Hydrogel combined reagent and tough hydrogel

Abstract

The invention belongs to the field of biomedical engineering and medical materials, and discloses a hydrogel combined reagent, tough hydrogel can be prepared based on the hydrogel combined reagent, the tough hydrogel is based on brand-new dual-network hydrogel, the network of the tough hydrogel is composed of a PEGMA / GelNB covalent cross-linked network and a PEGMA self-cross-linked network, norbornene serves as a rigid structure, and the hydrogel combined reagent can be used for preparing the tough hydrogel. The hydrogel composition reagent has the advantages that the hydrogel composition reagent has a spring-like effect in a gel network, the stretching and compression performance of the hydrogel can be remarkably improved, and the tough hydrogel prepared from the hydrogel composition reagent has good biological activity and excellent mechanical property and can be quickly gelatinized under mild conditions.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedical engineering and medical materials, and in particular relates to providing a hydrogel combination reagent and a strong and tough hydrogel. Background Art

[0002] PEG is a biomaterial with excellent safety. However, due to its high solubility in water and the low mechanical strength of the resulting hydrogels, additional manipulation is often required to achieve hydrogels with higher mechanical strength. For example, frozen hydrogels can be prepared through physical manipulations such as repeated freeze-thaw cycles, or chemical modification can be used to produce PEG derivatives with crosslinking sites. Although frozen hydrogels can achieve strong mechanical strength, their complex manipulation, limited preformation, and inability to load cells make them difficult to meet the requirements for in situ gelation for in vivo applications. Therefore, chemical modification to obtain in situ crosslinkable PEG has become a key application and research focus. PEGMA is a chemically modified PEG material with methacrylate functional groups that is injectable and undergoes free radical polymerization under the action of an initiator, forming a covalently crosslinked hydrogel. Due to the excellent spatiotemporal controllability of photocrosslinking, PEGMA hydrogels have been widely studied and used in previous studies. However, PEGMA molecules are linear polymers with few crosslinking sites. Even multi-arm derivatives (such as 4-arm-PEGMA) that provide multiple crosslinking sites still suffer from poor mechanical properties. In addition, PEGMA alone lacks biological activity. Therefore, inorganic materials (such as nanohydroxyapatite, nanosilica, graphene, carbon nanotubes, etc.) are currently used to mix and cross-link with PEGMA derivatives to achieve improved mechanical and biological properties. However, the above materials often greatly affect the transparency of the polymer solution, so that the degree of cross-linking in the middle of the material is insufficient, resulting in insufficient mechanical properties of the hydrogel; in addition, a large amount of unreacted acrylic monomers have high cytotoxicity, which has a serious impact on cell proliferation and differentiation. Some researchers have also mixed and cross-linked PEGMA with natural materials to achieve improved mechanical and biological properties. However, natural protein materials are often linear polymers that lack a rigid structure, resulting in poor mechanical properties of PEG / natural protein material hydrogels, limiting their application in the biomedical field.

[0003] Gelatin is derived from collagen, and because its peptide sequences (such as Arg-Gly-Asp, RGD) interact with cell integrins, it inherently supports cell adhesion, proliferation, and differentiation. Gelatin can be enzymatically degraded, and the amino acids and peptides produced by degradation can be utilized by tissues, making it an excellent biomaterial. Since pure gelatin hydrogels are mechanically weak and cannot form gels in situ, many studies have currently chemically modified gelatin to enable it to form gels in situ, such as the preparation of photocrosslinkable methacrylate-modified gelatin (GelMA) and norbornene-modified gelatin (GelNB). Studies have shown that GelMA may lose its bioactive sites due to nonspecific polymerization, while GelNB retains most of its biofunctional motifs and therefore has better biocompatibility. Currently, the mechanical properties of individual GelNB hydrogels are relatively weak. For example, the open literature "Gckler T, Haase S, Kempter X, et al. Tuning Superfast Curing Thiol-Norbornene-Functionalized Gelatin Hydrogels for 3D Bioprinting[J]. John Wiley & Sons, Ltd, 2021(14). DOI: 10.1002 / ADHM.202100206." uses GelNB and compounds containing thiol (-SH) groups to form hydrogels with good biocompatibility through a click reaction. However, the mechanical properties of these hydrogels are weak, which limits their application in the biomedical field. There are currently no reports of strong and tough hydrogels based on GelNB. Summary of the Invention

[0004] To address the existing problems of PEG's lack of biological activity and PEG / natural protein hydrogels' lack of sufficient toughness and mechanical stability, the present invention provides a hydrogel composition for preparing a strong hydrogel. The strong hydrogel is based on a novel dual-network hydrogel consisting of a covalently cross-linked network of PEGMA / GelNB and a self-cross-linked network of PEGMA. Norbornene, as a rigid structure, acts like a spring within the gel network, significantly improving the hydrogel's tensile and compressive properties. The strong hydrogel prepared from this hydrogel combination exhibits good biological activity and excellent mechanical properties, and can rapidly gel under mild conditions.

[0005] In order to achieve the above-mentioned objectives, in a first aspect, the technical solution adopted by the present invention is to provide a hydrogel combination reagent, which includes a hydrogel precursor solution and an initiator. The hydrogel precursor solution is prepared by dissolving component A, i.e., a natural polymer modified with norbornene, in PBS, physiological saline or deionized water to obtain solution A; adding solution A to solution B containing component B, i.e., multi-arm polyethylene glycol modified with acrylic acid groups to obtain the hydrogel precursor solution; adding the initiator to the hydrogel precursor solution and mixing evenly; under irradiation with a light source, the norbornene in component A reacts with the acrylic acid groups in component B under light excitation to form covalent crosslinks; the acrylic acid groups in component B undergo free radical reaction and crosslinking, and a hydrogel can be prepared based on the above two networks; in addition, norbornene, as a rigid structure, greatly improves the tensile and compressive properties of the hydrogel; wherein the molar ratio of component B to component A is 0.1 to 1 and is further preferably 0.2 to 0.5.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows:

[0007] The multi-arm polyethylene glycol modified with acrylic acid groups, i.e., PEGMA, includes but is not limited to two-arm, three-arm, four-arm, six-arm, eight-arm and ten-arm PEGMA, each arm having at least one methacrylate functional group; further preferably, the multi-arm polyethylene glycol modified with acrylic acid groups is 4-arm-PEGMA with a molecular weight Mw of 5000.

[0008] The norbornene-modified natural polymer is a natural polymer modified with a norbornene derivative group, wherein the norbornene derivative group is a structure containing a norbornene functional group and a covalent cross-linking group, and the covalent cross-linking group refers to a group that can be covalently cross-linked with an acrylic acid group; therefore, the norbornene derivative group includes but is not limited to 5-norbornene-2-carboxylic acid (2-hydroxyethyl) ester, 5-norbornene-2,2-dimethanol, 5-norbornene-2-carboxamide, 2-aldehyde-5-norbornene, 5-norbornene-2,3-dicarboximide, 5-norbornene-2-methylamine, 2-cyano-5-norbornene, 5-norbornene-2,3-dicarboxylic acid, and norbornene methyl glycidyl ether; the natural polymer includes but is not limited to hyaluronic acid, silk fibroin, chitosan, gelatin, collagen, acellular matrix, starch, and cellulose. The norbornene-modified natural polymer is preferably gelatin modified with 5-norbornene-2,3-dicarboxylic anhydride, hereinafter referred to as GelNB, wherein the norbornene functional group is grafted onto the gelatin main chain.

[0009] Further preferably, the preparation steps of GelNB include: dissolving nadic anhydride in 0.5M, pH=6 MES buffer as a first mixed solution, and the raw material configuration ratio of nadic anhydride to MES buffer is 368mg:20ml; adding EDC·HCl and NHS to the first mixed solution as a second mixed solution, and stirring the second mixed solution at 50°C for 15 minutes for activation, wherein the raw material configuration mass ratio of nadic anhydride: EDC·HCl: NHS is 0.368:1.02:0.31; and adding nadic anhydride with a molecular weight of 300g Bloomed gelatin was pre-dissolved in deionized water to form a gelatin solution, which was then added to the second mixed solution as a third mixed solution, wherein the mass volume ratio of gelatin:deionized water was 1g:10ml. The pH of the third mixed solution was adjusted to 7.5-7.8 with 10M NaOH to form a fourth mixed solution. The fourth mixed solution was stirred at 50°C for at least 12 hours and then centrifuged at 2000 rpm for 3 minutes. The supernatant was transferred to a dialysis bag with a molecular weight cutoff of 3500. The solution was dialyzed against dH2O at 40°C for 3 days. The purified solution was frozen at -80°C overnight and lyophilized in a freeze dryer for 3 days to obtain GelNB.

[0010] The initiator is used to initiate free radical polymerization of methacrylate groups, and the initiator includes but is not limited to lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP), Irgacure 2959, eosin Y, horseradish catalase / hydrogen peroxide and other biocompatible initiators.

[0011] Preferably, the hydrogel combination reagent further includes growth factors, peptides, nucleic acids, drugs or stem cells.

[0012] The first aspect is the use of the hydrogel combination reagent in the preparation of 3D bio-printing bio-ink, for example, for printing hydrogel microneedles.

[0013] In the second aspect, based on the above-mentioned hydrogel combination reagent, the present invention also provides a strong hydrogel, which is prepared by cross-linking component A and component B in the hydrogel precursor liquid in the above-mentioned hydrogel combination reagent under the initiator and light, and the light is ultraviolet light or visible light irradiation, with a duration of 10s to 120s and a wavelength range of 320nm to 450nm; the prepared strong hydrogel exhibits a compressive strength of at least 1MPa and a tensile strain of at least 50%.

[0014] The tough hydrogel of the second aspect can be made into hydrogel microneedles for sustained drug release, or prefabricated into hydrogel for cartilage, skin or bone regeneration.

[0015] The second aspect is the application of the tough hydrogel in the preparation of drug carriers. The tough hydrogel loads drugs through physical adsorption or chemical bonding to achieve sustained and controlled release of drugs. The targeted delivery of drugs is achieved by modifying the targeting ligand on the network structure of the tough hydrogel.

[0016] The second aspect is the use of the tough hydrogel in the preparation of cell delivery carriers. The tough hydrogel acts as a cell scaffold to support cell growth and differentiation, providing an ideal carrier for tissue engineering.

[0017] Beneficial effects:

[0018] 1. Strength and toughness: Figure 1 As shown in the figure, taking the chemical structures of 4-arm-PEGMA and GelNB as examples, it can be seen that the multi-arm polyethylene glycol modified with acrylic acid groups provides a soft network, which provides the basic mechanical strength required to maintain the structural integrity of the hydrogel; the natural polymer modified with norbornene provides a second layer of soft network, which improves the mechanical strength of the hydrogel to a certain extent; the norbornene functional group can form covalent cross-links with the multi-arm polyethylene glycol modified with acrylic acid groups, thereby increasing the cross-linking density of the hydrogel; in addition, norbornene has strong rigidity and plays a role similar to a spring, thereby greatly improving the mechanical strength of the hydrogel.

[0019] 2. Adjustable properties: By varying the concentrations of acrylic acid-modified multi-arm polyethylene glycol and norbornene-modified natural polymers, the stiffness, porosity, and degradation rate of the hydrogels can be tailored to specific applications, such as cartilage repair (hard hydrogels) or soft tissue engineering (elastic hydrogels).

[0020] 3. Bioactivity and compatibility: Multi-arm polyethylene glycol modified with acrylic groups provides cell adhesion sites and is similar in composition to the natural extracellular matrix. This composite hydrogel has both mechanical strength and biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the preparation of component A by modifying gelatin with norbornene derivatives and the preparation of the tough hydrogel, where the red dots represent the cross-linking points of PEG; the purple dots represent the cross-linking points of PEG and GelNB;

[0022] Figure 2 For gelatin modified with nadic anhydride 1 HNMR nuclear magnetic resonance spectrum;

[0023] Figure 3 This is the Fourier infrared image of gelatin modified with nadic anhydride;

[0024] Figure 4 is the Fourier infrared image of GP016 hydrogel;

[0025] Figure 5 is the Fourier infrared image of GBP016 hydrogel;

[0026] Figure 6 The following are the degradation test results of the hydrogels prepared in the examples and comparative examples;

[0027] Figure 7 These are photos of the hydrogel products prepared in the examples and comparative examples;

[0028] Figure 8 This is a photo of the microneedles of the GBP016 hydrogel prepared in Example 3;

[0029] Figure 9 The mechanical properties test results of the hydrogel product prepared in Comparative Example 1;

[0030] Figure 10 The mechanical properties test results of the hydrogel product prepared in Comparative Example 3;

[0031] Figure 11 The mechanical properties test results of the hydrogel product prepared in Comparative Example 6 are as follows;

[0032] Figure 12 The mechanical properties test results of the hydrogel product prepared in Comparative Example 4;

[0033] Figure 13 The mechanical properties test results of the hydrogel product prepared in Comparative Example 5;

[0034] Figure 14 The mechanical properties test results of the hydrogel product prepared in Example 2;

[0035] Figure 15 The mechanical properties test results of the hydrogel product prepared in Example 3;

[0036] Figure 16 1 is a comparison chart of the tensile strength test results of the hydrogel products prepared in each embodiment and comparative example;

[0037] Figure 17 1 is a comparison chart of the compressive strength test results of the hydrogel products prepared in each embodiment and comparative example. DETAILED DESCRIPTION

[0038] The wt% mentioned in this application refers to the weight percentage of the raw materials in the solution after preparation.

[0039] Norbornene diacid anhydride is 5-norbornene-2,3-dicarboxylic anhydride, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., item number: N814504-25g; gelatin (300g Bloom) was purchased from Sigma-Aldrich, item number V900863-500G; 4-arm-PEGMA was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., item number A850287; lithium phenyl-2,4,6-trimethylbenzoylphosphine was purchased from Beijing Inokai Technology Co., Ltd., item number A38873-5G.

[0040] Example 1 Preparation of GelNB

[0041] Preparation materials: gelatin (300 g bloom), nadic anhydride, EDC·HCl, NHS, NaOH, deionized water.

[0042] Example 1 provides a preferred preparation process for GelNB of the present invention: 326 μL, or 368 mg, of nadic anhydride is dissolved in 20 ml of 0.5 M MES buffer at a pH of 6; 1.02 g of EDC·HCl and 0.31 g of NHS are added for activation, and the mixture is stirred at 50°C for 15 minutes; a gelatin solution of 1 g dissolved in 10 ml of deionized water is added, and the pH is adjusted to 7.5 with 10 M NaOH; the reaction mixture is stirred at 50°C for 12 hours; the solution is centrifuged at 2000 rpm, and the supernatant is transferred to a dialysis bag with a molecular weight cutoff of 3500; the solution is dialyzed against dH2O at 40°C for 3 days; the purified solution is frozen at -80°C overnight and lyophilized in a freeze dryer for 3 days to obtain GelNB.

[0043] like Figure 2 Shown is the gelatin modified with nadic anhydride 1 From the H NMR spectrum, it can be seen that the olefin peaks at positions 4 and 5 in the structural formula of nadic anhydride and the proton peaks of the tertiary carbon at positions 3 and 6 appear at 6.25ppm and 3.25ppm, respectively. Compared with gelatin, the nadic anhydride-modified gelatin has new peaks at 6.25ppm and 3.25ppm, indicating that the norbornene groups in nadic anhydride are successfully grafted onto gelatin.

[0044] like Figure 3 The Fourier infrared image of gelatin modified with nadic anhydride is shown. It can be seen that after the gelatin modified with nadic anhydride, the wavelength at 1600 cm -1 There is an enhancement of the peak around 2800-2950cm, which is the stretching vibration absorption peak of C=C bond. In addition, -1The peak enhancement is the stretching vibration absorption peak of the CH bond. The above results show that nadic anhydride is successfully grafted onto gelatin.

[0045] Example 2 Preparation of GBP008 hydrogel

[0046] Materials: 4-arm-PEGMA (Mw 5000), GelNB, lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP), and deionized water.

[0047] The preparation process is as follows:

[0048] Preparation of the precursor solution: 45 wt% 4-arm-PEGMA and 3.6 wt% GelNB were dissolved in deionized water, and 1.2 wt% LAP was added as a photoinitiator.

[0049] Preparation of hydrogel: The precursor solution was exposed to ultraviolet light with a wavelength of 365 nm for 60 seconds to prepare a hydrogel;

[0050] Mechanical testing of hydrogel: observe whether the hydrogel is formed. If so, use a universal mechanical tester to measure the compressive strength and tensile strength.

[0051] Example 3 Preparation of GBP016 hydrogel

[0052] The only difference between Example 3 and Example 2 is that 7.2 wt % of GelNB is used in Example 3 to replace 3.6 wt % of GelNB in Example 2, and the other raw materials and operations are the same.

[0053] like Figure 5 Shown are GBP016 hydrogel and 4-arm-PEGMA at 1720 cm -1 The C=O vibration stretching peak of methacrylic acid appears around 1642 cm -1 、1540cm -1 and 1543cm -1 The stretching vibration absorption peaks of amide I, amide II and amide III of GelNB appeared at the top; the above results showed that GBP016 hydrogel was prepared by compounding GelNB and 4-arm-PEGMA.

[0054] Comparative Example 1: PEGMA Single Component Configuration Example

[0055] Materials: 4-arm-PEGMA (Mw 5000), lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP), deionized water;

[0056] The only difference between Comparative Example 1 and Example 2 is that the preparation process of the precursor solution in Comparative Example 1 is: 45 wt% 4-arm-PEGMA is directly dissolved in deionized water, and 1.2 wt% LAP is added as a photoinitiator, that is, GelNB is not added in the preparation of the precursor solution in Comparative Example 1.

[0057] Comparative Example 2: GelNB Single Component Configuration Example

[0058] Materials: GelNB, lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP), and deionized water.

[0059] The only difference between Comparative Example 2 and Example 2 is that the precursor solution in Comparative Example 2 is prepared by dissolving 45 wt % GelNB and 1.2 wt % LAP photoinitiator in deionized water, that is, 4-arm-PEGMA is not added in the preparation of the precursor solution in Comparative Example 2.

[0060] Comparative Example 3 Preparation of PG008 hydrogel

[0061] The only difference between Comparative Example 3 and Example 2 is that the preparation process of the precursor solution in Comparative Example 3 is: 3.6 wt% 4-arm-PEGMA and 45 wt% gelatin are dissolved in deionized water, and LAP (1.2 wt%) is added as a photoinitiator to prepare the precursor solution; the remaining raw materials and operations are the same.

[0062] Comparative Example 4 Preparation of GP008 Hydrogel

[0063] Materials: 4-arm-PEGMA (Mw 5000), gelatin (300 g bloom), lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP), deionized water.

[0064] The preparation process of Comparative Example 4 differs from that of Example 2 only in that the preparation process of the precursor solution in Comparative Example 4 is as follows: 45 wt% of 4-arm-PEGMA and 3.6 wt% of gelatin are dissolved in deionized water, and LAP (1.2 wt%) is added as a photoinitiator.

[0065] Comparative Example 5GP016 hydrogel

[0066] The only difference between Comparative Example 5 and Comparative Example 4 is that 7.2 wt % of gelatin is used in Comparative Example 5 to replace 3.6 wt % of gelatin in Comparative Example 4, and the other raw materials and operations are the same.

[0067] Figure 4 The Fourier infrared image of GP016 hydrogel shows that the peaks of 1720 cm-1 in the GBP016 hydrogel and 4-arm-PEGMA are -1The C=O vibration stretching peak of methacrylic acid appears around 1642 cm -1 、1540cm -1 and 1543cm -1 The stretching vibration absorption peaks of amide I, amide II and amide III of gelatin appeared at the top; the above results showed that GP016 hydrogel was prepared by compounding gelatin and 4-arm-PEGMA.

[0068] Comparative Example 6 Preparation of PGB008 hydrogel

[0069] The only difference between Comparative Example 6 and Example 2 is that in Comparative Example 6, 3.6 wt% 4-arm-PEGMA and 45 wt% GelNB were dissolved in deionized water, and LAP (1.2 wt%) was added as a photoinitiator to prepare a precursor solution; the remaining raw materials and operations were the same.

[0070] Table 1 Comparison of the composition of different hydrogels

[0071]

[0072] like Figure 7 Actual photos of the hydrogel products prepared in Examples 1 to 3 and Comparative Examples 1 to 6 are shown. Among them, PG008, PGB008 and GelNB hydrogels are yellow, with unclear edges, and cannot maintain the prefabricated shape after demolding, indicating that the above three hydrogels have weak mechanics and poor shaping properties, and are obviously not suitable for use as strong and tough hydrogels; GP008, GBP008, PEGMA, GP016 and GBP016 hydrogels are uniform in color, transparent or milky white, with clear edges, indicating that the above hydrogels have relatively good mechanics.

[0073] Test Example 1 Mechanical properties test of hydrogel products

[0074] The tensile and compressive properties of the hydrogels prepared in Examples 1 to 3 and Comparative Examples 1 and 3 to 6 were tested respectively. The results are as follows: Figure 9 、 Figure 10 、 Figure 11 、 Figure 12 、 Figure 13 、 Figure 14 、 Figure 15 As shown, Figures 9 to 15 The results were statistically analyzed and plotted Figure 16 and Figure 17 , Figure 16 1 is a comparison chart of the tensile strength test results of the hydrogel products prepared in each embodiment and comparative example; Figure 17The figure shows a comparison of the compressive strength test results of the hydrogel products prepared in each embodiment and comparative example. It can be seen that compared with PG008 and PEGMA hydrogels, the mechanical properties of other composite hydrogels are significantly improved; in addition, among the composite hydrogels of gelatin or gelatin derivatives with the same proportion, the tensile and compressive properties of the GBP group hydrogel are better than those of the GP group hydrogel.

[0075] Test Example 2: Test of Hydrogel Product Degradation Cycle

[0076] The degradation cycles of the hydrogel products prepared in Examples 1 to 3 and Comparative Examples 2 to 6 were measured. The specific testing process was as follows: the hydrogel materials of each preparation example were immersed in PBS at a temperature of 60° C., and the degradation time was calculated by observation and weighing;

[0077] like Figure 6 The degradation results of the hydrogels prepared in each preparation example are shown. GelNB and PG008 hydrogels showed zero residual mass after 7 days of degradation in PBS. The residual mass of GP008, GBP008, GP016, and GBP016 hydrogels gradually decreased over the 42-day degradation period, demonstrating that all prepared hydrogels were degradable. The greatest reduction in residual mass, exceeding 30%, occurred within the first 7 days of degradation. At the same time point after degradation began, the GBP008 and GBP016 hydrogels had slightly higher residual masses than the GP008 and GP016 hydrogels, respectively, indicating that the cross-linked network density within the GBP008 and GBP016 hydrogels was higher than that of the GP008 and GP016 hydrogels, respectively.

[0078] The combination of 4-arm-PEGMA and norbornene derivative-modified gelatin, provided by the present invention, produces a tough, elastic hydrogel with high biocompatibility and mechanical strength. This material represents a significant advancement in hydrogel design and offers a promising solution for applications requiring both mechanical strength and biological functionality.

[0079] Based on the hydrogel formulation in Example 3, a hydrogel microneedle preparation scheme in Example 4 is provided.

[0080] Example 4 Hydrogel Microneedles

[0081] Materials: 4-arm-PEGMA (Mw 5000), GelNB, lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP), and deionized water.

[0082] The preparation process is as follows:

[0083] 1. Preparation of precursor solution: 4-arm-PEGMA (45 wt %) and GelNB (7.2 wt %) were dissolved in deionized water, and LAP (1.2 wt %) was added as a photoinitiator.

[0084] 2. Preparation of hydrogel: The precursor liquid was injected into the microneedle mold, allowed to stand to remove bubbles, and then exposed to ultraviolet light (365nm) for 60 seconds. After shaping, the mold was removed to obtain hydrogel microneedles.

[0085] like Figure 8 The following are photos of a portion of the process for preparing microneedles of GBP016 hydrogel in Example 4. Figure 8 The upper left picture shows the hydrogel microneedles being formed in the microneedle mold; the upper right picture shows the finished hydrogel microneedles after demolding; the lower left picture is the main view of the finished hydrogel microneedle; the lower right picture is the side view of the finished hydrogel microneedle; the hydrogel solution can be photocrosslinked on the microneedle mold; in addition, the hydrogel can be completely demolded; the microneedles on the hydrogel surface have obvious shapes, clear edges and high resolution, indicating that GBP016 hydrogel has good mechanical properties and printing properties, and has the potential to be used as a carrier for health monitoring and drug controlled release.

Claims

1. A hydrogel combination reagent, characterized in that: The hydrogel combination reagent includes a hydrogel precursor solution and an initiator. The hydrogel precursor solution is prepared by dissolving component A, i.e., a natural polymer modified with norbornene, in PBS, physiological saline, or deionized water to obtain solution A, and then adding solution A to solution B containing component B, i.e., a multi-arm polyethylene glycol modified with acrylic acid groups. The initiator is used to initiate free radical polymerization of methacrylate groups. After the initiator is added to the hydrogel precursor solution and mixed evenly, a cross-linking reaction occurs under irradiation with a light source to form a hydrogel. The molar ratio of component B to component A is 0.1 to 1.

2. The hydrogel combination reagent according to claim 1, characterized in that: The multi-arm polyethylene glycol PEGMA modified with acrylic acid groups is a two-arm, three-arm, four-arm, six-arm, eight-arm or ten-arm PEGMA, and each arm has at least one methacrylate functional group.

3. The hydrogel combination reagent according to claim 1, characterized in that: The norbornene-modified natural polymer is a natural polymer modified with a norbornene derivative group, wherein the norbornene derivative group is a structure containing a norbornene functional group and a covalent crosslinking group, and the covalent crosslinking group is a group that can be covalently crosslinked with an acrylic acid group.

4. A hydrogel combination reagent according to claim 3, characterized in that: The norbornene derivative group is any one of 5-norbornene-2-carboxylic acid (2-hydroxyethyl) ester, 5-norbornene-2,2-dimethanol, 5-norbornene-2-carboxamide, 2-aldehyde-5-norbornene, 5-norbornene-2,3-dicarboximide, 5-norbornene-2-methylamine, 2-cyano-5-norbornene, 5-norbornene-2,3-dicarboxylic acid or norbornene methyl glycidyl ether; the natural polymer is any one of hyaluronic acid, silk fibroin, chitosan, gelatin, collagen, acellular matrix, starch, and cellulose.

5. The hydrogel combination reagent according to claim 1, characterized in that: The norbornene-modified natural polymer is gelatin GelNB modified with 5-norbornene-2,3-dicarboxylic anhydride.

6. The hydrogel combination reagent according to claim 1, characterized in that: The initiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP), Irgacure 2959, eosin Y, horseradish catalase / hydrogen peroxide or other biocompatible initiators.

7. The hydrogel combination reagent according to claim 1, characterized in that: The hydrogel combination reagent further includes any one of growth factors, peptides, nucleic acids, drugs or stem cells.

8. Use of the hydrogel combination reagent according to claim 1 in preparing biological ink for 3D bioprinting.

9. A tough hydrogel, prepared by crosslinking component A and component B in the hydrogel precursor solution of the hydrogel combination reagent according to claim 1 under the influence of an initiator and light, wherein the light is ultraviolet light or visible light irradiation with a duration of 10s to 120s and a wavelength range of 320nm to 450nm.

10. Use of the tough hydrogel according to claim 9 in the preparation of hydrogel microneedles, wherein the hydrogel microneedles are used for sustained drug release, or are prefabricated into hydrogels for cartilage, skin or bone regeneration.

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