Biomimetic immune regulation double-network hydrogel, preparation method and application thereof

Abstract

The application discloses a kind of bionic immune regulation double-network hydrogel and its preparation method and application, its preparation method is: GelMA-SPD and HAMA-NB are dissolved, join photo-crosslinking agent, crosslinking is carried out under light, obtain double-network hydrogel;GelMA-SPD is methacrylated gelatin-spermidine;HAMA-NB is methacrylated hyaluronic acid N-(2-aminoethyl)-4-[4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy]-butyramide.The hydrogel prepared in the application has good biocompatibility, and the preparation process is simple, and can be degraded.The hydrogel of the application can be applied to tissue repair material or tissue engineering scaffold, and is expected to solve the immune rejection problem in the process of tissue organ transplantation, and is conducive to reducing inflammatory reaction and promoting the rapid healing of tissue trauma.

Description

A biomimetic immune-regulating dual-network hydrogel, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical materials and related medical device research and development technology, and more specifically to a biomimetic immune-regulating hydrogel, its preparation method and application. Background Technology

[0002] Globally, hundreds of millions of people face various forms of organ and tissue damage each year, and with the increasing aging population, the number of patients suffering from chronic degenerative diseases is also rising. The spinal cord and brain tissue play crucial roles in nerve signal transmission and are key to normal physiological functions. Spinal cord injury (SCI) is a severe central nervous system traumatic disease. Injury to the spinal cord causes temporary or permanent loss of motor function, severely reducing the patient's quality of life. SCI patients not only face daily life obstacles caused by neurological dysfunction but also bear the burden of expensive treatments and endure immense psychological distress. Spinal cord injury is one of the most disabling and destructive neurological diseases. Due to the poor plasticity of the central nervous system and limited neuronal regeneration capacity, regeneration and repair after spinal cord injury are extremely weak.

[0003] Currently, clinical treatment for spinal cord injury is limited to surgical decompression, hormone pulse therapy, and neuroprotective measures, with minimal clinical efficacy. Patients mainly maintain residual neurological function through post-injury rehabilitation training, and there are no effective clinical treatments to significantly restore the neurological function lost after spinal cord injury. Spinal cord injury repair and functional reconstruction have become a major global medical challenge and a significant challenge facing the global health field.

[0004] Medical hydrogels, as a novel tissue engineering scaffold, have a three-dimensional network structure and contain a large amount of water, which can provide a suitable microenvironment for cell proliferation; at the same time, they can limit local inflammatory responses, inhibit cell apoptosis, and promote tissue repair and regeneration.

[0005] After tissue injury, its local microenvironment undergoes significant changes, transforming into a severely inflammatory microenvironment, which further exacerbates the loss of extracellular matrix and worsens the damage. Furthermore, local inflammation promotes the formation of glial scars, hindering axonal regeneration. Therefore, biomaterial scaffolds capable of modulating the local microenvironment offer hope for the repair and regeneration of spinal cord injuries. Currently, hyaluronic acid hydrogels, PLGA hydrogels, HEMA hydrogels, nanofibers, and self-assembled peptide hydrogels can structurally support cell migration and axonal regeneration. However, most hydrogels differ significantly from the autologous environment, failing to adequately meet the conditions for nerve regeneration, and exhibiting significant rejection in vivo, causing greater tissue damage. Cell-adaptive dynamic hydrogels can provide a better microenvironment for cell proliferation, differentiation, and the recruitment of immune cells. Therefore, developing a cell-adaptive, anti-rejection dual-network hydrogel can not only improve mechanical properties but also provide a favorable microenvironment for cell proliferation and differentiation, facilitating tissue repair and regeneration.

[0006] Therefore, how to provide a safe and immunomodulatory dual-network hydrogel is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] Therefore, this invention provides a method for preparing a biomimetic immune-regulating dual-network hydrogel. The hydrogel prepared using hyaluronic acid, gelatin, and spermidine exhibits good biocompatibility and immune-regulating effects. The hydrogel prepared by this invention has a simple preparation process, is biodegradable, and shows promising application prospects.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a biomimetic immune-regulating dual-network hydrogel involves dissolving GelMA-SPD and HAMA-NB, adding a photocrosslinking agent, and crosslinking under light irradiation to obtain a dual-network hydrogel; wherein GelMA-SPD is methacrylated gelatin-spermidine; and HAMA-NB is methacrylated hyaluronic acid-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide.

[0010] The hydrogel of this invention is based on a UV-curable dual-network structure design. Spermine is introduced into the dual-network hydrogel through dynamic covalent bonds. As the hydrogel degrades, spermine is released slowly, exerting a long-term biological effect. Under UV light, NB generates aldehyde groups, which then react with amino groups to form a primary network through a Schiff base reaction; a secondary network is formed through a free radical polymerization reaction of carbon-carbon double bonds. The two networks combine to form a dual-network hydrogel.

[0011] The Schiff base reaction and free radical polymerization reaction involved in this invention have the advantages of rapid reaction and mild reaction conditions. The preparation method of the hydrogel is simple, efficient, convenient, and easy to scale up, and has broad application prospects in the field of biomedical hydrogels. Furthermore, gelatin, hyaluronic acid, and spermidine are all endogenous biological substances, and their degradation products are non-toxic and easily excreted from the body. The dual-network hydrogel prepared by this invention can regulate immune responses, reduce inflammation, alleviate rejection reactions, and can be used as a tissue repair scaffold to promote tissue repair and regeneration.

[0012] Preferably, the preparation method of GelMA is as follows: gelatin is dissolved in a phosphate buffer solution, the pH is adjusted to 7.4–11.0, heated to 50–70°C until fully dissolved, then methacrylic anhydride is added, the reaction is stirred for 6–24 h, dialyzed for 48–96 h, and freeze-dried for 48–96 h to obtain methacrylated gelatin (GelMA); the concentration of gelatin in the phosphate buffer solution is 1 wt%–10 wt%, and the molar ratio of gelatin to methacrylic anhydride is 1:

[0013] (2~5): (2~5): (1~10).

[0014] Preferably, the preparation method of GelMA-SPD is as follows: GelMA is dissolved in distilled water at a concentration of 1wt% to 10wt%, 1-ethyl-3-(3-dimethylaminopropyl)carbodiammonium hydrochloride (EDC), N-hydroxysuccinimide (NHS), and spermidine (SPD) are added, with the molar ratio of EDC, NHS, GelMA, and SPD being 1:(1 to 10). The mixture is stirred for 6 to 72 hours, dialyzed for 48 to 96 hours, and freeze-dried for 48 to 96 hours to obtain GelMA-SPD.

[0015] Preferably, the preparation method of HAMA is as follows: hyaluronic acid HA is dissolved in distilled water at a concentration of 0.1wt% to 5wt%, then methacrylic anhydride is added and stirred for 12 to 48 hours, the molar ratio of HA to methacrylic anhydride is 1:(1 to 10), dialyzed for 48 to 96 hours, and freeze-dried for 48 to 96 hours to obtain methacrylated hyaluronic acid HAMA.

[0016] Preferably, the preparation method of HAMA-NB is as follows: HAMA is dissolved in distilled water at a concentration of 0.1wt% to 5wt%, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC, N-hydroxysuccinimide NHS, and 4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide NB are added. The molar ratio of HAMA, EDC, NHS and NB is 1:(1-5):(1-5):(1-5). The reaction is carried out for 6-24 hours, dialyzed for 48-96 hours, and freeze-dried for 48-96 hours to obtain HAMA-NB.

[0017] Preferably, GelMA-SPD and HAMA-NB are dissolved in distilled water at a concentration of 1wt% to 20wt%, and a photocrosslinking agent is added. The molar ratio of GelMA-SPD, HAMA-NB, and the photocrosslinking agent is (1 to 10).

[0018] (0.1~10): (0.1~1) crosslinked under light to obtain a double network hydrogel.

[0019] Preferably, the dialysis parameters during the preparation of GelMA-SPD and HAMA-NB are as follows: dialysis bags of 3kDa to 20kDa are used, then the bags are frozen in an environment of -10℃ to -80℃, and then dried in a freeze dryer.

[0020] Preferably, the photocrosslinking agent is selected from any one of I2959, LAP, TPO, MBP, benzophenone, thiopropoxythionone, benzoin, and fluorinated diphenyltitanium oxide, and the illumination conditions are: wavelength 250-800nm, power 2W-30W, and photocrosslinking time 5-30s.

[0021] The advantages of adopting the above technical solution are: the reaction is carried out at low temperature, the reaction conditions are mild, and the damage to gelatin is minimized.

[0022] This invention also provides an application of a biomimetic immune-regulating dual-network hydrogel in tissue repair materials or tissue engineering scaffolds.

[0023] The beneficial effects of this invention are as follows: This invention dissolves two polymeric materials in distilled water and prepares a double-network hydrogel through photocrosslinking. Furthermore, the slow release of spermidine through degradation reduces local concentration and mitigates toxic side effects, resulting in excellent bioactivity, biocompatibility, and biodegradability. Simultaneously, the chemical reaction involved in this invention has advantages such as rapid reaction speed and mild reaction conditions, making the hydrogel preparation method simple, efficient, convenient, and suitable for mass production, thus possessing broad application prospects in the field of biomedical hydrogels. The hydrogel of this invention can be applied to tissue repair materials or tissue engineering scaffolds, potentially solving the problem of immune rejection during tissue and organ transplantation, helping to reduce inflammatory responses, and promoting rapid healing of tissue wounds. Figure description:

[0024] Figure 1 shows the synthetic route of GelMA-SPD;

[0025] Figure 2 shows the synthetic route of HAMA-NB;

[0026] Figure 3 shows the preparation route of the biomimetic immune-regulating dual-network hydrogel;

[0027] Figure 4 shows the NMR spectra of GelMA-SPD and HAMA-NB;

[0028] Figure 5 shows a physical image of the biomimetic immune regulation dual-network hydrogel.

[0029] Figure 6 shows the SEM image of the biomimetic immune-regulating dual-network hydrogel.

[0030] Figure 7 is a statistical graph of cell activity of the endothelial cells (HUVECs) of the present invention in the biomimetic immune regulation dual-network hydrogel extract;

[0031] Figure 8 shows the migration rate of the endothelial cells (HUVECs) of the present invention on the surface of the biomimetic immune-regulating dual-network hydrogel;

[0032] Figure 9 shows the tube formation rate of the endothelial cells (HUVECs) of the present invention on the surface of the biomimetic immune-regulated dual-network hydrogel;

[0033] Figure 10 shows the wound repair of mouse skin damaged by the present invention at different time points on the surface of the biomimetic immune-regulating dual-network hydrogel.

[0034] Figure 11 shows the biocompatibility of the biomimetic immune-regulating dual-network hydrogel of the present invention after subcutaneous implantation. Detailed implementation method:

[0035] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings.

[0036] Example 1:

[0037] A method for preparing a biomimetic immune-regulating dual-network hydrogel includes the following steps:

[0038] S1: Dissolve 1 mol gelatin and 1 mol methacrylic anhydride in 100 mL phosphate solution, adjust the pH to 7.4, heat to 50 °C, react for 5 h under light-protected conditions, then add distilled water to terminate the reaction, dialyze for 72 h, and then freeze dry to obtain methacrylated gelatin (GelMA).

[0039] S2: Dissolve 1 mol GelMA in 100 mL of distilled water, add 2 mol 1-ethyl-3-(3-dimethylaminopropyl)carbodiammonium hydrochloride (EDC), 2 mol N-hydroxysuccinimide (NHS), and 1 mol spermidine (SPD), react at room temperature for 24 h, dialyze for 72 h, and freeze dry for 48 h to obtain GelMA-SPD;

[0040] S3: Dissolve 1 mol of hyaluronic acid and 1 mol of methacrylic anhydride in 100 mL of distilled water, react at room temperature for 24 h, dialyze for 48 h, and freeze dry for 48 h to obtain methacrylated hyaluronic acid (HAMA).

[0041] S4: 1 mol HAMA, 1 mol N-(2-aminoethyl)-4-(4-hydroxymethyl)-2-methoxy-5-nitrosoethoxy)butyramide (NB), 2 mol 1-ethyl-3-(3-dimethylaminopropyl)carbodiammonium hydrochloride (EDC), and 2 mol N-hydroxysuccinimide (NHS) were dissolved in 100 mL of distilled water, reacted at room temperature for 24 h, dialyzed for 48 h, and freeze-dried to obtain HAMA-NB;

[0042] S5: Mix and dissolve 0.1 mol LGelMA-SPD, 0.01 mol LHAMA-NB, and 0.001 mol LLAP in 10 mL of distilled water, stir and mix evenly at room temperature, let stand to degas, and crosslink under 405 nm blue light for 5 s to obtain hydrogel.

[0043] In steps S1-S4, the dialysis parameters are as follows: use a dialysis bag with a capacity of 3000 Da to 20 kDa for dialysis for 72 hours, and then freeze the bag at -10°C.

[0044] Example 2:

[0045] A method for preparing a biomimetic immune-regulating dual-network hydrogel includes the following steps:

[0046] S1: Dissolve 1 mol gelatin and 2 mol methacrylic anhydride in 100 mL phosphate solution, adjust the pH to 7.4, heat to 60 °C, react for 8 h under dark conditions, then add distilled water to terminate the reaction, dialyze for 96 h, and freeze dry to obtain methacrylated gelatin (GelMA).

[0047] S2: Dissolve 1 mol GelMA in 100 mL of distilled water, add 3 mol 1-ethyl-3-(3-dimethylaminopropyl)carbodiammonium hydrochloride (EDC), 3 mol N-hydroxysuccinimide (NHS), and 2 mol spermidine (SPD), react at room temperature for 48 h, dialyze for 96 h, and freeze dry for 72 h to obtain GelMA-SPD;

[0048] S3: Dissolve 1 mol of hyaluronic acid and 2 mol of methacrylic anhydride in 100 mL of distilled water, react at room temperature for 36 h, dialyze for 96 h, and freeze dry for 72 h to obtain methacrylated hyaluronic acid (HAMA).

[0049] S4: 1 mol HAMA, 3 mol N-(2-aminoethyl)-4-(4-hydroxymethyl)-2-methoxy-5-nitrosoethoxy)butyramide (NB), 3 mol 1-ethyl-3-(3-dimethylaminopropyl)carbodiammonium hydrochloride (EDC), and 3 mol N-hydroxysuccinimide (NHS) were dissolved in 100 mL of distilled water, reacted at room temperature for 72 h, dialyzed for 72 h, and freeze-dried to obtain HAMA-NB;

[0050] S5: Mix and dissolve 0.2 mol LGelMA-SPD, 0.1 mol LHAMA-NB, and 0.01 mol LLAP in 20 mL of distilled water, stir and mix evenly at room temperature, let stand to degas, and crosslink under 405 nm blue light for 10 s to obtain hydrogel.

[0051] In steps S1-S4, the dialysis parameters are as follows: use a dialysis bag with a capacity of 3000 Da to 20 kDa for dialysis for 72 hours, and then freeze the bag at -40°C.

[0052] Example 3:

[0053] A method for preparing a biomimetic immune-regulating dual-network hydrogel includes the following steps:

[0054] S1: Dissolve 1 mol gelatin and 2 mol methacrylic anhydride in 100 mL phosphate solution, adjust the pH to 11, heat to 70 °C, react for 12 h under light-protected conditions, then add distilled water to terminate the reaction, dialyze for 120 h, and freeze dry to obtain methacrylated gelatin (GelMA).

[0055] S2: Dissolve 1 mol GelMA in 100 mL of distilled water, add 5 mol 1-ethyl-3-(3-dimethylaminopropyl)carbodiammonium hydrochloride (EDC), 5 mol N-hydroxysuccinimide (NHS), and 10 mol spermidine (SPD), react at room temperature for 72 h, dialyze for 120 h, and freeze dry for 96 h to obtain GelMA-SPD;

[0056] S3: Dissolve 1 mol of hyaluronic acid and 5 mol of methacrylic anhydride in 200 mL of phosphate solution, adjust the pH to 11, react at room temperature for 48 h, dialyze for 120 h, and freeze dry for 120 h to obtain methacrylated hyaluronic acid (HAMA).

[0057] S4: 1 mol HAMA, 5 mol N-(2-aminoethyl)-4-(4-hydroxymethyl)-2-methoxy-5-nitrosoethoxy)butyramide (NB), 3 mol 1-ethyl-3-(3-dimethylaminopropyl)carbodiammonium hydrochloride (EDC), and 3 mol N-hydroxysuccinimide (NHS) were dissolved in 200 mL of distilled water and reacted at room temperature for 72 h. Then, the mixture was dialyzed for 120 h and freeze-dried for 96 h to obtain HAMA-NB.

[0058] S5: Mix and dissolve 0.1 mol LGelMA-SPD (10%), 0.11 mol LHAMA-NB, and 0.01 mol LLAP in 10 mL of distilled water, stir and mix evenly at room temperature, let stand to degas, and crosslink under 405 nm blue light for 60 s to obtain hydrogel.

[0059] In steps S1-S4, the dialysis parameters are as follows: use a dialysis bag with a capacity of 3000 Da to 20 kDa for dialysis for 120 hours, and then freeze the bag at -80°C.

[0060] Experimental example:

[0061] one, 1 HNMR analysis

[0062] Using deuterated water as a solvent, the GelMA-SPD and HAMA-NB prepared in Example 1 were tested using a Bruker 400 nuclear magnetic resonance spectrometer. The NMR spectra are shown in Figure 4. The spectral analysis confirmed the correctness of the chemical structures of the GelMA-SPD and HAMA-NB prepared in this invention.

[0063] II. Morphological Analysis

[0064] Figures 5 and 6 are the physical image and SEM image of the dual-network hydrogel prepared in Example 1, respectively. As shown in Figure 6, the dual-network hydrogel has a three-dimensional network structure inside.

[0065] III. Biocompatibility Testing

[0066] 1. In vitro cytotoxicity evaluation of hydrogels

[0067] The cytotoxic effect of the hydrogel on endothelial cells (HUVECs) was assessed using the CCK-8 assay. The dual-network hydrogel (1:1, 10%) prepared in Example 1 was co-cultured with cells for 2 h, followed by treatment with CCK-8 solution (10 μL, 5 mg / mL) for 4 h. The absorbance at 450 nm was measured, and its cytotoxicity was calculated by comparing it with untreated cells. As shown in Figure 7, the cell viability was above 90%, demonstrating that the dual-network hydrogel did not exhibit significant cytotoxicity against endothelial cells (HUVECs).

[0068] According to GB / T16886.5-2017, Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests, the experimental results show that the cell survival rate is not less than 70% and the cytotoxicity grade is I, indicating that the hydrogel has no cytotoxicity.

[0069] 2. Evaluation of the effect of hydrogels on cell migration

[0070] The double-network hydrogel prepared in Example 1 was spread on a culture dish, and then cells were seeded onto the culture dish and cultured for 24 hours. Then, cell scratches were gently made with a pipette, and the cells were placed in an incubator for culture. Cell migration was observed under an optical microscope (see Figure 8). The migration rate of the experimental group was twice that of the control group, indicating that the hydrogel can promote cell migration and is beneficial to wound healing.

[0071] 3. Evaluation of hydrogel-induced cell tube formation

[0072] The dual-network hydrogel prepared in Example 1 was coated on a cell culture plate, and then the cells were seeded on the culture plate and cultured in an incubator. The cell tube formation was then observed under an optical microscope (Figure 9). The hydrogel can promote cell tube formation, and there is no significant difference compared with the matrix gel, indicating that it has a good effect on cell tube formation.

[0073] 4. In vivo skin damage repair test

[0074] SD rats (8 weeks old, n=5) were general anesthetized and fixed on the operating table to create a full-thickness skin injury model. They were divided into a control group and an experimental group (using the double-network hydrogel prepared in Example 1). The wound repair was observed at different time points, as shown in Figure 10. After treatment with the hydrogel prepared in Example 1, the experimental group rats showed good wound recovery. This indicates that the hydrogel can promote skin wound repair. It also demonstrates that the hydrogel has a good ability to promote tissue repair.

[0075] 5. In vivo rejection test

[0076] The dual-network hydrogel prepared in Example 1 was implanted subcutaneously and showed good biocompatibility in vivo without significant fibrous capsule formation, while the control group showed significant fibrous capsule formation.

[0077] In summary, the prepared hydrogel exhibits good biocompatibility, promotes cell migration and tube formation, and facilitates tissue repair and regeneration, making it a promising biological scaffold.

[0078] The above description of the embodiments is provided to enable those skilled in the art to understand and apply the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the embodiments described herein, and any improvements and modifications made to the present invention by those skilled in the art based on the disclosure thereof should be within the scope of protection of the present invention.

Claims

1. A method for preparing a biomimetic immune-regulating dual-network hydrogel, characterized in that, S1: Dissolve 1 mol of gelatin and 1 mol of methacrylic anhydride in 100 mL of phosphate solution, adjust the pH to 7.4, heat to 50 °C, react for 5 h under light-protected conditions, then add distilled water to terminate the reaction, dialyze for 72 h, and then freeze-dry to obtain methacrylated gelatin (GelMA); S2: Dissolve 1 mol of GelMA in 100 mL of distilled water, add 2 mol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiamine hydrochloride (EDC), 2 mol of N-hydroxysuccinimide (NHS), and 1 mol of spermidine (SPD), react at room temperature for 24 h, dialyze for 72 h, and freeze-dry for 48 h to obtain GelMA-SPD; S3: Dissolve 1 mol of hyaluronic acid and 1 mol of methacrylic anhydride in 100 mL of distilled water, react at room temperature for 24 h, dialyze for 48 h, and freeze-dry for 48 h to obtain methacrylated hyaluronic acid (HAMA); S4: Dissolve 1 mol of HAMA, 1 2 mol N-(2-aminoethyl)-4-(4-hydroxymethyl)-2-methoxy-5-nitrosoethoxy)butyramide (NB), 2 mol 1-ethyl-3-(3-dimethylaminopropyl)carbodiammonium hydrochloride (EDC), and 2 mol N-hydroxysuccinimide (NHS) were dissolved in 100 mL of distilled water and reacted at room temperature for 24 h. Then, the mixture was dialyzed for 48 h and freeze-dried to obtain HAMA-NB. S5: 0.1 mol LGelMA-SPD, 0.01 mol HAMA-NB, and 0.001 mol LLAP were mixed and dissolved in 10 mL of distilled water. The mixture was stirred and mixed evenly at room temperature, allowed to stand to degas, and crosslinked under 405 nm blue light for 5 s to obtain a hydrogel. In steps S1-S4, the dialysis parameters were dialysis using a dialysis bag of 3000 Da~20 kDa for 72 h, and then the mixture was frozen at -10 ℃.

2. A biomimetic immune-regulating dual-network hydrogel prepared by the preparation method according to claim 1.

3. The application of the biomimetic immune-regulating dual-network hydrogel as described in claim 2 in the preparation of tissue repair materials or tissue engineering scaffolds.

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