A method of making and products of an electric field responsive self-assembling base photocurable 3D printing ink

Abstract

The application discloses a preparation method of an electric field response self-assembly based photo-curing 3D printing ink, comprising the following steps: (1) mixing a photo initiator, a photosensitive resin and water to obtain a photosensitive resin aqueous solution; (2) uniformly mixing a polypeptide or an amino acid derivative with the photosensitive resin aqueous solution to obtain a mixed solution, and preparing the electric field response self-assembly based photo-curing 3D printing ink after self-assembly; the structure of the polypeptide is shown in the following formula (I)-(IV), and the amino acid derivative is shown in the following formula (V); the printing ink prepared by the application can self-assemble 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, a structure with regular arrangement in macro and micro and enhanced mechanical properties can be obtained after 3D printing, so that the structure obtained by curing and forming the ink has excellent mechanical properties, good structural stability, a fidelity effect and good biocompatibility.

Description

Technical Field

[0001] This invention relates to the technical field of bio-3D printing, and more particularly to a method for preparing an electric field-responsive self-assembly-based photocurable 3D printing ink and a product thereof. Background Technology

[0002] Bioprinting is a cutting-edge technology combining 3D printing and biomaterials. It utilizes cells, biomaterials, and bioactive molecules as "ink," precisely stacking them layer by layer according to a pre-defined three-dimensional model to construct biological structures such as bioactive tissues and organs. However, to date, manufacturing organs or tissues truly suitable for in vivo implantation using 3D printing technology still faces significant challenges, with the development of suitable biomaterials for 3D printing being crucial. However, 3D bioprinting still faces the following challenges: existing materials, whether used alone or in combination, struggle to balance biocompatibility and mechanical properties. Therefore, enhancing the mechanical properties of bioinks, especially those used for soft tissue and organ biomimetic manufacturing, without altering their biological characteristics, is a pressing issue in the field of bioprinting.

[0003] Through a long process of evolution, nature has developed high-strength, multifunctional helical array-layered superstructures from simple biological organic materials, serving as framework structures to enhance the mechanical strength of arthropod shells and vertebrate skeletons. Small molecules such as amino acids and bases, as basic building blocks of living systems, have formed self-assembled biomaterials with specific ordered structures or functions, such as polypeptides, proteins, collagen, and DNA. To improve the mechanical properties of these structures, mimicking the arraying of microstructures in high-strength materials in nature, and achieving patterned supramolecular morphology and integrated properties, holds promise for the biomimetic manufacture or de novo design of various supramolecular structural components that balance biocompatibility and mechanical performance.

[0004] However, current self-assembling biomaterials suitable for 3D printing mainly focus on the macroscopic design and fabrication of diverse and customized array structures. For example, Chinese patent document CN 115887763A discloses a hyaluronic acid-based bio-ink reinforced with peptide dendritic macromolecules and its preparation method. The method involves dissolving thiol-modified hyaluronic acid in phosphate buffer to obtain a first prepolymer; dissolving peptide dendritic macromolecules in phosphate buffer to obtain a second prepolymer; mixing the first and second prepolymers to obtain a first mixture; adding a crosslinking agent and a photoinitiator to the first mixture and mixing thoroughly to perform a photocrosslinking reaction, thus obtaining the bio-ink. While the high mechanical properties of the bio-ink disclosed in this technology are based on the structure of peptide dendritic macromolecules, the choice of raw materials is limited, resulting in several constraints. Furthermore, although the mechanical properties of the printed product are improved compared to those printed with methacryloyl hyaluronic acid as the bio-ink, further improvements are still needed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention discloses an electric field-responsive self-assembly-based photocurable 3D printing ink. By using 3D printing and an electric field to arrange the ink at both macroscopic and microscopic levels, a printed component with enhanced mechanical properties, high biocompatibility, excellent printing accuracy and shape fidelity can be obtained, which can be widely used in 3D bioprinting.

[0006] The specific technical solution is as follows:

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

[0008] (1) A photoinitiator, photosensitive resin and water are mixed to obtain an aqueous solution of photosensitive resin;

[0009] (2) Mix the polypeptide or amino acid derivative with the photosensitive resin aqueous solution to obtain a mixture, and then prepare an electric field responsive self-assembled photocurable 3D printing ink after self-assembly.

[0010] The structural formulas of the polypeptide are shown in formulas (Ⅰ) to (Ⅳ) below:

[0011]

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

[0013]

[0014] This invention discloses a method for preparing an electric field-responsive self-assembly-based photocurable 3D printing ink. After extensive experimentation, specific peptide or amino acid derivatives were screened and obtained. These derivatives can not only achieve rapid self-assembly in a photosensitive resin system to form self-assemblies with different microstructures, but also exhibit electric field responsiveness. Under the action of an electric field, they can be regularly arranged to form directional structures, significantly improving mechanical properties.

[0015] In step (1):

[0016] In this invention, there are no special requirements for the type of photoinitiator, which 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] The present invention does not have any special requirements for the type of photosensitive resin, which can be selected from conventional types in the art, such as one or more of polyethylene glycol diacrylate, polylactide, and polyglycolic acid.

[0018] Preferred:

[0019] The concentration of the photosensitive resin in the aqueous solution 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 within the range, and more preferably (5-10) wt%.

[0020] The mass percentage of the photoinitiator, based on the mass of the photosensitive resin, is (0.3-5.0)%, specifically 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 within this range, and more preferably (1-5)%.

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

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

[0023] Step 1: Dissolve the amino acid with the protecting group in an organic solvent to obtain an amino acid solution; dissolve the activator in an organic solvent to obtain an activator solution; dissolve the activating base in an organic solvent to obtain an activating base solution; dissolve the deprotecting agent in an organic solvent to obtain a deprotecting agent solution; install each solution onto a peptide synthesizer accordingly, and add resin as a carrier to synthesize a resin-coated peptide;

[0024] Step 2: Prepare acetylation reagent. When preparing polypeptides of structure (I), it is necessary to use acetylation reagent to acetylate the resin-containing polypeptides prepared in step 1; when preparing polypeptides of structures (II) to (IV), step 2 is not required.

[0025] Step 3: Prepare the lysis buffer. Mix the lysis buffer with the resin-containing polypeptide prepared in Step 1, or with the resin-containing polypeptide after acetylation in Step 2, and then lyse it to obtain the polypeptide.

[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 may 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 this range. If multiple amino acids are used, the concentrations of the solutions for different types of amino acids can 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, benzotriazole-1-yl-oxytripyrrolidinephosphide hexafluorophosphate, 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, etc.

[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 this range.

[0031] The activating base is selected from common types in the art, such as ethyl 2-oxime cyanoacetate, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, N-methylmorpholine, etc.

[0032] Preferably, the concentration of the activated alkaline 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 this range.

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

[0034] Preferably, the concentration of the deprotectant 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, all the above raw material solutions use the same solvent, which can be selected from N,N-dimethylformamide, N-methylpyrrolidone, dichloromethane, methanol, acetonitrile:ethanol (3:1) mixed solvent, etc.

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

[0037] In step two, the acetylation reagent is selected from conventional types in the art, such as acetic anhydride, acetyl chloride, glacial acetic acid, etc.

[0038] Preferably, a mixture of 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 in the reaction system; 1-hydroxybenzotriazole can improve the reaction activity and reduce the occurrence of side reactions.

[0039] When performing the acetylation reaction, the amount of acetylation reagent added is related to the amount of resin-containing polypeptide prepared in step one as follows: 1g of resin-containing polypeptide is added to 4-20mL of acetylation reagent.

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

[0041] In step three, 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 lysis reaction is carried out under the following conditions: continuous shaking on a shaker at room temperature for more than 2 hours. When the peptide with resin contains side chain protecting groups, the lysis time needs to be extended appropriately.

[0044] The crude product after pyrolysis still needs to undergo post-processing.

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

[0046] In step (2), the self-assembly refers to incubation at room temperature for no less than 24 hours.

[0047] This invention also discloses an electric field-responsive self-assembly-based photocurable 3D printing ink prepared according to the above method. This 3D printing ink can self-assemble into certain microstructures (such as tubes, fibers, belts, spheres, etc.), and under the action of an electric field, after 3D printing, it can obtain a structure with a regular arrangement at both the macroscopic and microscopic levels and enhanced mechanical properties. The structure obtained by curing the ink exhibits excellent mechanical properties, good structural stability, high fidelity, and good biocompatibility.

[0048] Preferably, the structure of the polypeptide is as shown in formula (I), (III) or (IV) above; more preferably, the structure of the polypeptide is as shown in formula (III) above.

[0049] With continuous optimization of the structural formula of the above-mentioned peptides, the final 3D printing ink obtained after 3D printing 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 base photocurable 3D printing ink of the present invention is photocurable 3D printing under the assistance of an electric field to obtain a dual arrangement effect on both the macroscopic and microscopic levels, which effectively enhances 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 biological materials with excellent biocompatibility and biodegradability, which are beneficial to 3D bioprinting. Attached Figure Description

[0054] Figure 1 The matrix-assisted laser desorption / ionization time-of-flight mass spectrum (MALDI-TOF) of the polypeptide prepared in Example 1;

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

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

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

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

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

[0060] Figure 7 The images show the microstructure of the 3D printing bio-inks prepared in Examples 1-5 after being aligned by an electric field. Examples 1-4 are atomic force microscopy images, and Example 5 is a TEM image.

[0061] Figure 8 The images show actual photos of the 3D printing bio-inks prepared in Examples 1-5 (ink before curing) and the inks cured by ultraviolet light (ink after curing);

[0062] Figure 9 These are 3D printed objects printed using the 3D printing bio-inks prepared in Examples 1-5, respectively.

[0063] Figure 10 The rheological characterization of the 3D printing inks prepared in Examples 1 to 5 after 3D printing is given, and the rheological characterization of the 3D printing ink (pure resin) prepared in Comparative Example 2 after 3D printing is given as a comparison. Detailed Implementation

[0064] The present invention will be described in further detail below with reference to embodiments and comparative examples, but the implementation of the present invention is not limited thereto.

[0065] Example 1

[0066] (1) Under light-protected conditions, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, polyethylene glycol diacrylate and water were mixed to obtain an aqueous solution of polyethylene glycol diacrylate with a concentration of 8 wt%. The mass percentage of ethyl 2,4,6-trimethylbenzoylphenylphosphonate was 3% based on the mass of polyethylene glycol diacrylate. The solution was stored for later use.

[0067] (2) Weigh out 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 through a 0.2 μm polyvinylidene fluoride filter membrane and store them for later use.

[0068] (3) Measure N,N′-diisopropylcarbodiimide into N,N-dimethylformamide to prepare a 0.5 mol / L activator solution and store it for later use;

[0069] (4) Weigh out ethyl 2-oxime cyanoacetate and dissolve it in N,N-dimethylformamide to prepare a 1 mol / L activated alkaline solution, and store it for later use;

[0070] (5) Measure a certain volume of piperidine into N,N-dimethylformamide to prepare a 20% volume fraction deprotecting agent solution and store it for later use;

[0071] (6) Install the reagents from steps (2) to (5) onto the peptide synthesizer, add a sufficient amount of the main solvent N,N-dimethylformamide, and add resin to the reaction vessel. After ensuring sufficient nitrogen capacity, input the designed sequence Ac-IIIIK-NH2 into the computer. After the airtightness check is passed, synthesize the peptide with resin, wash with N,N-dimethylformamide, dry it, and store it at -80℃ for later 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. After mixing evenly, the acetylation reagent is obtained.

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

[0074] (9) Trifluoroacetic acid: triisopropylsilane: ultrapure water were added in sequence according to the volume ratio of trifluoroacetic acid: triisopropylsilane: ultrapure water of 95:2.5:2.5, and the mixture was stirred to obtain the lysis solution.

[0075] (10) Add 11 mL of the lysis buffer obtained in step (9) to the peptide with resin prepared in step (8), and shake continuously on a shaker at room temperature for 2.5 h to lyse the peptide from the resin.

[0076] (11) After washing the sample obtained in step (10) with a small amount of DCM and filtering it several times, the remaining liquid is concentrated under a nitrogen blower until 2 mL of liquid remains.

[0077] (12) Add the concentrated liquid obtained in step (11) dropwise to 8 times the residual liquid volume of ice-cold ether, while pouring and rinsing with ether to disperse the polypeptide precipitated in the ether as much as possible.

[0078] (13) After slowly adding ether to produce a white flocculent precipitate, centrifuge (temperature 4℃, speed 8000rpm, time 8min), discard the supernatant and leave the solid. After centrifuging 8 times, a white precipitate with a pH of about neutral is obtained. Place it in a fume hood to allow the ether to evaporate naturally.

[0079] (14) Dissolve the precipitate in ultrapure water, shake well in a vortex mixer, pre-freeze at -80℃ for 2 hours, freeze-dry for 3 days to obtain purified polypeptide, and store at -20℃ for later use.

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

[0081]

[0082] (15) Weigh the polypeptide obtained in step (14) and dissolve it in the 8 wt% polyethylene glycol diacrylate aqueous solution obtained in step (1) at a concentration of 8 mM. After vortex dissolution, incubate at room temperature for 24 h to obtain 3D printing ink.

[0083] Figure 1 The matrix-assisted laser desorption / ionization time-of-flight mass spectrum (MALDI-TOF) of the peptide prepared in this embodiment shows a distinct proton peak with a molecular weight of 640.4687, corresponding to the peptide Ac-IIIIK-NH2, which is consistent with the theoretical molecular weight of 639.8700. Furthermore, the content of impurity peaks or fragment ion peaks in the mass spectrum is very low, indicating that the synthesized short peptide is the target molecule we need.

[0084] Example 2

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

[0086] (6) Install the reagents from steps (2) to (5) onto the peptide synthesizer, add a sufficient amount of the main solvent N,N-dimethylformamide, and add resin to the reaction vessel. After ensuring sufficient nitrogen capacity, input the designed sequence Fmoc-IIIIK-NH2 into the computer. After the airtightness check is passed, synthesize the peptide with resin, wash with N,N-dimethylformamide, dry it, and store it at -80℃ for later use.

[0087] (7) Trifluoroacetic acid: triisopropylsilane: ultrapure water were added in sequence according to the volume ratio of trifluoroacetic acid: triisopropylsilane: ultrapure water of 95:2.5:2.5, and the mixture was stirred to obtain the lysis solution.

[0088] (8) Add 11 mL of the lysis buffer obtained in step (7) to the resin-bound polypeptide prepared in step (6) to lyse the polypeptide from the resin.

[0089] (9) After washing the sample obtained in step (8) with a small amount of DCM and filtering it several times, the remaining liquid is concentrated under a nitrogen blower until 2 mL of liquid remains.

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

[0091] (11) After slowly adding ether to produce a white flocculent precipitate, centrifuge (temperature 4℃, speed 8000rpm, time 8min), discard the supernatant and leave the solid. After centrifuging 8 times, a white precipitate with a pH of about neutral is obtained. Place it in a fume hood to allow the ether to evaporate naturally.

[0092] (12) Dissolve the precipitate in ultrapure water, shake well in a vortex mixer, pre-freeze at -80℃ for 2 hours, freeze-dry for 3 days to obtain purified polypeptide, and store at -20℃ for later use.

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

[0094]

[0095] (13) Weigh the polypeptide obtained in step (12) and dissolve it in the 8wt% polyethylene glycol diacrylate aqueous solution obtained in step (1) at a concentration of 3mM. After vortex dissolution, incubate at room temperature for 24h to obtain 3D printing ink.

[0096] Figure 2The MALDI-TOF mass spectrum of the peptide prepared in this embodiment shows a distinct proton peak with a molecular weight of 820.5301, corresponding to the peptide Fmoc-IIIIK-NH2, which matches the theoretical molecular weight of 820.0721. Furthermore, the content of impurity peaks or fragment ion peaks in the mass spectrum is very low, indicating that the synthesized short peptide is the target molecule we need.

[0097] Example 3

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

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

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

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

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

[0103]

[0104] Figure 3 The MALDI-TOF mass spectrum of the peptide prepared in this embodiment shows a distinct proton peak with a molecular weight of 662.3256, corresponding to the peptide Fmoc-FFK-NH2, which matches the theoretical molecular weight of 661.7892. Furthermore, the content of impurity peaks or fragment ion peaks in the mass spectrum is very low, indicating that the synthesized short peptide is the target molecule we need.

[0105] Example 4

[0106] The preparation process is basically the same as in 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 with isoleucine, aspartic acid and tyrosine.

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

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

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

[0111]

[0112] Figure 4 The image shows the electrospray ionization mass spectrum (ESI-MS) of the peptide prepared in this embodiment. Observing the image, the molecular weight is 1413.59, corresponding to the peptide 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 out the peptide nucleic acid PNA (commercially available) and dissolve it in the 8wt% polyethylene glycol diacrylate aqueous solution obtained in step (1) at a concentration of 4mM. After adjusting the pH to 5, place it in boiling water at 100℃ for 20min, cool it to room temperature, and incubate it at room temperature for 24h to obtain 3D printing ink.

[0116] Comparative Example 1

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

[0118] In step (2), the concentration of the prepared amino acid solution is still 0.2 mol / L, but the amino acid types are replaced with 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 peptide prepared for this comparative example shows a distinct proton peak with a molecular weight of 482.2724, corresponding to the peptide Ac-FFK-NH2, which is consistent with the theoretical molecular weight of 481.5872. Furthermore, the content of impurity peaks or fragment ion peaks in the mass spectrum is very low, indicating that the synthesized short peptide is the target molecule we need.

[0123] Comparative Example 2

[0124] Prepare an 8wt% aqueous solution of polyethylene glycol diacrylate, and then dissolve it by vortexing to obtain 3D printing ink.

[0125] TEM images of the 3D printing bio-inks prepared in Examples 1-5 and Comparative Example 1 are shown below. 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-assemblies were observed in the product prepared in Comparative Example 1. The self-assemblies in the 3D printing bio-inks prepared in Examples 1 and 4 are long fibers, the self-assemblies in the 3D printing bio-ink prepared in Example 2 are short and thick sheets, the self-assemblies in the 3D printing bio-ink prepared in Example 3 are relatively thin tubes, and the self-assemblies in the 3D printing bio-ink prepared in Example 5 are spherical.

[0126] The 3D printing bio-inks prepared in each embodiment were arranged in an electric field of 5 kV / cm for 60 s, and the resulting microstructure images are shown below. Figure 7 As shown, observations revealed that self-assembled bodies of various morphologies can be arranged in a regular manner to form directional structures.

[0127] Performance testing:

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

[0129] Physical images of the 3D printing bio-inks prepared in each embodiment are shown below. Figure 8 As shown in the upper middle image, the liquid before curing is a flowable liquid. A 385nm ultraviolet light source was used for curing, and the curing time was 30 seconds. The image of the cured product is shown below. Figure 8 As shown in the lower figure, the bio-ink solidifies after being irradiated with ultraviolet light, indicating that it can be used for digital light processing 3D printing.

[0130] The 3D printing bio-inks prepared in Examples 1-5 were used for digital light processing (DLP) 3D printing. The curing light source was 385nm ultraviolet light, the layer thickness was 0.02mm, and the curing time was 3s, resulting in printed components of various shapes, such as... Figure 9 As shown.

[0131] Test Example 2: Mechanical property testing of 3D printed components.

[0132] The 3D printing inks prepared in each embodiment were subjected to mechanical property testing after 3D printing. The storage modulus (G') and loss modulus (G”) of each 3D printing ink were tested at 37°C within a strain range of 0.1–100%. Specific values ​​are listed in Table 1 below, and the rheological characterization diagrams are shown in the figure. Figure 10 As shown in the figure, the self-assembled 3D printing inks prepared in each embodiment showed a significant improvement in mechanical properties compared to pure resin (polyethylene glycol diacrylate) after photopolymerization 3D printing. The tubular self-assembled body prepared in Example 3 provided the greatest improvement in mechanical properties, followed by the fibrous self-assembled bodies prepared in Examples 1 and 4, while the spherical self-assembled body prepared in Example 5 provided the least improvement 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-described embodiments are preferred embodiments, but the scope of protection of the present invention is not limited thereto. Those skilled in the art can easily understand the spirit of the present invention based on the above embodiments and make different extensions and variations, but as long as they do not depart from the spirit of the present invention, they are all within the scope of protection of the present invention.

Claims

1. An electric field responsive self-assembled photo-base 3D printed component, characterized in that, The electric field response self-assembly based light-curing 3D printing ink is used as raw material, digital light processing 3D printing is adopted, the curing light source is 385 nm ultraviolet light, the layer thickness is 0.02 mm, and the curing time is 3 s; The storage modulus of the electric field response self-assembly based light-curing 3D printing component is 5826.14648 Pa, and the loss modulus is 501.13718 Pa; The self-assembly body in the electric field response self-assembly based light-curing 3D printing ink is tubular; The preparation method of the electric field response self-assembly based light-curing 3D printing ink comprises the following steps: (1) under the light shielding condition, ethyl 2,4,6-trimethylbenzoyl phenyl phosphonate and polyethylene glycol diacrylate are mixed with water to obtain a polyethylene glycol diacrylate aqueous solution with a concentration of 8 wt%, the mass ratio of ethyl 2,4,6-trimethylbenzoyl phenyl phosphonate to polyethylene glycol diacrylate is 3%, and the solution is stored for standby; (2) phenylalanine and lysine are weighed and dissolved in N,N-dimethylformamide to prepare amino acid solutions with a concentration of 0.2 mol / L respectively, and the solutions are filtered by using a 0.2 μm polyvinylidene fluoride filter film after solid dissolution and then stored for standby; (3) N,N'-diisopropyl carbodiimide is weighed and dissolved in N,N-dimethylformamide to prepare an activator solution with a concentration of 0.5 mol / L, and the solution is stored for standby; (4) 2-hydroximoyl cyanacetic acid ethyl ester is weighed and dissolved in N,N-dimethylformamide to prepare an activated base solution with a concentration of 1 mol / L, and the solution is stored for standby; (5) a certain volume of piperidine is weighed and dissolved in N,N-dimethylformamide to prepare a deprotection agent solution with a volume fraction of 20%, and the solution is stored for standby; (6) the reagents in steps (2)-(5) are correspondingly installed on a polypeptide synthesizer, sufficient amount of a main solvent N,N-dimethylformamide is added, a resin is added in a reaction container, nitrogen gas is ensured to have sufficient capacity, a designed sequence Fmoc-FFK-NH2 is input into a computer, after passing the airtightness check, the polypeptide with the resin is synthesized, the polypeptide is cleaned and dried by using N,N-dimethylformamide, and then the polypeptide is stored at-80 ℃ for standby; (7) according to the volume ratio of trifluoroacetic acid: triisopropylsilane: ultrapure water 95:2.5:2.5, ultrapure water, triisopropylsilane and trifluoroacetic acid are sequentially added, and a lysis solution is obtained after mixing; (8) 11 mL of the lysis solution obtained in step (7) is added to the polypeptide with the resin prepared in step (6), and the polypeptide is cleaved from the resin; (9) the sample obtained in step (8) is cleaned and filtered by using a small amount of DCM for several times, and then the liquid is left, and concentrated under a nitrogen blower to 2 mL of liquid; (10) the liquid obtained by concentration in step (9) is added dropwise into 8 times the volume of ice ether, and the polypeptide precipitated in the ether is dispersed as much as possible; (11) after slowly adding ether to produce white flocculent precipitate, centrifugation is carried out at a temperature of 4 ℃, a rotation speed of 8000 rpm and a time of 8 min, the supernatant is discarded, and the white precipitate with neutral pH is obtained after centrifugation for 8 times, and the ether is naturally volatilized in a fume hood; (12) The precipitate was dissolved in ultrapure water, vortexed, and pre-frozen at -80℃ for 2 h. After 3 days of freeze-drying, the purified polypeptide was obtained and stored at -20℃ for later use; The polypeptide is Fmoc-FFK-NH2, and the structural formula is as follows: ； (13) The polypeptide obtained in step (12) was weighed and dissolved in the 8wt% polyethylene glycol diacrylate aqueous solution obtained in step (1) at a concentration of 8 mM. After vortex dissolving, incubation was performed at room temperature for 24 h to obtain a 3D printing ink.

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