.

[0065] Example 1

[0066] (1) in a light-proof condition, mixing ethyl 2,4,6-trimethylbenzoylphenylphosphonate, polyethylene glycol diacrylate and water to obtain an aqueous solution of polyethylene glycol diacrylate with a concentration of 8 wt %, wherein the mass proportion of ethyl 2,4,6-trimethylbenzoylphenylphosphonate is 3 % based on the mass of polyethylene glycol diacrylate, and storing for later use;

[0067] (2) Weigh isoleucine and lysine and dissolve them in N,N-dimethylformamide to prepare amino acid solutions with a concentration of 0.2 mol / L. After the solids are dissolved, filter them using a 0.2 μm polyvinylidene fluoride filter membrane and store them for later use;

[0068] (3) weighing N,N′-diisopropylcarbodiimide and dissolving it in N,N-dimethylformamide to prepare a 0.5 mol / L activator solution, and storing it for later use;

[0069] (4) Weighing ethyl 2-oximecyanoacetate and dissolving it in N,N-dimethylformamide to prepare a 1 mol / L activated alkaline solution, and storing it for later use;

[0070] (5) taking a certain volume of piperidine and dissolving it in N,N-dimethylformamide to prepare a 20% volume fraction deprotection agent solution, and storing it for later use;

[0071] (6) Install the reagents in steps (2) to (5) on the peptide synthesizer, add a sufficient amount of the main solvent N,N-dimethylformamide, and add resin to the reaction container. After ensuring that the nitrogen capacity is sufficient, input the designed sequence Ac-IIIIK-NH2 into the computer. After passing the airtightness check, synthesize the peptide with resin, wash it with N,N-dimethylformamide, drain it, and store it at -80°C for future use;

[0072] (7) Weigh 1-hydroxybenzotriazole and dissolve it in N,N-dimethylformamide to prepare a solution with a concentration of 0.015 mol / L, then add acetic anhydride and N,N-diisopropylethylamine in sequence, wherein the volume fraction of acetic anhydride is 20% and the concentration of N,N-diisopropylethylamine is 0.125 mol / L, and mix them evenly to obtain an acetylation reagent;

[0073] (8) Add 15 mL of the acetylation reagent prepared in step (7) to the polypeptide with resin prepared in step (6), wash once with N,N-dimethylformamide, and then filter with dichloromethane (DCM) several times to clean the powder, and store it at -80°C for later use;

[0074] (9) adding ultrapure water, triisopropylsilane, and trifluoroacetic acid in a ratio of trifluoroacetic acid: triisopropylsilane: ultrapure water of 95:2.5:2.5 by volume, and mixing to obtain a cleavage solution;

[0075] (10) Add 11 mL of the lysate obtained in step (9) to the polypeptide with resin prepared in step (8), and shake on a shaker at room temperature for 2.5 h to cleave the polypeptide from the resin;

[0076] (11) The sample obtained in step (10) was washed with a small amount of DCM and filtered several times to leave the liquid, which was then concentrated under a nitrogen blower until 2 mL of liquid remained;

[0077] (12) Add the concentrated liquid obtained in step (11) dropwise into 8 times the residual volume of ice ether using a dropper, while pouring and washing with ether to disperse the polypeptide precipitated in the ether as much as possible;

[0078] (13) Slowly add ether to produce a white flocculent precipitate, then centrifuge (temperature 4°C, speed 8000 rpm, time 8 min), pour off the supernatant and keep the solid, centrifuge 8 times to obtain a white precipitate with a pH of approximately neutral, and place in a fume hood to evaporate the ether naturally;

[0079] (14) The precipitate was dissolved in ultrapure water, shaken on a vortexer, and pre-frozen at -80°C for 2 h. The purified polypeptide was obtained after freeze-drying for 3 days and stored at -20°C for future use;

[0080] The polypeptide Ac-IIIIK-NH2 prepared in this example has the following structural formula:

[0081]

[0082] (15) The polypeptide obtained in step (14) was weighed and dissolved in the 8 wt % polyethylene glycol diacrylate aqueous solution obtained in step (1) at a concentration of 8 mM. After vortexing to dissolve, the mixture was incubated at room temperature for 24 h to obtain a 3D printing ink.

[0083] Figure 1 The matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF) of the polypeptide prepared in this example shows that an obvious proton peak appears in the figure with a molecular weight of 640.4687, corresponding to the polypeptide Ac-IIIIK-NH2, which is consistent with the theoretical molecular weight of 639.8700, and the contents of impurity peaks or fragment ion peaks in the mass spectrum are very low, which indicates that the synthesized short peptide is the target molecule we need.

[0084] Example 2

[0085] Steps (1) to (5) are exactly the same as those in Example 1;

[0086] (6) Install the reagents in steps (2) to (5) on the peptide synthesizer, add a sufficient amount of the main solvent N,N-dimethylformamide, and add resin to the reaction container. After ensuring that the nitrogen capacity is sufficient, input the designed sequence Fmoc-IIIIK-NH2 into the computer. After passing the airtightness check, synthesize the peptide with resin, wash it with N,N-dimethylformamide, drain it, and store it at -80°C for future use;

[0087] (7) adding ultrapure water, triisopropylsilane, and trifluoroacetic acid in a ratio of trifluoroacetic acid: triisopropylsilane: ultrapure water of 95:2.5:2.5 by volume, and mixing to obtain a cleavage solution;

[0088] (8) adding 11 mL of the lysate obtained in step (7) to the polypeptide with resin prepared in step (6) to cleave the polypeptide from the resin;

[0089] (9) The sample obtained in step (8) was washed with a small amount of DCM and filtered several times to leave the liquid, which was then concentrated under a nitrogen blower until 2 mL of liquid remained;

[0090] (10) Add the concentrated liquid obtained in step (9) dropwise into 8 times the residual volume of ice ether using a dropper, while pouring and rinsing with ether to disperse the polypeptide precipitated in the ether as much as possible;

[0091] (11) Slowly add ether to produce a white flocculent precipitate, then centrifuge (temperature 4°C, speed 8000 rpm, time 8 min), pour off the supernatant and keep the solid, centrifuge 8 times to obtain a white precipitate with a pH of approximately neutral, and place in a fume hood to evaporate the ether naturally;

[0092] (12) The precipitate was dissolved in ultrapure water, shaken on a vortexer, and pre-frozen at -80°C for 2 h. The purified polypeptide was obtained after freeze-drying for 3 days and stored at -20°C for future use;

[0093] The polypeptide Fmoc-IIIIK-NH2 prepared in this example has the following structural formula:

[0094]

[0095] (13) The polypeptide obtained in step (12) was weighed and dissolved in the 8 wt % polyethylene glycol diacrylate aqueous solution obtained in step (1) at a concentration of 3 mM. After vortexing to dissolve, the mixture was incubated at room temperature for 24 h to obtain a 3D printing ink.

[0096] Figure 2This is the MALDI-TOF mass spectrum of the polypeptide prepared in this example. Observing the figure, there is an obvious proton peak in the figure, and the molecular weight is 820.5301, which corresponds to the polypeptide Fmoc-IIIIK-NH2, consistent with the theoretical molecular weight of 820.0721, and the contents of impurity peaks or fragment ion peaks in the mass spectrum are very low, which shows that the synthesized short peptide is the target molecule we need.

[0097] Example 3

[0098] The preparation process is basically the same as that of Example 2, except that:

[0099] In step (2), the concentration of the prepared amino acid solution is still 0.2 mol / L, but the types of amino acids are replaced by phenylalanine and lysine;

[0100] In step (6), the designed sequence is Fmoc-FFK-NH2;

[0101] In step (13), the peptide concentration was replaced with 8 mM.

[0102] The polypeptide Fmoc-FFK-NH2 prepared in this example has the following structural formula:

[0103]

[0104] Figure 3 This is the MALDI-TOF mass spectrum of the polypeptide prepared in this example. Observing the figure, there is an obvious proton peak in the figure, and the molecular weight is 662.3256, which corresponds to the polypeptide Fmoc-FFK-NH2, consistent with the theoretical molecular weight of 661.7892, and the content of impurity peaks or fragment ion peaks in the mass spectrum is very low, which shows that the synthesized short peptide is the target molecule we need.

[0105] Example 4

[0106] The preparation process is basically the same as that of Example 2, except that:

[0107] In step (2), the concentration of the prepared amino acid solution is still 0.2 mol / L, but the types of amino acids are replaced by isoleucine, aspartic acid and tyrosine;

[0108] In step (6), the designed sequence is KYDYKYDYKK;

[0109] In step (13), the peptide concentration was replaced with 5 mM.

[0110] The polypeptide KYDYKYDYKK prepared in this example has the following structural formula:

[0111]

[0112] Figure 4 This is the electrospray ionization mass spectrometry (ESI-MS) of the polypeptide prepared in this example. Observing the figure, the molecular weight in the figure is 1413.59, corresponding to the polypeptide KYDYKYDYKK, which is consistent with the theoretical molecular weight of 1413.57, indicating that the synthesized short peptide is the target molecule we need.

[0113] Example 5

[0114] Step (1) is exactly the same as in Example 1;

[0115] (2) Weigh peptide nucleic acid (PNA) (commercially available) and dissolve it in the 8 wt % polyethylene glycol diacrylate aqueous solution obtained in step (1) at a concentration of 4 mM. Adjust the pH to 5 and boil it in boiling water at 100° C. for 20 min. After cooling to room temperature, incubate it at room temperature for 24 h to obtain 3D printing ink.

[0116] Comparative Example 1

[0117] The preparation process is basically the same as that of Example 1, except that:

[0118] In step (2), the concentration of the prepared amino acid solution is still 0.2 mol / L, but the types of amino acids are replaced by phenylalanine and lysine;

[0119] In step (6), the designed sequence is Ac-FFK-NH2.

[0120] The polypeptide Ac-FFK-NH2 prepared in this comparative example has the following structural formula:

[0121]

[0122] Figure 5 The MALDI-TOF mass spectrum of the polypeptide prepared in this comparative example shows that an obvious proton peak appears in the figure with a molecular weight of 482.2724, corresponding to the polypeptide Ac-FFK-NH2, which is consistent with the theoretical molecular weight of 481.5872, and the contents of impurity peaks or fragment ion peaks in the mass spectrum are very low, which indicates that the synthesized short peptide is the target molecule we need.

[0123] Comparative Example 2

[0124] An 8 wt % polyethylene glycol diacrylate aqueous solution was prepared and vortexed to dissolve to obtain a 3D printing ink.

[0125] The TEM images of the 3D printing bio-inks prepared in Examples 1 to 5 and Comparative Example 1 are as follows: Figure 6 As shown, observe Figure 6It can be seen that the 3D printing bio-inks prepared in each embodiment can achieve self-assembly, while no self-assembly was observed in the product prepared in Comparative Example 1. The self-assembly in the 3D printing bio-inks prepared in Example 1 and Example 4 was in the form of long fibers, the self-assembly in the 3D printing bio-ink prepared in Example 2 was in the form of short and thick sheets, the self-assembly in the 3D printing bio-ink prepared in Example 3 was in the form of relatively thin tubes, and the self-assembly in the 3D printing bio-ink prepared in Example 5 was in the form of spheres.

[0126] The 3D printed bio-ink prepared in each example was placed under an electric field strength of 5 kV / cm for 60 seconds, and the obtained microscopic morphology is shown in FIG. Figure 7 As shown, it was observed that self-assemblies of various morphologies can be arranged regularly to form directional structures.

[0127] Performance Test:

[0128] Test Example 1: 3D printing of self-assembly-based 3D printing bio-ink.

[0129] The actual pictures of 3D printing bio-inks prepared in each embodiment are as follows: Figure 8 As shown in the upper middle picture, it is a flowing liquid before curing. The curing light source is 385nm ultraviolet light, and the curing time is 30s. The actual picture after curing is as follows Figure 8 As shown in the lower middle figure, after being exposed to ultraviolet light, the bio-ink solidifies, indicating that it can be used for digital light processing 3D printing.

[0130] The 3D printing bio-inks prepared in Examples 1 to 5 were respectively printed using digital light processing (DLP) 3D printing, with a curing light source of 385 nm ultraviolet light, a layer thickness of 0.02 mm, and a curing time of 3 s to obtain printed components of various shapes such as Fig. 9 shown.

[0131] Test Example 2: Mechanical properties test of 3D printed components.

[0132] The mechanical properties of the 3D printing inks prepared in each embodiment were tested after 3D printing. The storage modulus (G') and loss modulus (G") of each 3D printing ink were tested at 37°C in the strain range of 0.1-100%. The specific values ​​are listed in Table 1 below. The rheological characterization diagram is shown in Fig.10 As shown in the figure, it can be seen that the mechanical properties of the self-assembled 3D printing ink prepared in each embodiment are greatly improved compared with the pure resin (polyethylene glycol diacrylate) after photocuring 3D printing, and the tubular self-assembly prepared in Example 3 helps the most in improving the mechanical properties, the fibrous self-assembly prepared in Examples 1 and 4 helps the second most in improving the mechanical properties, and the spherical self-assembly prepared in Example 5 helps the least in mechanical properties.

[0133] Table 1

[0134] serial number Storage modulus (G') / Pa Loss modulus (G") / Pa Example 1 4702.29004 404.04996 Example 2 3883.20825 307.16061 Example 3 5826.14648 501.13718 Example 4 5448.56152 368.26816 Example 5 1663.83875 167.29962 Comparative Example 2 538.95062 20.19775

[0135] The above-mentioned embodiments are preferred embodiments, but the protection scope of the present invention is not limited thereto. A person skilled in the art can easily understand the spirit of the present invention based on the above-mentioned embodiments and make different extensions and changes. However, as long as they do not deviate from the spirit of the present invention, they are all within the protection scope of the present invention.

Claims

1. A method for preparing electric field responsive self-assembly-based photocurable 3D printing ink, It is characterized in that The steps include: (1) mixing a photoinitiator, a photosensitive resin and water to obtain a photosensitive resin aqueous solution; (2) uniformly mixing the polypeptide or amino acid derivative with the photosensitive resin aqueous solution to obtain a mixed solution, and preparing an electric field responsive self-assembly-based photocurable 3D printing ink after self-assembly; The structural formula of the polypeptide is shown in the following formulas (I) to (IV): The amino acid derivative is shown in the following formula (V):

2. The method for preparing the electric field responsive self-assembly-based photocurable 3D printing ink according to claim 1, It is characterized in that In step (1): The photoinitiator is selected from one or more of 2,4,6-trimethylbenzoylphenylphosphonic acid ethyl ester, 2,4,6-trimethylbenzoyldiphenylphosphine oxide, Irgacure 2022, benzoin dimethyl ether, and 1-hydroxycyclohexylphenyl ketone; The photosensitive resin is selected from one or more of polyethylene glycol diacrylate, polylactide and polyglycolide.

3. The method for preparing the electric field responsive self-assembly-based photocurable 3D printing ink according to claim 1, It is characterized in that In step (1): In the photosensitive resin aqueous solution, the concentration of the photosensitive resin is (3-20) wt%; Based on the mass of the photosensitive resin, the mass proportion of the photoinitiator is (0.3-5.0)%.

4. The method for preparing the electric field responsive self-assembly-based photocurable 3D printing ink according to claim 1, It is characterized in that In step (2), the concentration of the polypeptide or amino acid derivative in the mixed solution is 0.1 mmol / L to the maximum saturation concentration of the polypeptide or amino acid derivative in the mixed solution.

5. The method for preparing the electric field responsive self-assembly-based photocurable 3D printing ink according to claim 1, It is characterized in that In step (2), the preparation of the polypeptide comprises: Step 1, dissolving an amino acid with a protecting group in an organic solvent to obtain an amino acid solution, dissolving an activating agent in an organic solvent to obtain an activating agent solution, dissolving an activating base in an organic solvent to obtain an activating base solution, and dissolving a deprotecting agent in an organic solvent to obtain a deprotecting agent solution; installing each solution on a peptide synthesizer, adding a resin as a carrier, and synthesizing a peptide with the resin; Step 2: preparing an acetylation agent. When preparing a polypeptide of formula (I), an acetylation agent is required to perform an acetylation reaction on the polypeptide with resin prepared in step 1. When preparing a polypeptide of formula (II) to (IV), step 2 is not required. Step 3: prepare a lysis solution, mix the lysis solution with the polypeptide with resin prepared in step 1, or with the polypeptide with resin after acetylation reaction prepared in step 2, and obtain the polypeptide after lysis.

6. The method for preparing the electric field responsive self-assembly-based photocurable 3D printing ink according to claim 5, It is characterized in that In step one: The concentration of the amino acid solution is 0.04-0.20 mol / L; The concentration of the activator solution is 0.05-1.00 mol / L; The concentration of the activated alkali solution is 0.05-1.00 mol / L; The concentration of the deprotecting agent solution is (4-20) vol%.

7. The method for preparing the electric field responsive self-assembly-based photocurable 3D printing ink according to claim 5, It is characterized in that In step 3, the crude product after cleavage needs to be post-processed; The post-treatment includes concentration, precipitation, washing until the pH value is close to neutral, and freeze-drying.

8. An electric field responsive self-assembly-based photocurable 3D printing ink prepared according to the method of claims 1 to 7.

9. The electric field responsive self-assembly-based photocurable 3D printing ink according to claim 8, It is characterized in that The structural formula of the polypeptide is as above formula (I), (III) or (IV).

10. The electric field responsive self-assembly-based photocurable 3D printing ink according to claim 8, It is characterized in that The structural formula of the polypeptide is as shown in the above formula (III).

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