A method for rheological programming of gel-based biomaterials and applications thereof

Abstract

The application belongs to the technical field of biomedical materials, and particularly discloses a method for preparing a gel-based biomaterial based on rheological programming and control and application thereof, which comprises the following steps: taking a high polymer with a phenylboronic acid group and a photo-curing reaction group, a polymer containing an o-diol group, a photo-initiator and a hydrochloric acid solution containing urea as raw materials to obtain a gel precursor system; alkalinizing the gel precursor system, controlling the alkalinization time, and obtaining a gel body with a preset storage modulus; the preset storage modulus comprises a low-grade storage modulus, a medium-grade storage modulus and a high-grade storage modulus; and applying light to the gel body with the preset storage modulus to crosslink, so that a gel-based biomaterial is obtained. The method can realize programmable control of rheology, and can prepare various gel-based biomaterials such as a gel microneedle, an injectable gel and a gel scaffold.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method and application for preparing gel-based biomaterials based on rheological programming control. Background Technology

[0002] As a processable hydrogel precursor material, biogel ink can achieve the required fluidity and printability by controlling its rheological behavior and curing process. Through in-situ casting, direct-write 3D printing, or injection molding, microneedles, scaffolds, or injectable gels with corresponding mechanical support and structural stability can be constructed at the target site. It has important application potential in the repair of tissues such as diabetic chronic wounds and bone defects.

[0003] Among them, phenylboronic acid-modified methacrylic acid polysaccharide and polyvinyl alcohol are widely used as biogel inks in dual-network hydrogels. These dual-network hydrogels are based on the formation of dynamic borate ester bonds to construct the primary network and the secondary network is constructed through photocrosslinking, exhibiting certain adhesive properties and mechanical properties. Patent application 202511704861.2, "An Immunomodulatory Hydrogel Based on Ginsenoside Rd, Preparation Method and Application," discloses a double cross-linked structure using a natural polysaccharide modified with phenylboronic acid and a protein-based biopolymer modified with methacrylic anhydride as the gel matrix. This structure can load the bioactive ingredient ginsenoside and exhibits injectability, self-healing properties, and glucose responsiveness. Patent application 202410165850.0, "A Hydrogel for Endometrial Repair and Its Preparation Method," discloses a hydrogel for endometrial repair. This hydrogel is obtained by mixing HAMA-PBA and PVA solutions, forming a primary network under the action of calcium ions, and then cross-linking it under light irradiation. It further discloses that UV light irradiation can increase the modulus of the hydrogel, thereby further improving its self-healing properties. During their research, the inventors discovered that existing biogel ink systems mainly suffer from the following problems: difficulty in achieving controllable curing, and the tendency to premature gel, which can affect the sufficiency of mold cavity filling or the smoothness of printing; current preparation processes for gel-based biomaterials are usually based on the matching of precursor and application end morphology, which cannot be compatible with multiple molding processes; when used as drug carriers, they often suffer from problems such as unstable drug release, uneven triggering, or difficulty in control, which affect the continuous regulation and repair effect on the oxidative stress microenvironment.

[0004] Develop a biogel ink platform with a controllable curing window and programmable rheological control capabilities to meet the application needs of multi-morphological medical material preparation and oxidative stress-related tissue repair. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method and application for preparing gel-based biomaterials based on rheological programming control, which addresses the shortcomings of the prior art. This method can realize programmable control of rheology and can prepare a variety of gel-based biomaterials such as gel microneedles, injectable gels and gel scaffolds.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] On the one hand, a method for preparing gel-based biomaterials based on rheological programming is provided, including:

[0008] A gel precursor system was obtained using a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea as raw materials.

[0009] The gel precursor system is alkalized, and the alkalization time is controlled to obtain a gel with a preset level of energy storage modulus; the preset level of energy storage modulus includes low-level energy storage modulus, medium-level energy storage modulus and high-level energy storage modulus;

[0010] Light conditions are applied to a gel with a preset energy storage modulus to induce cross-linking, thereby obtaining a gel-based biomaterial.

[0011] On the other hand, a method for preparing gel microneedles is provided, comprising:

[0012] A gel precursor system was obtained using a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea as raw materials.

[0013] The gel precursor system was infused into a microneedle mold;

[0014] The microneedle mold containing the gel precursor system was transferred into a sealed environment and heated to alkalize it, thus obtaining a low-grade energy storage modulus gel.

[0015] By applying light, cross-linking is achieved, resulting in gel microneedles.

[0016] On the other hand, a method for preparing an injectable gel is provided, comprising:

[0017] A gel precursor system was obtained by mixing a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea in a preset ratio.

[0018] The gel precursor system was alkalized in a closed reaction vessel to obtain a gel with a medium storage modulus.

[0019] Light conditions are applied to the medium-grade energy storage modulus gel to cause cross-linking, resulting in an injectable gel.

[0020] Furthermore, a method for preparing a gel scaffold is provided, comprising:

[0021] A gel precursor system was obtained by mixing a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea in a preset ratio.

[0022] The gel precursor system was alkalized in a closed reaction vessel to obtain a gel with a high energy storage modulus.

[0023] The high-grade energy storage modulus gel was printed using a 3D printing device to obtain a scaffold structure;

[0024] The scaffold structure is subjected to light irradiation to induce cross-linking, thereby obtaining a gel scaffold.

[0025] On the other hand, this paper provides an application of gel-based biomaterials prepared by the above-mentioned method based on rheological programming in the preparation of medical materials.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] 1. This invention is based on a strategy of hierarchical control of key rheological parameters to achieve stable switching between "low modulus high flowability - medium modulus injectability - high modulus shape retention", and realizes a preparation process compatible with multiple gel-based biomaterials on the same platform.

[0028] 2. This invention provides graded control of key rheological parameters by introducing a hydrochloric acid solution containing urea into the precursor system. This inhibits premature complete cross-linking in the precursor stage and triggers the first cross-linking through heating and alkalization, thus maintaining the processability of the system during molding stages such as pouring / injection / printing. This reduces the risk of mold failure, incomplete defoaming, printing needle blockage, and structural collapse caused by premature gelation.

[0029] 3. This invention can achieve controllable energy storage modulus through simple control of component concentration and alkalization time, and realize the coverage of three types of morphological requirements: microneedles, injectable gels and 3D printed scaffolds on the same platform.

[0030] 4. The method of the present invention includes two stages: reversible cross-linking followed by photocuring. After alkalization, a second network is formed by photocuring, thereby improving the demolding integrity and stability of the gel-based biomaterial.

[0031] 5. The gel prepared by the method of the present invention has a stronger cross-linking structure. Compared with the unalkalized gel, the drug-loaded gel-based biomaterial of the present invention can achieve long-term sustained release under physiological conditions, locking the drug in the gel network. Under oxidative stress, it can respond to the inflammatory microenvironment and increase the drug release. The drug-loaded gel-based biomaterial of the present invention has a stronger responsiveness to ROS and / or pH changes, and can achieve controlled release of the drug, which is suitable for the local repair needs related to oxidative stress.

[0032] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the process for preparing the drug-loaded gel-based biomaterial in Example 8;

[0034] Figure 2 A schematic diagram showing the pH change of the HAMA-PBA precursor solution when the urea concentration is changed.

[0035] Figure 3 This is a schematic diagram showing the storage modulus of the gels in Examples 2-4 and Comparative Examples 1-3;

[0036] Figure 4 This is a schematic diagram of the microneedle morphology in Example 2, wherein... Figure 4 The left image is a schematic diagram of the microneedle tip structure. Figure 4 The right image shows the overall morphology of the microneedles;

[0037] Figure 5 This is a schematic diagram of the morphology of the injectable gel after injection molding in Example 3;

[0038] Figure 6 The image shows the morphology of the gel obtained in Example 4;

[0039] Figure 7 This is a schematic diagram of the cumulative drug release curve of the drug-loaded gel-based biomaterial in Example 8 under ROS / pH stimulation;

[0040] Figure 8 This is a schematic diagram illustrating the evaluation of the repair effect of drug-loaded gel-based biomaterials in a diabetes wound model related to oxidative stress, as described in Example 8. Figure 8 Figure A is a flowchart of the modeling and drug administration timeline. Figure 8 Image B shows the wound healing status after repair. Detailed Implementation

[0041] The technical solution will now be clearly and completely described with reference to the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0042] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.

[0043] In the following description, the terms “including,” “containing,” “having,” and “containing” are open-ended terms, meaning that they include but are not limited to.

[0044] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0045] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] The technical principle employed in this invention is as follows: The gel system of a dual-network hydrogel, based on a primary network constructed from dynamic borate ester bonds and a secondary network constructed from photocrosslinking, exhibits high reactivity, resulting in immediate sol-gel transformation and uniform gelation. However, this makes it difficult to meet the requirements for molding into different gel-based biomaterials. This invention addresses this by introducing a urea-containing hydrochloric acid solution into the precursor stage, which inhibits premature dynamic crosslinking of the gel precursor, maintaining good fluidity and processability. Subsequently, the system is alkalized under heating conditions, leading to the formation of dynamic borate ester bonds and obtaining a first network with a preset storage modulus. Finally, photocuring forms the second network, enabling programmable adjustment of the gel system's storage modulus, molding performance, and final structural stability. This allows for injectability, self-healing, or specific tissue repair, resulting in drug-loaded gel-based biomaterials that respond to oxidative stress microenvironments and stably release drugs.

[0048] On the one hand, a method for preparing gel-based biomaterials based on rheological programming is provided, including:

[0049] A gel precursor system was obtained using a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea as raw materials.

[0050] The gel precursor system is alkalized, and the alkalization time is controlled to obtain a gel with a preset level of energy storage modulus; the preset level of energy storage modulus includes low-level energy storage modulus, medium-level energy storage modulus and high-level energy storage modulus, the energy storage modulus range of low-level energy storage modulus is 280~320 Pa, the energy storage modulus range of medium-level energy storage modulus is 430~490 Pa, and the energy storage modulus range of high-level energy storage modulus is 600~700 Pa;

[0051] Light is applied to a gel with a preset energy storage modulus to induce cross-linking, thereby obtaining a gel-based biomaterial; the wavelength of the light is 365 nm; the gel-based biomaterial includes gel microneedles, gel scaffolds, or injectable gels.

[0052] On the other hand, a method for preparing gel microneedles is provided, comprising:

[0053] A gel precursor system was obtained using a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea as raw materials.

[0054] The gel precursor system was infused into a microneedle mold;

[0055] The microneedle mold containing the gel precursor system was transferred into a sealed environment and heated to alkalize it, thus obtaining a low-grade energy storage modulus gel.

[0056] By applying light, cross-linking is achieved, resulting in gel microneedles.

[0057] This invention provides a method for preparing gel microneedles, comprising first injecting a gel precursor system into a microneedle mold, then heating to alkalize it to obtain a gel, and then curing it by light to obtain gel microneedles. The gel precursor system has high reactivity and is very easy to gel. When injected into the microneedle mold, it is easy to have difficulty in filling it completely or to have incomplete degassing. This invention separates the sol-gel stage by means of the above method, which can keep the system in a sol-like form with good fluidity before injection, and perform alkalization and light curing after injection to form gel in situ.

[0058] On the other hand, a method for preparing an injectable gel is provided, comprising:

[0059] A gel precursor system was obtained by mixing a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea in a preset ratio.

[0060] The gel precursor system was alkalized in a closed reaction vessel to obtain a gel with a medium storage modulus.

[0061] Light conditions are applied to the medium-grade energy storage modulus gel to cause cross-linking, resulting in an injectable gel.

[0062] Furthermore, a method for preparing a gel scaffold is provided, comprising:

[0063] A gel precursor system was obtained by mixing a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea in a preset ratio.

[0064] The gel precursor system was alkalized in a closed reaction vessel to obtain a gel with a high energy storage modulus.

[0065] The high-grade energy storage modulus gel was printed using a 3D printing device to obtain a scaffold structure;

[0066] The scaffold structure is subjected to light irradiation to induce cross-linking, thereby obtaining a gel scaffold.

[0067] In some embodiments, the polymer having phenylboronic acid groups and photocurable reactive groups is one or more of the following: phenylboronic acid-modified methacrylamide hyaluronic acid (HAMA-PBA), phenylboronic acid-modified methacrylamide gelatin (GelMA-PBA), phenylboronic acid-modified methacrylamide silk fibroin (SilMA-PBA), and phenylboronic acid-modified methacrylamide alginate (AlgMA-PBA). In some preferred embodiments, the polymer having phenylboronic acid groups and photocurable reactive groups is phenylboronic acid-modified methacrylamide hyaluronic acid (HAMA-PBA); the polymer containing vicinal diol groups is polyvinyl alcohol (PVA), and the molecular weight Mw of the polyvinyl alcohol is approximately 31,000; the concentration of HCl in the urea-containing hydrochloric acid solution is 10 mM to 20 mM, and the concentration of urea is 0.2 to 0.8 g / mL; the photoinitiator is LAP.

[0068] In the method for preparing gel-based biomaterials based on rheological programming of this invention, the gel precursor system is a polymer solution containing phenylboronic acid groups and photocurable reactive groups and a polymer solution containing vicinal diol groups, which forms a gel precursor system with good fluidity. Under the action of hydrochloric acid solution containing urea, reversible cross-linking is achieved by heating and alkalization to obtain a first network with a preset level of energy storage modulus. Subsequently, after photocuring, a covalent second network is formed, resulting in a double-network hydrogel with optimal shape retention.

[0069] The phenylboronic acid-modified methacrylic acid hyaluronic acid (HAMA-PBA) and polyvinyl alcohol (PVA) dual-network hydrogel is a hydrogel with certain adhesive and mechanical properties based on the primary network constructed by dynamic borate ester bonds and the secondary network constructed by photocrosslinking. However, the HAMA-PBA+PVA gel system has high reactivity, making it difficult to achieve injectability, self-healing and specific tissue repair and application and molding requirements respectively. Based on this, the present invention further optimizes the polymer with phenylboronic acid groups and photocurable reactive groups as phenylboronic acid-modified methacryloyl hyaluronic acid (HAMA-PBA). Based on the above-mentioned urea-containing hydrochloric acid solution to control the fluidity and processability of the gel precursor, controllable crosslinking of the gel precursor is achieved to obtain gels with different levels of storage modulus.

[0070] The role of the urea-containing hydrochloric acid solution is to inhibit premature complete cross-linking before the formation of the first network, so that the gel precursor system remains flowable and processable before the formation of the first network. During the alkalization process, urea decomposes, the pH of the reaction system is adjusted upward, and the formation of the first network is promoted.

[0071] In some embodiments, the alkalization temperature is 40~80°C, preferably 55~65°C, and more preferably 60°C.

[0072] The gel precursor system is heated and alkalized to form a first network. By controlling the alkalization time, a gel with a preset energy storage modulus is obtained. This invention achieves reversible cross-linking of the precursor under the control of a urea-containing hydrochloric acid solution. By controlling the energy storage modulus in advance and coordinating with subsequent photocuring, compared with photocuring only or photocuring before reversible cross-linking, it can realize the customized generation of biogel inks on the same platform, meeting the different needs of various applications such as infusion, injection, or printing.

[0073] In some embodiments, the mass ratio of the photoinitiator to the polymer having phenylboronic acid groups and photocurable reactive groups is 0.5~3:16~18.

[0074] In the preparation method of the present invention, the photoinitiator is added to the gel precursor system in advance, and photocuring is carried out after alkalization to the preset storage modulus, which effectively avoids premature photocuring from affecting molding processes such as pouring, injection or printing.

[0075] In some embodiments, the wavelength of the light under the illumination conditions is 365 nm, and the light intensity is 5~20 mW / cm². 2 The illumination time is 30~300 s.

[0076] In some preferred embodiments, when the preset storage modulus of the gel is a low-level storage modulus, the mass ratio of PVA to HAMA-PBA in the gel precursor system is 3:8, the alkalization temperature is 60°C, and the alkalization time is 18~24h, preferably 18h; when the preset storage modulus of the gel is a medium-level storage modulus, the mass ratio of PVA to HAMA-PBA in the gel precursor system is 3:10, the alkalization temperature is 60°C, and the alkalization time is 4~18h, preferably 18h; when the preset storage modulus of the gel is a high-level storage modulus, the mass ratio of PVA to HAMA-PBA in the gel precursor system is 3:12, the alkalization temperature is 60°C, and the alkalization time is 6~18h, preferably 18h.

[0077] In some embodiments, the gel precursor system further includes a nanomedicine or a drug delivery system, wherein the nanomedicine includes one or more of resveratrol, curcumin, epigallocatechin gallate, insulin, and norfloxacin; and the drug delivery system is obtained by encapsulating or loading the nanomedicine onto a nanocarrier, wherein the nanocarrier includes hydrogenated soybean phosphatidylcholine (HSPC) liposomes and / or phospholipid-polyethylene glycol (DSPE-PEG) micelles.

[0078] In some specific embodiments, a gel precursor system is obtained by mixing a polymer having phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a urea-containing hydrochloric acid solution according to a preset ratio, comprising:

[0079] A polymer containing phenylboronic acid groups and photocurable reactive groups is dissolved in a urea-containing hydrochloric acid solution to obtain a polymer solution containing phenylboronic acid groups and photocurable reactive groups; the concentration of the polymer containing phenylboronic acid groups and photocurable reactive groups in the polymer solution is 4~20 wt%.

[0080] The polymer solution containing phenylboronic acid groups and photocurable reactive groups is mixed with a polymer solution containing vicinal diol groups, and a photoinitiator is added to obtain a gel precursor system; or, the photoinitiator is added to the polymer solution containing phenylboronic acid groups and photocurable reactive groups and the polymer solution containing vicinal diol groups respectively, and mixed to obtain a gel precursor system.

[0081] Preferably, the photoinitiator LAP is added in advance to the hyaluronic acid derivative solution and / or the mixed gel precursor system, but the 365 nm light irradiation step is performed after alkalization is completed or the target storage modulus is reached, so as to avoid premature photocuring affecting infusion, injection or printing.

[0082] In some embodiments, the method further includes molding, which includes microneedle molding or three-dimensional molding; when the gel is a low-level energy storage modulus gel, the alkalization is performed after placing the gel precursor system in a molding die to achieve microneedle formation; when the gel is a high-level energy storage modulus gel, the gel is used as a direct-write printing material for direct-write printing, followed by photocuring. Medium-level energy storage modulus gels do not require additional molding steps and can be applied directly after photocuring.

[0083] The third aspect of this application provides an application of gel-based biomaterials obtained by the above-described method for preparing gel-based biomaterials based on rheological programming in the preparation of medical materials. The gel-based biomaterials include at least one of gel microneedles, biological scaffolds, and injectable gels. The medical materials are used for the repair of oxidative stress-related tissues, with preferred application scenarios including the repair of chronic diabetic wounds and bone defects.

[0084] Prior to the application for this invention, a series of experiments were conducted. Some of the experimental results are listed below to provide a more detailed description of the invention. The following is a detailed description in conjunction with the embodiments.

[0085] Example 1

[0086] This embodiment provides a method for preparing gel-based biomaterials based on rheological programming, including:

[0087] Step 1: Preparation of hyaluronic acid derivative solution: Dissolve HAMA-PBA in a urea-containing hydrochloric acid solution, add LAP and mix well to obtain a HAMA-PBA precursor solution; the concentration of HCl in the urea-containing hydrochloric acid solution is 10 mM, and the concentration of urea is 0.3 g / ml; the mass ratio of LAP to HAMA-PBA is 1:16; the concentration of HAMA-PBA in the HAMA-PBA precursor solution is 4 wt%.

[0088] Step 2: Prepare PVA solution: Provide a 3 wt% PVA aqueous solution; the molecular weight Mw of the PVA is approximately 31,000;

[0089] Step 3: Mixing to obtain the gel precursor system: Mix the PVA solution and the HAMA-PBA precursor solution at a volume ratio of 1:2 to obtain a gel precursor system with good flowability;

[0090] Step 4, Injection Molding: The gel precursor system is placed in a container and heated at 60°C for a preset time to allow the system to complete alkalization and form a first network, obtaining a gel with the target storage modulus. When the heating time is 18 h, the storage modulus of the gel is 320 Pa; when the heating time is 28 h, the storage modulus of the gel is 430 Pa; when the heating time is 36 h, the storage modulus of the gel is 620 Pa. When the target storage modulus is low, the container is a microneedle mold; when the target storage modulus is medium or high, the container is a closed reaction vessel that can hold the reaction raw materials.

[0091] Step 5, Secondary photocuring: Apply 365 nm light with an intensity of 20 mW / cm² to the gel. 2 Irradiation with light causes LAP to initiate secondary cross-linking of methacryloyl groups on HAMA to form a second network, resulting in gel-based biomaterials.

[0092] The preparation method of HAMA-PBA described in this embodiment includes:

[0093] Step 101: Dissolve sodium hyaluronate (4 g, molecular weight 5 kDa) completely in 100 mL of deionized water to obtain a sodium hyaluronate solution. Under ice bath conditions (0~4℃), add 1.6 mL of methacrylic anhydride (MA) to the above sodium hyaluronate solution at one time to obtain a mixed solution.

[0094] Step 102: Add 3.2 M NaOH (5 mol) solution dropwise to the above mixed solution using a syringe pump. The dropwise addition time shall not be less than 10 min, and the reaction shall be carried out under ice bath conditions for 4 hours.

[0095] Step 103: After the reaction is complete, the reaction system is placed in a dialysis bag (MWCO 3500) to remove small molecules. The dialysis solution is then freeze-dried to obtain a white solid HAMA.

[0096] Step 104: Dissolve 5.3 g HAMA in 530 mL PBS (pH 7.4), add 1.9981 g 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM) as a condensing agent, and stir for 30 minutes to activate the carboxyl group; the CAS number of DMTMM is 3945-69-5;

[0097] Step 105: Add 265 mL of 3-aminomethylphenylboronic acid (1.2667 g) aqueous solution to the activated system, adjust the pH to 5.0~6.0, and react at room temperature for 16 hours. After the reaction is complete, add the reaction system to a dialysis bag (MWCO 3500) to remove small molecules. Freeze-dry the dialysis solution to obtain a white solid HAMA-PBA.

[0098] Example 2

[0099] This embodiment provides a method for preparing gel-based biomaterials based on rheological programming, including:

[0100] Step 1: Preparation of hyaluronic acid derivative solution: Dissolve HAMA-PBA in a urea-containing hydrochloric acid solution, add LAP and mix well to obtain a HAMA-PBA precursor solution; the concentration of HCl in the urea-containing hydrochloric acid solution is 10 mM, and the concentration of urea is 0.3 g / mL; the mass ratio of LAP to HAMA-PBA is 1:16; the concentration of HAMA-PBA in the HAMA-PBA precursor solution is 4 wt%.

[0101] Step 2: Prepare PVA solution: Provide a 3 wt% PVA aqueous solution; the molecular weight Mw of the PVA is approximately 31,000;

[0102] Step 3: Mixing to obtain the gel precursor system: Mix the PVA solution and the HAMA-PBA precursor solution at a volume ratio of 1:2 to obtain a gel precursor system with good flowability;

[0103] Step 4, Infusion Molding: The gel precursor system is infused into the microneedle mold cavity, and necessary defoaming / negative pressure assisted filling is performed; the microneedle mold specifications and dimensions are 15*15 array, needle height 800 µm, patch size 13.2*13.2 mm;

[0104] Step 5: Trigger the first network: Place the entire mold after injection in a sealed environment and heat it at 60°C for 18 hours to allow the system to complete alkalization and form the first network, thereby obtaining a gel with the target energy storage modulus of 320 Pa.

[0105] Step Six, Secondary Photocuring: After demolding the above gel, and after alkalization, apply light at 365 nm with an intensity of 20 mW / cm² to the system. 2 Irradiation for 90 seconds causes LAP to initiate secondary cross-linking of methacryloyl groups on HAMA to form a second network, thereby improving needle strength and shape retention.

[0106] Step 7, Characterization: Evaluate the integrity, consistency, and shape retention of the microneedle tip.

[0107] Comparative Example 1

[0108] This comparative study investigates the effect of urea on the properties of the gel-based biomaterial and provides a method for preparing gel-based biomaterials, which is the same as in Example 2, except that step one is: preparing a hyaluronic acid derivative solution: dissolving HAMA-PBA in hydrochloric acid solution, adding LAP and mixing well to obtain a HAMA-PBA precursor solution; the concentration of HCl in the hydrochloric acid solution is 10 mM; the mass ratio of LAP to HAMA-PBA is 1:16.

[0109] Example 3

[0110] This embodiment provides a method for preparing gel-based biomaterials based on rheological programming, including:

[0111] Step 1: Prepare the gel precursor system according to steps 1-3 of Example 2, wherein the concentration of PVA solution is 3 wt% and the concentration of HAMA-PBA in the HAMA-PBA precursor solution is 5 wt%.

[0112] Step 2: Place the gel precursor system in a sealed reaction vessel and heat it at 60°C for 18 h to complete alkalization and form a first network, thereby obtaining a medium-grade storage modulus gel; the medium-grade storage modulus is 450 Pa.

[0113] Step 3: Immediately place the gel in a 365 nm light source with an intensity of 20 mW / cm². 2 Expose the gel to light for 60 seconds to allow it to solidify and form a second network, thereby enhancing its shape retention and resistance to deformation.

[0114] Step 4: Evaluate its injectability, gel stability, and retention for defect filling.

[0115] Comparative Example 2

[0116] This comparative study investigates the effect of urea on the properties of the gel-based biomaterial and provides a method for preparing gel-based biomaterials, which is the same as in Example 3, except that step one is: preparing a hyaluronic acid derivative solution: dissolving HAMA-PBA in hydrochloric acid solution, adding LAP and mixing well to obtain a HAMA-PBA precursor solution; the concentration of HCl in the hydrochloric acid solution is 10 mM; the mass ratio of LAP to HAMA-PBA is 1:16.

[0117] Example 4

[0118] This embodiment provides a method for preparing gel-based biomaterials based on rheological programming, including:

[0119] Step 1: Prepare the gel precursor system according to steps 1-3 of Example 2, wherein the concentration of PVA solution is 3 wt% and the concentration of HAMA-PBA in the HAMA-PBA precursor solution is 6 wt%.

[0120] Step 2: Place the gel precursor system in a sealed reaction vessel and heat it at 60°C for 18 h to complete alkalization and form a first network, thereby obtaining a gel with a high-level energy storage modulus; the high-level energy storage modulus is 650 Pa.

[0121] Step 3: The gel is loaded into a direct-write 3D printing device for printing to obtain a porous grid support structure; the nozzle model is 22 G, the air pressure is 100 kPa, the printing speed is 6 mm / s, the layer height is 0.30 mm, the line spacing is 0.60 mm, the printing platform temperature is room temperature, and the printing path is an alternating 0° / 90° grid structure.

[0122] Step 4: Place the scaffold structure under light at 365 nm and an intensity of 20 mW / cm. 2 The scaffold is subjected to 60 seconds of light exposure to allow it to solidify and form a second network, thereby improving interlayer stability and long-term structural retention, resulting in a 3D scaffold.

[0123] Step 5: Evaluate structural stability indicators such as print continuity, collapse height / line width retention, and compression springback.

[0124] Comparative Example 3

[0125] This comparative study investigates the effect of urea on the properties of the gel-based biomaterial and provides a method for preparing gel-based biomaterials, which is the same as in Example 4, except that step one is: preparing a hyaluronic acid derivative solution: dissolving HAMA-PBA in hydrochloric acid solution, adding LAP and mixing well to obtain a HAMA-PBA precursor solution; the concentration of HCl in the hydrochloric acid solution is 10 mM; the mass ratio of LAP to HAMA-PBA is 1:16.

[0126] Example 5

[0127] This embodiment provides a method for preparing gel-based biomaterials based on rheological programming, including:

[0128] Step 1: Dissolve GelMA-PBA in a urea-containing hydrochloric acid solution, add LAP and mix well to obtain a GelMA-PBA precursor solution; the concentration of HCl in the urea-containing hydrochloric acid solution is 10 mM, and the concentration of urea is 0.4 g / ml; the mass ratio of LAP to GelMA-PBA is 1:12; the concentration of GelMA-PBA in the GelMA-PBA precursor solution is 10 wt%.

[0129] Step 2: Prepare PVA solution: Provide a 3 wt% PVA aqueous solution; the molecular weight Mw of the PVA is approximately 31,000;

[0130] Step 3: Mixing to obtain the gel precursor system: Mix the PVA solution and the GelMA-PBA precursor solution at a volume ratio of 1:2 to obtain a gel precursor system with good flowability;

[0131] Step 4: Place the gel precursor system in a sealed reaction vessel and heat it at 60°C for 20 h to complete alkalization and form a first network, thereby obtaining a medium-grade storage modulus gel; the medium-grade storage modulus is 462 Pa.

[0132] Step 5: Immediately place the gel under light at 365 nm and an intensity of 20 mW / cm². 2 Irradiation under light for 60 seconds allows for secondary curing, forming a second network to enhance gel shape retention and resistance to deformation, resulting in an injectable gel.

[0133] This embodiment provides a dual-network gel preparation method based on GelMA-PBA+PVA, which can achieve programmable transformation of the gel precursor between the processable state, the target energy storage modulus state and the final cured state through rheological programming, and can be adapted to different application requirements such as casting molding, injection molding or 3D printing.

[0134] Example 6

[0135] This embodiment provides a method for preparing injectable gels based on rheological programming, including:

[0136] Step 1: Dissolve SilMA-PBA in a urea-containing hydrochloric acid solution, add LAP and mix well to obtain a SilMA-PBA precursor solution; the concentration of HCl in the urea-containing hydrochloric acid solution is 10 mM, and the concentration of urea is 0.4 g / ml; the mass ratio of LAP to SilMA-PBA is 1:18; the concentration of SilMA-PBA in the SilMA-PBA precursor solution is 20 wt%.

[0137] Step 2: Prepare PVA solution: Provide a 3 wt% PVA aqueous solution; the molecular weight Mw of the PVA is approximately 31,000;

[0138] Step 3: Mixing to obtain the gel precursor system: Mix the PVA solution and the SilMA-PBA precursor solution at a volume ratio of 1:2 to obtain a gel precursor system with good flowability;

[0139] Step 4: Place the gel precursor system in a sealed reaction vessel and heat at 60 °C for 22 h to complete alkalization and form the first network, obtaining a medium-grade storage modulus gel; the medium-grade storage modulus is 482 Pa.

[0140] Step 5: Immediately place the gel under light at 365 nm and an intensity of 20 mW / cm². 2 Irradiation under light for 60 seconds allows for secondary curing, forming a second network to enhance gel shape retention and resistance to deformation, resulting in an injectable gel.

[0141] Example 7

[0142] This embodiment provides a method for preparing injectable gels based on rheological programming, including:

[0143] Step 1: Dissolve AlgMA-PBA in a urea-containing hydrochloric acid solution, add LAP and mix well to obtain an AlgMA-PBA precursor solution; the concentration of HCl in the urea-containing hydrochloric acid solution is 10 mM, and the concentration of urea is 0.4 g / ml; the mass ratio of LAP to AlgMA-PBA is 1:18; the concentration of AlgMA-PBA in the AlgMA-PBA precursor solution is 4 wt%.

[0144] Step 2: Prepare PVA solution: Provide a 3 wt% PVA aqueous solution; the molecular weight Mw of the PVA is approximately 31,000;

[0145] Step 3: Mixing to obtain the gel precursor system: Mix the PVA solution and the AlgMA-PBA precursor solution at a volume ratio of 1:2 to obtain a gel precursor system with good flowability;

[0146] Step 4: Place the gel precursor system in a sealed reaction vessel and heat it at 60 °C for 20 h to complete alkalization and form the first network, thereby obtaining a gel with a medium-grade storage modulus; the medium-grade storage modulus is 470 Pa.

[0147] Step 5: Immediately place the gel under light at 365 nm and an intensity of 20 mW / cm². 2 Irradiation under light for 60 seconds allows for secondary curing, forming a second network to enhance gel shape retention and resistance to deformation, resulting in an injectable gel.

[0148] Example 8

[0149] This embodiment provides a method for preparing drug-loaded gel-based biomaterials, the process of which is illustrated below. Figure 1 As shown, the preparation method is the same as that in Examples 2-7 or Comparative Examples 1-3, except that in step one, when preparing the hyaluronic acid derivative solution, the drug is added at the same time as LAP, and the mass percentage of the drug in the resulting precursor solution is 0.1%; the drug is norfloxacin or resveratrol.

[0150] We recorded changes in the gel network and drug release behavior in simulated ROS-increased environments and different pH buffer systems to verify its ability to release drugs in response to oxidative stress-related microenvironments.

[0151] Performance Evaluation

[0152] Figure 2 The diagram illustrates the pH change of the HAMA-PBA precursor solution when the urea concentration is varied. The preparation methods for each sample are the same as step one of Example 2, except that the urea concentration in the urea-containing hydrochloric acid solution is 0–8 g / mL. The corresponding HAMA-PBA precursor solutions were used as samples and heated at 60°C. It can be seen that when the HAMA-PBA precursor solution does not contain urea and is only a hydrochloric acid solution, the pH of the system does not change with increasing heating time. When the urea concentration in the urea-containing hydrochloric acid solution is 0.2 g / mL–0.8 g / mL, the pH of the system increases with increasing heating time, showing a change from acidic to alkaline. This indicates that the urea-containing hydrochloric acid solution plays a role in regulating the pH of the system during the reaction, and the rate of pH change accelerates with increasing urea concentration in the urea-containing hydrochloric acid solution.

[0153] Figure 3The diagram shows the storage modulus of the gels in Examples 2-4 and Comparative Examples 1-3. Figure A corresponds to Example 2 and Comparative Example 1, where the precursor system is 4% HAMA-PBA + 3% PVA; Figure B corresponds to Example 3 and Comparative Example 2, where the precursor system is 5% HAMA-PBA + 3% PVA; Figure C corresponds to Example 4 and Comparative Example 3, where the precursor system is 6% HAMA-PBA + 3% PVA; and Figure D is a summary diagram of the storage modulus of the gels in Examples 2-4 and Comparative Examples 1-3. It can be seen that the gels obtained by alkalization treatment in a urea-containing hydrochloric acid solution using the present invention have significantly improved storage modulus, and this improvement is universal in all precursor solution systems.

[0154] Figure 4 This is a schematic diagram of the microneedle morphology in Example 2. It can be seen that the needle tip is complete, the needle body structure is clear, and the uniformity is high. Figure 5 This is a schematic diagram of the morphology of the injectable gel after injection molding in Example 3. It can be seen that it has the characteristics of being injectable and capable of in-situ molding, with stable gel formation and high shape retention. Figure 6 The image shows the morphology of the gel obtained in Example 4. It can be seen that it can be printed continuously and can be used for direct-write 3D printing, which can meet the requirements of printing continuity and structural preservation in 3D printing.

[0155] Figure 7 This is a schematic diagram of the cumulative drug release curve of the drug-loaded gel-based biomaterial in Example 8 under ROS / pH stimulation. The gel-based biomaterial is an injectable gel (HAMA-PBA+PVA). The test method includes: placing 1 g of the drug-loaded gel-based biomaterial in 10 mL of the corresponding buffer solution, placing it in a shaker at 37°C, taking a 1 mL sample, and adding 1 mL of fresh medium. The formula for calculating the cumulative release rate is: Q n =(C n *V0+(C1+C2+C3+……+C n-1 )*V) / Q0. Where, Q n C is the cumulative drug release at the nth sampling point; n Vn is the concentration at the nth sampling point; V0 is the volume of the release medium; V is the sampling volume each time; Q0 is the drug loading. The results show that, in a near-normal physiological environment (pH 7.4), the cumulative release rate of the drug-loaded gel-based biomaterial of this invention is consistently lower than that of the untreated drug-loaded gel-based biomaterial. In an acidic microenvironment (pH 5.5), the drug-loaded gel-based biomaterial of this invention can sustain release for 70 hours with a cumulative release rate exceeding 80%, demonstrating excellent pH-responsive drug release. In a hydrogen peroxide environment, the cumulative release rate of the drug-loaded gel-based biomaterial of this invention is significantly higher than that of the comparative example, demonstrating excellent selective drug release under oxidative stress.

[0156] Figure 8This is a schematic diagram illustrating the effect of drug-loaded gel-based biomaterials on repairing diabetic wounds in Example 8. The method for constructing the diabetic wound model was as follows: Type 1 diabetes was established using 6-8 week old male C57BL / 6J mice. Streptozotocin (STZ) was administered intraperitoneally (30 mg / kg) for 7 consecutive days to induce diabetes. During and after modeling, mouse weight and fasting blood glucose levels were monitored regularly. When fasting blood glucose remained ≥16.7 mmol / L for 4 weeks after STZ injection, the diabetic model was considered successfully established and included in subsequent experiments. Before the experiment, the mice were anesthetized by intraperitoneal injection of 2.5% tribromoethanol (TBE, 200 mg / kg), their back hair was shaved, and routine disinfection was performed. Subsequently, a 10 mm diameter incision was prepared on the back skin. A full-thickness skin defect of mm was treated with resveratrol-loaded microneedles (HAMA-PBA+PVA). The wound was photographed and recorded at preset times to document the wound healing process. The results showed that the resveratrol microneedle group showed a significant reduction in wound area on the 7th day of repair, and the wound was almost completely healed on the 14th day. The drug-loaded gel-based material of this invention can effectively promote the healing of diabetic wounds and can be applied to oxidative stress-related diseases.

[0157] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing gel-based biomaterials based on rheological programming, characterized in that, include: A gel precursor system is obtained using a polymer having phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a urea-containing hydrochloric acid solution as raw materials; the polymer having phenylboronic acid groups and photocurable reactive groups is one or more of phenylboronic acid-modified methacrylamide hyaluronic acid, phenylboronic acid-modified methacrylamide gelatin, phenylboronic acid-modified methacrylamide silk fibroin, and phenylboronic acid-modified methacrylamide alginate; the polymer containing vicinal diol groups is polyvinyl alcohol; The gel precursor system is heated and alkalized, and the alkalization time is controlled to obtain a gel with a preset level of energy storage modulus; the preset level of energy storage modulus includes low-level energy storage modulus, medium-level energy storage modulus and high-level energy storage modulus; the energy storage modulus range of low-level energy storage modulus is 280~320 Pa, the energy storage modulus range of medium-level energy storage modulus is 430~490 Pa, and the energy storage modulus range of high-level energy storage modulus is 600~700 Pa; Light conditions are applied to a gel with a preset energy storage modulus to induce cross-linking, thereby obtaining a gel-based biomaterial.

2. The method for preparing gel-based biomaterials based on rheological programming control according to claim 1, characterized in that, The photoinitiator is LAP.

3. The method for preparing gel-based biomaterials based on rheological programming control according to claim 1, characterized in that, The concentration of HCl in the urea-containing hydrochloric acid solution is 10 mM to 20 mM, and the concentration of urea is 0.2 to 0.8 g / mL; and / or, the alkalization temperature is 40 to 80°C; the mass ratio of the photoinitiator to the polymer having phenylboronic acid groups and photocuring reactive groups is 0.5 to 3:16 to 18; and / or, the light wavelength of the illumination conditions is 365 nm, the light intensity is 5 to 20 mW / cm², and the illumination time is 30 to 300 s.

4. The method for preparing gel-based biomaterials based on rheological programming according to claim 1, characterized in that, The gel-based biomaterial includes gel microneedles, injectable gels, or gel scaffolds; when the gel-based biomaterial is a gel microneedle, the preset energy storage modulus is a low-level energy storage modulus; when the gel-based biomaterial is an injectable gel, the preset energy storage modulus is a medium-level energy storage modulus; when the gel-based biomaterial is a gel scaffold, the preset energy storage modulus is a high-level energy storage modulus.

5. The method for preparing gel-based biomaterials based on rheological programming control according to claim 4, characterized in that, The method further includes molding, which includes microneedle molding or three-dimensional molding; when the gel is a low-level energy storage modulus gel, the alkalization is performed after the gel precursor system is placed in the molding mold; when the gel is a high-level energy storage modulus gel, the gel is used as a direct-write printing material for direct-write printing, followed by photocuring.

6. The method for preparing gel-based biomaterials based on rheological programming control according to claim 1, characterized in that, Using polymers with phenylboronic acid groups and photocurable reactive groups, polymers containing vicinal diol groups, photoinitiators, and urea-containing hydrochloric acid solutions as raw materials, a gel precursor system was obtained comprising: A polymer containing phenylboronic acid groups and photocurable reactive groups is dissolved in a hydrochloric acid solution containing urea to obtain a polymer solution containing phenylboronic acid groups and photocurable reactive groups. The polymer solution containing phenylboronic acid groups and photocurable reactive groups is mixed with a polymer solution containing vicinal diol groups, and a photoinitiator is added to obtain a gel precursor system; or, the photoinitiator is added to the polymer solution containing phenylboronic acid groups and photocurable reactive groups and the polymer solution containing vicinal diol groups respectively, and mixed to obtain a gel precursor system. And / or, the gel precursor system further includes a nanomedicine or a drug delivery system, wherein the nanomedicine includes one or more of resveratrol, curcumin, epigallocatechin gallate, insulin, and norfloxacin; and the drug delivery system is obtained by encapsulating or loading the nanomedicine onto a nanocarrier, wherein the nanocarrier includes hydrogenated soybean phosphatidylcholine liposomes and / or phospholipid-polyethylene glycol micelles.

7. A method for preparing gel microneedles, characterized in that, include: A gel precursor system was obtained using a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea as raw materials. The gel precursor system was infused into a microneedle mold; The microneedle mold containing the gel precursor system was transferred into a sealed environment and heated to alkalize it, thus obtaining a low-grade energy storage modulus gel. Applying light to crosslink the gel microneedles; the low-level storage modulus ranges from 280 to 320 Pa; the polymer with phenylboronic acid groups and photocurable reactive groups is one or more of phenylboronic acid-modified methacryloyl hyaluronic acid, phenylboronic acid-modified methacryloyl gelatin, phenylboronic acid-modified methacryloyl silk fibroin, and phenylboronic acid-modified methacryloyl alginate; the polymer containing vicinal diol groups is polyvinyl alcohol.

8. A method for preparing an injectable gel, characterized in that, include: A gel precursor system was obtained by mixing a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea in a preset ratio. The gel precursor system was heated and alkalized in a closed reaction vessel to obtain a gel with a medium storage modulus. Light is applied to the medium-grade storage modulus gel to cause crosslinking, resulting in an injectable gel; the storage modulus of the medium-grade storage modulus ranges from 430 to 490 Pa; the polymer having phenylboronic acid groups and photocurable reactive groups is one or more of phenylboronic acid-modified methacryloyl hyaluronic acid, phenylboronic acid-modified methacryloyl gelatin, phenylboronic acid-modified methacryloyl silk fibroin, and phenylboronic acid-modified methacryloyl alginate; the polymer containing vicinal diol groups is polyvinyl alcohol.

9. A method for preparing a gel scaffold, characterized in that, include: A gel precursor system was obtained by mixing a polymer with phenylboronic acid groups and photocurable reactive groups, a polymer containing vicinal diol groups, a photoinitiator, and a hydrochloric acid solution containing urea in a preset ratio. The gel precursor system was heated and alkalized in a closed reaction vessel to obtain a gel with a high energy storage modulus. The high-grade energy storage modulus gel was printed using a 3D printing device to obtain a scaffold structure; Apply light to the scaffold structure to induce cross-linking, thereby obtaining a gel scaffold; The high-level energy storage modulus ranges from 600 to 700 Pa; the polymer with phenylboronic acid groups and photocurable reactive groups is one or more of the following: phenylboronic acid-modified methacryloyl hyaluronic acid, phenylboronic acid-modified methacryloyl gelatin, phenylboronic acid-modified methacryloyl silk fibroin, and phenylboronic acid-modified methacryloyl alginate; the polymer containing vicinal diol groups is polyvinyl alcohol.

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