A three-dimensional printing photo-crosslinking helical polyisocyanide hydrogel matrix and a preparation method thereof

Abstract

The application provides a three-dimensional printing photo-crosslinking spiral polyisocyanide hydrogel matrix and a preparation method thereof. Ultraviolet crosslinking can induce free radical chain reaction between acrylamide double bonds at the end of a PIC side chain, effectively improving the rigidity of the PIC hydrogel. The liquid can be directly crosslinked at low temperature to form a weak non-fibrillar structure; crosslinking at room temperature or higher temperature can form covalent bonds between polymer molecules on the basis of the fibrillar structure formed by temperature sensitivity, significantly improving the mechanical strength of the system, and overcoming the problem of low rigidity of the hydrogel in the prior art.

Description

Technical Field

[0001] This invention relates to the field of biomaterial preparation technology, specifically to a thermosensitive photocrosslinked helical polyisocyanate hydrogel matrix (PIC) that can be used for UV curing 3D printing and its preparation method. Background Technology

[0002] PIC hydrogel is a synthetic extracellular matrix (ECM) material that closely resembles natural extracellular matrix structural protein gels. The PIC polymer consists of a semi-flexible polyisocyanate backbone with polypeptide-ethylene glycol side chains. PIC hydrogel exhibits extremely low gelation concentration (commonly used in cell culture at 0.1% w / v), excellent biocompatibility, temperature sensitivity to both low-temperature liquid and high-temperature gelation, and stress-stiffening mechanical characteristics similar to biogels. These highly biomimetic and controllable physicochemical properties have led to its numerous applications in the biomedical field, such as wound dressings, drug delivery, and three-dimensional cell culture.

[0003] Although PIC hydrogels have been widely used in biomedical engineering, their inherent stiffness is relatively low, typically less than 1 kPa, and their self-supporting properties are limited, which restricts their application in bio-additive manufacturing. Therefore, effectively improving the stiffness of PIC hydrogels and expanding their tissue engineering applications is an urgent problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to solve the problem mentioned in the background art that the inherent stiffness of PIC hydrogels is usually less than 1 kPa and the self-support is limited, which leads to the very limited application of PIC hydrogels in bio-additive manufacturing.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The first aspect of this invention is to provide a method for preparing a three-dimensional printed photocrosslinked helical polyisocyanate hydrogel matrix, comprising the following steps:

[0007] S1: Synthesize a helical polyisocyanate polypeptide with azide groups modified on the side chain, namely PIC-N3;

[0008] S2: Add the cross-linking agent to the helical polyisocyanate peptide solution to obtain a double bond modified helical polyisocyanate peptide;

[0009] S3: Mix the double-bond modified helical polyisocyanate peptide with a photoinitiator solution to obtain the three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix under ultraviolet light.

[0010] UV photocrosslinking can initiate free radical chain reactions between the acrylamide double bonds at the ends of the PIC side chains, effectively improving the stiffness of the PIC hydrogel. At low temperatures, the liquid state can be directly crosslinked to form a weaker, non-fibrous structure; at room temperature or higher, crosslinking can form covalent bonds between polymer molecules on the basis of the already thermosensitive fibrous structure, significantly improving the mechanical strength of the system. The above preparation method is simple and easy to operate, and the prepared photocrosslinked helical polyisocyanate hydrogel matrix (helical polyisocyanate polypeptide, PIC) can achieve 3D printing with a pre-shaped structure before photocrosslinking. The final mechanical strength of the hydrogel can be adjusted by controlling the molecular weight and concentration of PIC. Furthermore, the entire crosslinking process is mild, the selected photoinitiator has good biocompatibility, and the dosage is extremely small, so it will not affect the subsequent cell survival. It fully utilizes the synergistic effect of the hydrogen bond-mediated physical gelation properties of PIC and the double bond chemical crosslinking mediated by phenyl-lithium phosphonate (LAP). The entire hydrogel system is very suitable as a matrix for UV curing in 3D material printing. The above preparation method is simple and suitable for a variety of biomedical applications; moreover, the materials used are all artificially synthesized, which is highly controllable, highly reproducible, and can support stable results in bio-3D printing.

[0011] Preferably, in step S1, a catalyst, such as nickel perchlorate hexahydrate, is added to the helical polyisocyanate polypeptide with azide groups modified on the side chain, thereby changing the average chain length of the helical polyisocyanate polypeptide and thus changing the final mechanical strength of the hydrogel.

[0012] Preferably, in step S1, the ratio of the added catalyst nickel perchlorate hexahydrate to the helical polyisocyanate polypeptide is in the range of 1:1000-1:5000. Within this ratio range, the chain length of the helical polyisocyanate polypeptide is suitable, and the prepared hydrogel has strong rigidity.

[0013] Preferably, the photoinitiator in step S3 includes lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959), both of which have good water solubility and high cell compatibility, and can be used for the polymerization of hydrogels or other polymer materials. Because of its higher water solubility, faster polymerization rate under 365 nm light, and absorbance at 400 nm (enabling visible light polymerization), this photoinitiator is superior to Irgacure 2959 and is more suitable for biological applications.

[0014] Preferably, in step S3, the concentration range of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP) is 0.2-0.5% w / v. Within this concentration range, the photocrosslinking effect is better, resulting in better performance of the photocrosslinked helical polyisocyanate hydrogel matrix.

[0015] Preferably, the reaction temperature is adjusted after mixing in step S3 to regulate the rigidity of the photocrosslinked helical polyisocyanate hydrogel matrix.

[0016] Preferably, the crosslinking agent in step S2 includes a thiol-based reactive agent containing acrylamide and dibenzocyclooctylene, such as DBCO-PEG4-Acrylamide; this type of crosslinking agent has good biocompatibility and grafting effect. The entire crosslinking process is conducted under mild conditions, the selected photoinitiator has good biocompatibility, and the amount used is extremely small and will not affect the subsequent cell survival. The synergistic effect of PIC hydrogen bond-mediated physical gelation properties and phenyl(2,4,6-trimethylbenzoyl)phosphonate lithium (LAP)-mediated double bond chemical crosslinking is fully utilized. The entire hydrogel system is very suitable as a matrix for UV curing in 3D material printing.

[0017] Preferably, the reaction time after adding the crosslinking agent in step S2 is 10-20 minutes. Within this time range, the crosslinking process is completed smoothly, and the efficiency of preparing PIC hydrogel is optimal.

[0018] Preferably, in step S2, the proportion of double bonds in the side chains of the double-bond modified helical polyisocyanate polypeptide is 0-3.3%, which can regulate the final mechanical strength of the PIC hydrogel.

[0019] Preferably, the density of azide groups in the helical polyisocyanate polypeptide is adjusted before step S1. The final mechanical strength of the hydrogel can be adjusted by adjusting the density of azide groups in PIC-N3.

[0020] A second aspect of the present invention is to provide a three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix prepared by any of the above methods, the photocrosslinked helical polyisocyanate hydrogel matrix having a fibrous structure.

[0021] A third aspect of the present invention is to provide the application of the above-mentioned three-dimensional printed photocrosslinked helical polyisocyanate hydrogel matrix in a three-dimensional cell culture matrix model.

[0022] The thermosensitive nature of helical polyisocyanate hydrogels allows them to gel at room temperature, enabling solid-state UV photocrosslinking. After localized photocrosslinking, cooling allows the uncrosslinked portions to liquefy, facilitating removal. By grafting acrylamide groups onto helical polyisocyanate peptides of varying molecular weights, a UV-curable hydrogel matrix for 3D printing / tissue engineering is obtained. Helical polyisocyanate peptides with double bond modifications on their side chains exhibit thermosensitive properties, allowing the hydrogel matrix to maintain its basic structure at room temperature and higher temperatures, enabling solid-state photocrosslinking printing. This photocrosslinked helical polyisocyanate hydrogel matrix demonstrates good cell compatibility and possesses a biomimetic fibrous hierarchical structure and nonlinear mechanical characteristics reminiscent of the natural extracellular matrix, providing a sufficient support environment for cell growth and promoting cell adhesion, proliferation, and targeted functional expression.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This application proposes a photocrosslinked helical polyisocyanate (PIC) hydrogel matrix for 3D printing and its preparation method. By grafting acrylamide groups onto PIC molecules of different molecular weights, a UV-curable hydrogel matrix for 3D printing / tissue engineering is obtained. UV photocrosslinking can initiate free radical chain reactions between the acrylamide double bonds at the end of the PIC side chains, effectively improving the hydrogel stiffness. At low temperatures, the liquid state can be directly crosslinked to form a weak, non-fibrous structure; at room temperature or higher, crosslinking can form covalent bonds between polymer molecules on the basis of the already temperature-sensitive fibrous structure, significantly improving the mechanical strength of the system. The final mechanical strength of the hydrogel can be adjusted by controlling the molecular weight and concentration of PIC. The entire crosslinking process is mild, the selected photoinitiator has good biocompatibility, and the amount used is minimal and will not affect subsequent cell survival. The synergistic effect of the hydrogen bond-mediated physical gelation properties of PIC and the double bond chemical crosslinking mediated by phenyl-lithium phosphonate (LAP) is fully utilized. The entire hydrogel system is very suitable as a UV-curable matrix for 3D material printing. The crosslinking agent used in the preparation method of this invention is a large molecule with good biocompatibility and gelling effect, resulting in a hydrogel three-dimensional scaffold with low toxicity and good cell compatibility. While providing the three-dimensional environment required for cell growth, it also promotes cell adhesion, growth, and proliferation. Furthermore, the preparation method is simple and suitable for various biomedical applications. In addition, all materials used are artificially synthesized, ensuring high controllability and reproducibility. This preparation method is unique and highly innovative compared to existing photocuring systems. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The diagram shows the molecular structure of a PIC polymer (a) and a small molecule crosslinking agent (b).

[0027] Figure 2 This invention relates to a method for preparing a three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix;

[0028] Figure 3 shows the photocrosslinking rheological curves of the 3D printed photocrosslinked spiral polyisocyanate hydrogel matrix at low temperature (5℃);

[0029] Figure 4 shows the photocrosslinking rheological curves of the 3D printed photocrosslinked helical polyisocyanate hydrogel matrix at room temperature (25℃);

[0030] Figure 5 shows the photocrosslinking rheological curves of the 3D printed photocrosslinked helical polyisocyanate hydrogel matrix at body temperature (37℃). Detailed Implementation

[0031] The technical solution of this patent will be further described in detail below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] PIC hydrogels have been widely used in biomedical engineering; however, their inherent stiffness is relatively low, typically less than 1 kPa, and their self-supporting properties are limited, restricting their application in bio-additive manufacturing. Therefore, effectively improving the stiffness of PIC hydrogels and expanding their tissue engineering applications is a pressing problem to be solved.

[0033] To address the aforementioned issues, this application provides a three-dimensional printing photocrosslinked helical polyisocyanate (PIC) hydrogel matrix and its preparation method. Acrylamide groups are grafted onto helical PIC molecules of different molecular weights to prepare UV-curable three-dimensional printing hydrogel matrices of varying concentrations. This hydrogel matrix fully utilizes the inherent temperature-sensitive properties of PIC hydrogels; it is liquid at low temperatures (5°C) and transforms into a gel state upon reaching room temperature or higher (25°C), allowing for UV covalent crosslinking of PIC inks in different states at different temperatures. This not only solves the problem of the hydrogel's inherent low stiffness but also provides a biomimetic fibrous microenvironment for cells. Click chemistry can be used to modify bioactive peptides, thereby promoting cell adhesion, proliferation, and other functional expressions.

[0034] The technical solution of this patent will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the methods used in the embodiments are conventional methods.

[0035] Example 1: A 3D Printed Photocrosslinked Helical Polyisocyanate Hydrogel Matrix

[0036] This embodiment introduces a three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix, including a helical polyisocyanate polypeptide (PIC) with double bond modification on the side chain.

[0037] The thermosensitive nature of PIC hydrogels allows them to gel at room temperature, enabling solid-state UV photocrosslinking. Cooling after photocrosslinking allows the uncrosslinked portions to liquefy and be easily removed. By grafting acrylamide groups onto PIC molecules of varying molecular weights, UV-curable hydrogel matrices for 3D printing / tissue engineering are obtained. Helical polyisocyanate peptides with double-bond side chains possess thermosensitive properties, enabling the hydrogel matrix to maintain its basic structure at room temperature and higher temperatures, achieving photocrosslinking printing in the solid state. This photocrosslinked helical polyisocyanate hydrogel matrix exhibits good cell compatibility and possesses a biomimetic fibrous hierarchical structure and nonlinear mechanical characteristics similar to the natural extracellular matrix, providing a sufficient support environment for cell growth and promoting cell adhesion, proliferation, and targeted functional expression.

[0038] This photocrosslinked helical polyisocyanate hydrogel matrix utilizes the inherent temperature-sensitive properties of PIC (low-temperature liquid state, high-temperature curing) to undergo photocrosslinking in both liquid and gel states. The stiffness of the hydrogel can be controlled by adjusting the polymer concentration, crosslinking density, and polymerization temperature.

[0039] Furthermore, the chain length, azide density, photocrosslinking density, and concentration of PIC are adjustable, thereby adjusting the stiffness of the photocrosslinked helical polyisocyanate hydrogel matrix, making the prepared photocrosslinked helical polyisocyanate hydrogel matrix suitable for different photocrosslinking application scenarios.

[0040] Furthermore, photoinitiators are used to interact with the photocrosslinked helical polyisocyanate hydrogel matrix to generate photocrosslinks for three-dimensional shaping. The photoinitiators include lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure2959). The concentrations of both are adjustable, they have good biocompatibility, and cause less damage to cells, which is conducive to the application of photocrosslinked helical polyisocyanate hydrogel matrix.

[0041] By grafting acrylamide groups onto PIC molecules of varying molecular weights, a UV-curable hydrogel matrix for 3D printing / tissue engineering was obtained. Utilizing the temperature-sensitive properties of PIC, the hydrogel matrix can achieve a basic structure at room temperature and higher temperatures, enabling photocrosslinking printing in a solid state. The resulting hydrogel 3D scaffold exhibits good cell compatibility and possesses a biomimetic fibrous hierarchical structure and nonlinear mechanical characteristics similar to the natural extracellular matrix, providing a sufficient support environment for cell growth and promoting cell adhesion, proliferation, and targeted functional expression. The entire PIC hydrogel system is highly suitable as a UV-curable matrix for 3D material printing.

[0042] The aforementioned 3D-printed photocrosslinked helical polyisocyanate hydrogel matrix alters the original linear structure, acquiring a fibrous structure. This overcomes the free radical chain reaction between the acrylamide double bonds at the end of the PIC side chains that can be initiated through UV photocrosslinking, effectively improving the hydrogel stiffness. At low temperatures, it can directly crosslink in the liquid state to form a weaker, non-fibrous structure; at room temperature or higher, crosslinking can form covalent bonds between polymer molecules on the basis of the already temperature-sensitive fibrous structure, significantly improving the mechanical strength of the system and overcoming the problem of low hydrogel stiffness in existing technologies. The 3D-printed photocrosslinked helical polyisocyanate hydrogel matrix has extremely high application value in the preparation of 3D culture substrate models.

[0043] Example 2: Preparation method of 3D printed photocrosslinked helical polyisocyanate hydrogel matrix

[0044] This embodiment provides a method for preparing the three-dimensional printing photocrosslinked spiral polyisocyanate hydrogel matrix described in Example 1. The photocrosslinked spiral polyisocyanate hydrogel matrix is ​​a UV-curable nano-photocrosslinked spiral polyisocyanate hydrogel matrix that can be used for three-dimensional printing.

[0045] Please see the appendix Figure 2 This invention relates to a three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix and its preparation method, specifically including the following steps:

[0046] S1: Synthesize a helical polyisocyanate polypeptide with azide groups modified on the side chain, namely PIC-N3;

[0047] S2: Adding nickel perchlorate hexahydrate as a catalyst to the helical polyisocyanate polypeptide with azide groups on the side chain changes the average chain length of PIC-N3, thereby changing the final mechanical strength of the hydrogel.

[0048] S3: Dissolve PIC-N3 in MilliQ to obtain a PIC-N3 solution with a concentration of 10 mg / ml;

[0049] S4: Add the crosslinking agent to the PIC-N3 solution. For example, dissolve the crosslinking agent DBCO-PEG4-acrylamide in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / ml, and add the crosslinking agent dropwise to the PIC solution in an appropriate ratio. After stirring evenly, place it on ice to react for at least 15 minutes. Modify the double bond to the PIC side chain through a ring-strain-promoted azide-acetylenite cycloaddition (SPAAC) reaction to obtain the double-bond modified helical polyisocyanate polypeptide. Please refer to the appendix. Figure 1 , is the molecular formula for double bond modification of the PIC side chain.

[0050] S5: The double-bond modified PIC-N3 and photoinitiator solution are mixed, and a three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix is ​​obtained under ultraviolet light; wherein, the final concentration of each component is:

[0051] The ratio of the catalyst nickel perchlorate hexahydrate to the spiral polyisocyanate peptide is 1:1000 or 1:5000;

[0052] PIC concentration is 1 mg / ml or 8 mg / ml;

[0053] Double bonds account for 3.3% of the side chain;

[0054] The concentration of the photoinitiator was 0.5% w / v.

[0055] In a further embodiment, the density of the azide groups in PIC-N3 in step S1 can be adjusted, and the final mechanical strength of the hydrogel can be adjusted by adjusting the density of the azide groups in PIC-N3.

[0056] In a further embodiment, the photoinitiator in step S5 includes lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959), both of which have good water solubility and high cell compatibility, and can be used for the polymerization of hydrogels or other polymer materials. Because of its higher water solubility, faster polymerization rate under 365 nm light, and absorbance at 400 nm (enabling visible light polymerization), this photoinitiator is superior to Irgacure 2959 and is more suitable for biological applications.

[0057] In a further embodiment, the concentration range of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP) in step S3 is 0.2-0.5% w / v. Within this concentration range, the photocrosslinking effect is better, resulting in better performance of the photocrosslinked helical polyisocyanate hydrogel matrix.

[0058] In a further embodiment, the crosslinking agent in step S4 includes a thiol-based reactive agent containing acrylamide and dibenzocyclooctylene, such as DBCO-PEG4-Acrylamide. This type of crosslinking agent exhibits good biocompatibility and gelling effect, resulting in a hydrogel 3D scaffold with low toxicity and good cell compatibility. While providing the necessary 3D environment for cell growth, it also promotes cell adhesion, growth, and proliferation. The entire crosslinking process is conducted under mild conditions, and the selected photoinitiator exhibits good biocompatibility and is used in minimal amounts, thus not affecting subsequent cell survival. The synergistic effect of PIC hydrogen bond-mediated physical gelling properties and phenyl(2,4,6-trimethylbenzoyl)phosphonate lithium (LAP)-mediated double bond chemical crosslinking is fully utilized. The entire hydrogel system is highly suitable as a matrix for UV curing in 3D material printing.

[0059] In a further embodiment, in step S1, the ratio of the added catalyst nickel perchlorate hexahydrate to PIC-N3 is in the range of 1:1000-1:5000. Within this ratio range, the chain length of PIC-N3 is suitable, and the prepared hydrogel has strong rigidity.

[0060] In a further embodiment, the reaction time after adding the crosslinking agent in step S4 is 10-20 minutes. Within this time range, the crosslinking process is successfully completed, and the efficiency of preparing PIC hydrogel is optimal.

[0061] In a further embodiment, the proportion of double bonds in the side chains of the double-bond modified PIC-N3 in step S4 is 0-3.3%. PIC hydrogels with this double bond ratio have better rigidity and are more convenient for application.

[0062] In a further embodiment, the reaction temperature is adjusted after mixing in step S5 to regulate the rigidity of the photocrosslinked helical polyisocyanate hydrogel matrix.

[0063] This method fully utilizes the temperature-sensitive properties of PIC (Polyisocyanate-based chromatograph), enabling the hydrogel matrix to achieve a basic, shaped structure at room temperature and higher temperatures, and achieving photocrosslinking printing in a solid state. The resulting three-dimensional hydrogel scaffold exhibits good cell compatibility and possesses a biomimetic fibrous hierarchical structure and nonlinear mechanical characteristics similar to the natural extracellular matrix, providing a sufficient support environment for cell growth and promoting cell adhesion, proliferation, and targeted functional expression. The temperature-sensitive nature of the helical polyisocyanate hydrogel allows it to gel at room temperature, making solid-state UV photocrosslinking possible. After photocrosslinking, cooling can transform the uncrosslinked portion into a liquid state for easy removal.

[0064] The crosslinking agent used in the preparation method of this invention is a large molecule with good biocompatibility and gelling effect, resulting in a hydrogel three-dimensional scaffold with low toxicity and good cell compatibility. While providing the three-dimensional environment required for cell growth, it also promotes cell adhesion, growth, and proliferation. The preparation method of this invention is simple and suitable for various biomedical applications. All materials used in the above preparation method are artificially synthesized, offering high controllability and reproducibility. The preparation method of this invention is simple and rapid, using novel materials with biomimetic and controllable properties, making it suitable for widespread application in various biomedical fields.

[0065] This preparation method produces a photocrosslinked helical polyisocyanate hydrogel matrix that enables 3D printing with a pre-defined shape before photocrosslinking. The final mechanical strength of the hydrogel can be adjusted by controlling the molecular weight and concentration of PIC. Furthermore, the entire crosslinking process is conducted under mild conditions, the selected photoinitiator exhibits good biocompatibility, and its minimal dosage does not affect subsequent cell survival. It fully leverages the synergistic effect of PIC's hydrogen-bonded physical gelation properties and phenyl-phosphonate (LAP)-mediated double-bond chemical crosslinking. The entire hydrogel system is highly suitable as a matrix for UV curing in 3D material printing. This preparation method is suitable for various biomedical applications; moreover, all materials used are artificially synthesized, offering high controllability and reproducibility, which helps reduce production costs for enterprises.

[0066] Example 3: Performance Testing of 3D Printed Photocrosslinked Helical Polyisocyanate Hydrogel Matrix

[0067] In this embodiment, the performance of the three-dimensional printed photocrosslinked spiral polyisocyanate hydrogel matrix constructed by this scheme is tested through a series of experiments.

[0068] The mixture was thoroughly mixed on ice to obtain a UV-curable hydrogel matrix precursor suitable for 3D printing. Temperature scanning and photocuring tests were then performed on it using a rheometer. Anton Paar MCR302 rheometer and... The S1500 UV point light source curing system is set to a light intensity of 50%.

[0069] Low-temperature crosslinking: UV irradiation was performed at 5℃ for 100 s, followed by a temperature scan ranging from 5 to 37℃. The heating rate was 1.0℃ / min, the strain was 1%, and the frequency was 1.0 Hz. The results are shown in Figure 3. Figure 3A -D is the rheological result of PIC low-temperature (5℃) photocrosslinking. The horizontal axis shows temperature, and the vertical axis shows the storage modulus G'. The black squares show the change of storage modulus with temperature. Blue light crosslinking was performed at 5℃, followed by heating to 37℃. The PIC chain length (controlled by the ratio of catalyst nickel perchlorate hexahydrate to monomer, 1:1000 or 1:5000, abbreviated as 1k or 5k) and concentration (1 or 8 mg / ml) both affect the storage modulus G' before and after crosslinking.

[0070] Room temperature crosslinking: First, a temperature scan was performed, ranging from 5 to 25°C, with a heating rate of 1.0°C / min, a strain of 1%, and a frequency of 1.0 Hz. Then, UV irradiation was applied at 25°C for 100 s, followed by heating to 37°C using the same parameters. The results are shown in Figure 4. Figure 4A -D is the rheological result of PIC photocrosslinking at room temperature (25℃). The horizontal axis shows temperature, and the vertical axis shows storage modulus G'. The black squares show the change of storage modulus with temperature. The arrows indicate the direction of temperature change; upward arrows indicate heating, and downward arrows indicate cooling. The material was first heated from 5℃ to 25℃, then crosslinked with blue light at 25℃, followed by heating to 37℃, and finally cooling to 5℃. The PIC chain length (controlled by the ratio of catalyst nickel perchlorate hexahydrate to monomer, 1:1000 or 1:5000, abbreviated as 1k or 5k) and concentration 1 (or 8 mg / ml) both affect the storage modulus G' before and after crosslinking.

[0071] Temperature-based crosslinking: First, a temperature scan was performed, ranging from 5 to 37°C, with a heating rate of 1.0°C / min, a strain of 1%, and a frequency of 1.0 Hz. Then, the sample was irradiated with UV light at 37°C for 100 seconds, followed by cooling to 5°C using the same parameters. The results are shown in Figure 5. Figure 5A -D shows the rheological results of PIC photocrosslinking at body temperature (37℃). The horizontal axis represents temperature, and the vertical axis represents storage modulus G'. The black squares show the change in storage modulus with temperature. The arrows indicate the direction of temperature change; upward arrows indicate heating, and downward arrows indicate cooling. The material was first heated from 5℃ to 37℃, then subjected to UV photocrosslinking at 37℃, and subsequently cooled to 5℃. The PIC chain length (controlled by the ratio of catalyst nickel perchlorate hexahydrate to monomer, 1:1000 or 1:5000, abbreviated as 1k or 5k) and concentration (1 or 8 mg / ml) both affect the storage modulus G' before and after crosslinking.

[0072] This application proposes a photocrosslinked helical polyisocyanate (PIC) hydrogel matrix for 3D printing and its preparation method. By grafting acrylamide groups onto PIC molecules of different molecular weights, a UV-curable hydrogel matrix for 3D printing / tissue engineering is obtained. UV photocrosslinking can initiate free radical chain reactions between the acrylamide double bonds at the end of the PIC side chains, effectively improving the hydrogel stiffness. At low temperatures, the liquid state can be directly crosslinked to form a weak, non-fibrous structure; at room temperature or higher temperatures, crosslinking can form covalent bonds between polymer molecules on the basis of the already temperature-sensitive fibrous structure, significantly improving the mechanical strength of the system. The final mechanical strength of the hydrogel can be adjusted by controlling the molecular weight and concentration of PIC. The entire crosslinking process is conducted under mild conditions, the selected photoinitiator has good biocompatibility, and the amount used is minimal and will not affect subsequent cell survival. The synergistic effect of the hydrogen bond-mediated physical gelation properties of PIC and the double bond chemical crosslinking mediated by phenyl (2,4,6-trimethylbenzoyl) and lithium phosphonate (LAP) is fully utilized. The entire hydrogel system is highly suitable as a UV-curable matrix for 3D material printing. The crosslinking agent used in the preparation method of this invention is a large molecule with good biocompatibility and gelling effect, resulting in a hydrogel three-dimensional scaffold with low toxicity and good cell compatibility. While providing the three-dimensional environment required for cell growth, it also promotes cell adhesion, growth, and proliferation. This preparation method is simple and suitable for various biomedical applications; moreover, all materials used are artificially synthesized, offering high controllability and reproducibility. This preparation method is unique and highly innovative compared to existing photocuring systems.

[0073] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix, characterized in that, Includes the following steps: S1: Synthesize helical polyisocyanate peptides with azide groups modified on the side chains; S2: Add the cross-linking agent DBCO-PEG4-Acrylamide to the helical polyisocyanate polypeptide solution to obtain double bond modified helical polyisocyanate polypeptide; S3: Mix the double-bond modified helical polyisocyanate peptide with a photoinitiator solution to obtain the three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix under ultraviolet light.

2. The method as described in claim 1, characterized in that, In step S1, a catalyst is added to the helical polyisocyanate polypeptide with azide groups modified on the side chain, thereby changing the average chain length of the helical polyisocyanate polypeptide.

3. The method as described in claim 2, characterized in that, In step S1, the ratio of the catalyst nickel perchlorate hexahydrate to the spiral polyisocyanate polypeptide is in the range of 1:1000-1:5000.

4. The method as described in claim 1, characterized in that, The photoinitiator in step S3 includes lithium phenyl-2,4,6-trimethylbenzoylphosphinate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.

5. The method as described in claim 4, characterized in that, In step S3, the concentration range of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate is 0.2-0.5% w / v.

6. The method as described in claim 1, characterized in that, In the double-bond modified helical polyisocyanate polypeptide described in step S2, the proportion of double bonds in the side chain is 0-3.3%.

7. The method as described in claim 1, characterized in that, After mixing in step S3, the reaction temperature is adjusted to 5-37℃ to control the rigidity of the photocrosslinked helical polyisocyanate hydrogel matrix.

8. A three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix, characterized in that, Prepared by any one of the methods described in claims 1-7.

9. The three-dimensional printing photocrosslinked helical polyisocyanate hydrogel matrix as described in claim 8, characterized in that, It has a fibrous structure.

10. The application of the three-dimensional printed photocrosslinked helical polyisocyanate hydrogel matrix as described in claim 8 in a three-dimensional cell culture matrix model.

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