Injectable hydrogel as well as preparation method and application thereof in neural restoration

Abstract

The invention discloses injectable hydrogel and a preparation method and application thereof in nerve repair, the hydrogel is medical hydrogel of poly-beta amino ester loaded with indole-3-propionic acid, and the hydrogel comprises the following raw materials: polyethylene glycol diacrylate (PEGDA) as a scaffold material; a molecule containing a double primary amine group as a cross-linking agent, the cross-linking agent being cross-linked with polyethylene glycol diacrylate (PEGDA) via an acrylate bond; and indole-3-propionic acid (IPA) as an active ingredient. The injectable hydrogel is adjustable in mechanical property and excellent in biocompatibility, can slowly release IPA, and is used for filling nerve injury areas, inhibiting oxidative stress and promoting neuron regeneration and functional recovery.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to an injectable hydrogel, a preparation method thereof, and an application thereof in nerve repair. Background Art

[0002] Spinal cord injury (SCI) is a serious central nervous system disease that often causes loss of motor, sensory, and visceral organ function, and can even cause dysfunction of the respiratory and intestinal regulatory systems, leading to permanent disability and damage, and placing a heavy economic and psychological burden. Current clinical treatments (such as surgery, medication, and rehabilitation training) have limited effectiveness. This is mainly because after spinal cord injury occurs, damage to neurons, glial cells, and blood vessels causes abnormal ion regulation and transduction of apoptotic signals, leading to neuronal apoptosis, inflammatory cell activation, overexpression of collagen fibrillary acidic protein, oxidative stress response, axonal damage, and glial scar formation. Over time, these secondary spinal cord injuries create a more unfavorable pathological microenvironment, which in turn aggravates spinal cord damage and further hinders nerve regeneration. There is an urgent need to develop new treatments to promote the repair of spinal cord injury.

[0003] Hydrogels are three-dimensional cross-linked polymer networks with a high water content. Because they can act as an extracellular matrix for neural tissue, they are widely used in tissue engineering and drug delivery. In existing technologies, the mechanical modulus of natural hydrogels is too soft to meet the modulus requirements of spinal cord tissue. While synthetic hydrogels can regulate their properties, they lack bioactive ingredients that promote nerve regeneration. Indole-3-propionic acid (IPA), a tryptophan metabolite, significantly enhances the regeneration of injured sciatic nerve axons after delivery to the injury site, promoting the recovery of sensory function.

[0004] Therefore, there is an urgent need to develop a hydrogel material that has mechanical adjustability, biocompatibility and sustained-release IPA function to promote nerve injury repair. Summary of the Invention

[0005] The purpose of the present invention is to provide an injectable hydrogel, a preparation method thereof, and an application in nerve repair. The injectable hydrogel has adjustable mechanical properties, excellent biocompatibility, and can slowly release IPA. The injectable hydrogel is used to fill nerve damage areas, inhibit oxidative stress, and promote neuronal regeneration and functional recovery.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an injectable hydrogel.

[0008] An injectable hydrogel, wherein the hydrogel is a medical hydrogel of poly-β-amino ester loaded with indole-3-propionic acid, and the raw materials thereof include:

[0009] polyethylene glycol diacrylate (PEGDA) as scaffold material;

[0010] A molecule containing a diprimary amine group as a crosslinker that crosslinks with polyethylene glycol diacrylate (PEGDA) via an acrylate bond;

[0011] Indole-3-propionic acid (IPA) as the active ingredient.

[0012] Preferably, the molecule containing a diprimary amine group is selected from at least one of lysine (Lys), arginine (Arg), cystamine or ethylenediamine.

[0013] Preferably, the swelling rate of the hydrogel after immersion in phosphate buffered saline (PBS) with a pH of 7.4 at 37° C. for 4 hours is 21.04±8.23% to 90.14±53.01%, and the residual mass after immersion for 28 days is 62.26±8.55% to 74.88±5.56% of the original mass.

[0014] Preferably, the Young's modulus of the hydrogel is 1.9-6.1 KPa, which is achieved by controlling the total molar concentration of polyethylene glycol diacrylate and the cross-linking agent in the reaction system to be within the range of 0.6-0.9 M.

[0015] Preferably, the hydrogel has a porous structure, and the cumulative release of indole-3-propionic acid (IPA) in phosphate buffer (PBS) with a pH of 7.4 at 37° C. in vitro for 72 hours is 33.51±0.52%.

[0016] Preferably, the hydrogel has shear thinning properties, with viscosity decreasing as shear rate increases, and can be delivered to the injury site via a syringe.

[0017] In a second aspect, the present invention provides a method for preparing an injectable hydrogel.

[0018] A method for preparing the injectable hydrogel comprises the following steps:

[0019] (a) adding indole-3-propionic acid (IPA) to triple-distilled water to obtain an indole-3-propionic acid (IPA) solution;

[0020] (b) Polyethylene glycol diacrylate (PEGDA) and the cross-linking agent were dissolved in triple-distilled water, mixed, and then indole-3-propionic acid (IPA) solution was added;

[0021] (c) the mixed solution obtained in step (b) is subjected to a Michael addition reaction to form a hydrogel loaded with indole-3-propionic acid (IPA);

[0022] (d) soaking the hydrogel obtained in step (c) in distilled water to fully remove unreacted small molecules.

[0023] Preferably, in step (a), sodium hydroxide is used to adjust the pH until the indole-3-propionic acid is completely dissolved. Preferably, the pH value is 5.0-7.4.

[0024] Preferably, in step (b), the total molar concentration of polyethylene glycol diacrylate (PEGDA) and the cross-linking agent is controlled to be 0.6-0.9 M to regulate the Young's modulus, swelling ratio and degradation rate of the hydrogel.

[0025] Preferably, in step (c), the reaction is carried out at 50-80° C. for 8-12 hours.

[0026] Preferably, in step (d), the soaking time is 24-48 hours.

[0027] In a third aspect, the present invention provides a use of the injectable hydrogel.

[0028] Application of the injectable hydrogel in preparing biomaterials for repairing spinal cord injuries.

[0029] Preferably, the hydrogel is injected into the spinal cord injury area to inhibit oxidative stress and cell apoptosis; promote axon regeneration and motor function recovery; and reduce the formation of glial scars.

[0030] Beneficial effects:

[0031] (1) The medical hydrogel of poly-β-amino ester loaded with indole-3-propionic acid in the embodiment of the present invention was successfully prepared by Michael addition reaction of polyethylene glycol diacrylate, lysine, arginine, cystamine, ethylenediamine and other molecules containing diprimary amino groups with indole-3-propionic acid. The mechanical properties of the drug-loaded hydrogel are adjustable, with a Young's modulus ranging from 1.9 to 6.1 kPa; the drug-loaded hydrogel has good hydrophilicity, swelling, and degradation properties; and the in vitro release of indole-3-propionic acid reaches 33.51±0.52% within 72 hours. The physical support of the hydrogel and the biological activity of IPA synergistically promote nerve regeneration after spinal cord injury and promote the regeneration of cells in the injured area.

[0032] (2) The indole-3-propionic acid-loaded hydrogel provided by the present invention not only has good blood and cell compatibility, but also can slowly release IPA to scavenge reactive oxygen species, significantly reducing cell apoptosis caused by oxidative stress. In vitro cell experiments showed that the 3.4 kPa drug-loaded hydrogel is most suitable for nerve cell adhesion and proliferation, and can reduce cell apoptosis caused by oxidative stress.

[0033] (3) The hydrogel provided in the embodiment of the present invention can promote behavioral recovery and the growth of new neurons after spinal cord injury in rats. H&E staining of viscera showed that it was non-toxic and suitable for long-term implantation.

[0034] (4) The medical hydrogel provided in the embodiment of the present invention has shear thinning properties and is therefore injectable, which is conducive to the delivery of the hydrogel through minimally invasive methods.

[0035] (5) The preparation method of the medical hydrogel provided in the embodiment of the present invention is simple and controllable. The degradation rate of the medical hydrogel can be controlled by changing the dosage ratio of the three raw materials, polyethylene glycol diacrylate, lysine / arginine (Arg) / cystamine / ethylenediamine and indole-3-propionic acid, or the concentration of the reaction system. The swelling properties, hydrophilicity, mechanical properties, etc. of the medical hydrogel can also be controlled by changing the concentration of the reaction system, so that the medical hydrogel is more conducive to the repair and functional recovery of the nerve injury site, providing a promising biochemical material for in-depth research in the field of nerve regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Figure 2 shows the macroscopic state and scanning electron microscope images (showing the porous structure) of the indole-3-propionic acid-loaded hydrogel before and after gelation according to an embodiment of the present invention. Figure A shows the liquid state of four groups of hydrogels before and after gelation; Figure B shows the scanning electron microscope images of the hydrogels, which are 0.6M, 0.7M, 0.8M, and 0.9M from left to right, respectively. Scale Bar = 20 μm.

[0037] Figure 2 This is an embodiment of the present invention and the nuclear magnetic resonance hydrogen spectrum of the raw materials (PEGDA, Lys, hydrogel raw material mixed solution and drug-loaded hydrogel) and infrared analysis (PEGDA, Lys, IPA and drug-loaded hydrogel).

[0038] Figure 3 The rheological properties (gelation time) according to one embodiment of the present invention, wherein Figures AD are the rheological analysis of the gelation time of 0.6M, 0.7M, 0.8M, and 0.9M drug-loaded hydrogels; Figure E is the rheological properties of the hydrogel after gelation.

[0039] Figure 4 This is a compression mechanics analysis according to an embodiment of the present invention, wherein Figure A is a stress-strain curve; Figure B is a quantitative statistics of Young's modulus, n=5.

[0040] Figure 5 This is a thixotropy analysis according to one embodiment of the present invention, wherein Figures AD are rheological properties characterizations of 0.6M, 0.7M, 0.8M, and 0.9M drug-loaded hydrogels under high strain (500%) and low strain (1%) changes.

[0041] Figure 6 1 and 2 are curves of elastic deformation under 60% strain according to four embodiments of the present invention.

[0042] Figure 7 Figure 1 shows the hydrophilicity of four examples according to the present invention, wherein Figure A is a representative image of the contact angles of 0.6M, 0.7M, 0.8M, and 0.9M drug-loaded hydrogels; Figure B is the quantitative statistics of the contact angles of the hydrogels, n=5.

[0043] Figure 8 This is a 4-hour swelling performance test obtained according to four examples of the present invention.

[0044] Figure 9 The degradation curves of the medical hydrogels of poly (β-amino ester) loaded with indole-3-propionic acid according to four embodiments of the present invention at pH = 7.4 for 28 days.

[0045] Figure 10 Validation of indole-3-propionic acid for scavenging hydrogen peroxide in vitro, n = 6, ****P < 0.0001.

[0046] Figure 11 This is a syringeability analysis performed according to Example 3 of the present invention.

[0047] Figure 12 Figure A is an in vitro release test of indole-3-propionic acid according to an embodiment of the present invention, wherein Figure A is a standard curve of indole-3-propionic acid (ultraviolet absorption spectrum at 270 nm, R 2 =0.9863); Figure B shows the in vitro release curve of indole-3-propionic acid from the drug-loaded hydrogel within 72 hours, n=3.

[0048] Figure 13 Figure 1 is a biocompatibility evaluation obtained according to four embodiments of the present invention, wherein Figure A is an image of blood cells treated with triple-distilled water, normal saline, and four drug-loaded hydrogels (scale bar = 200 μm); Figure B is a macroscopic image of blood cells treated with triple-distilled water, normal saline, and four drug-loaded hydrogels; Figure C is a quantitative statistical analysis of the hemolysis rate of the hydrogel, n = 6, ***P < 0.001; Figure D is the CCK-8 result of L929 cells after hydrogel treatment (days 1 and 3), n = 8; Figure E is the CCK-8 result of ND7 / 23 cells after hydrogel treatment (days 1 and 3), n = 8.

[0049] Figure 14Figures 1, 2, 3, and 4 show the results of cell live and dead staining obtained according to Examples 1, 2, 3, and 4 of the present invention, wherein Figure A shows the live and dead staining results of ND7 / 23 cells cultured on the drug-loaded hydrogel for 1 day (scale bar = 200 μm); Figure B shows the live and dead staining results of ND7 / 23 cells cultured on the drug-loaded hydrogel for 3 days (scale bar = 200 μm); Figures C and D show the live and dead staining statistics of ND7 / 23 cells cultured on the drug-loaded hydrogel for 1 and 3 days, n = 8; Figure E shows the quantitative statistical results of the number of live and dead cells cultured on the drug-loaded hydrogel for 3 days, n = 8.

[0050] Figure 15 Figure 2 is a schematic diagram of the oxidative stress cell model and cell viability statistics obtained according to Example 2 of the present invention and the comparative example, wherein Figure A is a schematic diagram of the oxidative stress model; Figure B is the cell viability statistics of the negative control, positive control, blank hydrogel group (Gel), indole-3-propionic acid drug group (IPA), and indole-3-propionic acid-loaded hydrogel group (IPA@Gel) after oxidative stress stimulation, n=5, *P<0.05, ****P<0.0001.

[0051] Figure 16 Animal behavioral analysis at two and four weeks after spinal cord injury according to an embodiment of the present invention and a comparative example: A) BBB evaluation results, n=5; B) thermal pain detection results, where -1 day represents the preoperative thermal pain level, n=5.

[0052] Figure 17 Figures AB show representative gait patterns and statistical results for a spinal cord injury control group (SCI), a blank hydrogel (Gel), a drug group (IPA), and a drug-loaded hydrogel (IPA@Gel) at two and four weeks after spinal cord injury, according to one embodiment of the present invention and a comparative example. Figures AB show representative hindlimb gait patterns (LH: left hind; RH: right hind) for each group at two and four weeks. Figure C shows quantitative statistics of the maximum ground contact area on the injured side at two and four weeks. n = 3, *P < 0.05, **P < 0.01, ****P < 0.0001.

[0053] Figure 18Figure 1 shows the spinal cord recovery results and area statistics of rats at two and four weeks, obtained according to an embodiment of the present invention and a comparative example. Figure A shows the spinal cord recovery results of rats with spinal cord injury (SCI) in the control group, blank hydrogel (Gel), drug group (IPA), and drug-loaded hydrogel (IPA@Gel) at two and four weeks; Figure B shows the spinal cord injury area statistics of rats with spinal cord injury (SCI) in the control group, blank hydrogel (Gel), drug group (IPA), and drug-loaded hydrogel (IPA@Gel) at two and four weeks, n=5, *P<0.05, **P<0.01.

[0054] Figure 19 The immunohistochemical staining of GFAP and NF-200 in the injured spinal cord of a spinal cord injury control group (SCI), a blank hydrogel (Gel), a drug group (IPA), and a drug-loaded hydrogel (IPA@Gel) at the 2nd and 4th week time points according to an embodiment of the present invention and a comparative example is shown, wherein Figure A is a representative image of tissue immunochemistry at the 2nd week time point, red: NF-200, green: GFAP, blue: DAPI (scale bar = 50 μm); Figure B is a representative image of tissue immunochemistry at the 4th week time point, red: NF-200, green: GFAP, blue: DAPI (scale bar = 50 μm); Figure C is a quantitative statistical analysis of the grayscale value of NF-200 in the injured area at the 2nd and 4th week time points, n = 3, *P < 0.05; D) Quantitative statistical analysis of the grayscale value of GFAP in the injured area at the 2nd and 4th week time points, n = 3.

[0055] Figure 20 Figure 1 shows the immunohistochemical staining of Tuj-1 in the injured spinal cord of a control group (SCI), a blank hydrogel (Gel), a drug group (IPA), and a drug-loaded hydrogel (IPA@Gel) at two and four weeks, respectively, according to an embodiment of the present invention and a comparative example. Figure A shows a representative immunohistochemical image of the tissue at two weeks (red: Tuj-1, blue: DAPI (scale bar = 50 μm); Figure B shows a representative immunohistochemical image of the tissue at four weeks (red: Tuj-1, blue: DAPI (scale bar = 50 μm); Figure C shows the quantitative statistics of the grayscale values ​​of Tuj-1 in the injured area at two and four weeks (n = 3, *P < 0.05).

[0056] Figure 21 These are the H&E staining results of the heart, liver, spleen, lung, and kidney of rats in the spinal cord injury control group (SCI), blank hydrogel (Gel), drug-loaded hydrogel (IPA@Gel), and drug group (IPA) obtained according to an embodiment of the present invention and a comparative example. DETAILED DESCRIPTION

[0057] The technical solution of the present invention will be further described in detail below through examples and in conjunction with the accompanying drawings. In the specification, the same or similar reference numerals indicate the same or similar components. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the overall inventive concept of the present invention and should not be construed as limiting the present invention.

[0058] The reagents used in the examples are described in Table 1 below.

[0059] Table 1

[0060]

[0061]

[0062] The present invention provides an injectable hydrogel, which is a medical hydrogel of poly-β-amino ester loaded with indole-3-propionic acid, and its raw materials include:

[0063] polyethylene glycol diacrylate (PEGDA) as scaffold material;

[0064] A molecule containing a diprimary amine group as a crosslinker that crosslinks with polyethylene glycol diacrylate (PEGDA) via an acrylate bond;

[0065] Indole-3-propionic acid (IPA) as the active ingredient.

[0066] Preferably, the molecule containing a diprimary amine group is selected from at least one of lysine (Lys), arginine (Arg), cystamine or ethylenediamine.

[0067] The present invention provides a method for preparing an injectable hydrogel, which comprises the following steps: adding indole-3-propionic acid to triple-distilled water, adjusting the pH to 5.0-7.4 with sodium hydroxide until the indole-3-propionic acid is completely dissolved. Polyethylene glycol diacrylate (PEGDA) and a cross-linking agent are dissolved in triple-distilled water, and the mixture is stirred for 1 h. Figure 1 Indole-3-propionic acid, PEGDA, and lysine were thoroughly mixed in a suitable ratio and allowed to react in an oven at 50-80°C for 8-12 hours to prepare the drug-loaded hydrogel. The prepared hydrogel was then soaked in distilled water for 24-48 hours to remove any unreacted small molecules.

[0068] In the embodiments of the present invention, the performance of the medical hydrogel can be controlled by adjusting the dosage ratio of polyethylene glycol diacrylate (PEGDA), indole-3-propionic acid (IPA) and lysine (Lys) / arginine (Arg) / cystamine / ethylenediamine or the concentration of the reaction system.

[0069] In some embodiments, the Young's modulus of the medical hydrogel can be regulated by changing the concentration of the reactants. Specifically, the Young's modulus of the medical hydrogel can be reduced by increasing the concentration of the reactants, or the Young's modulus of the medical hydrogel can be increased by decreasing the concentration of the reactants.

[0070] In some embodiments, the maximum compressive strength, swelling ratio, etc. of the medical hydrogel can be regulated by changing the concentration of the reactants. Specifically, by increasing the concentration of the reactants, the maximum compressive strength and swelling ratio of the medical hydrogel are increased.

[0071] In some embodiments, the hydrophilicity of the medical hydrogel can be controlled by changing the concentration of the reactants. Furthermore, by increasing the concentration of the reactants, the contact angle of the medical hydrogel is reduced, thereby enhancing the hydrophilicity of the medical hydrogel.

[0072] The specific preparation dosage data of IPA@Gel in one embodiment of the present invention are shown in Table 2.

[0073] Table 2

[0074] IPA@Gel 0.6M 0.7M 0.8M 0.9M Lys(g) 0.0870 0.1022 0.1160 0.1314 PEGDA(g) 0.84 0.98 1.12 1.26 IPA (mg) 3 3 3 3 Water (mL) 1 1 1 1

[0075] Example 1

[0076] 3 mg of indole-3-propionic acid (IPA) was added to 1 mL of triple-distilled water, and the pH was adjusted with sodium hydroxide until it was completely dissolved. 0.84 g of polyethylene glycol diacrylate (PEGDA) and 0.0870 g of lysine (Lys) were dissolved in 1 mL of triple-distilled water to obtain a mixed solution, which was placed in an 80°C oven for 8 hours to fully react to form an IPA-loaded hydrogel. The hydrogel was soaked in distilled water for 24 hours to fully remove unreacted small molecules, and a drug-loaded hydrogel with a total molar concentration of PEGDA+Lys of 0.6 M was obtained. The surface structure scanning electron microscopy image is shown below. Figure 1 As shown in a of part B.

[0077] Example 2

[0078] Add 3 mg of indole-3-propionic acid (IPA) to 1 mL of triple-distilled water, and use sodium hydroxide to adjust the pH until it is completely dissolved. Dissolve 0.98 g of polyethylene glycol diacrylate (PEGDA) and 0.1022 g of lysine (Lys) in 1 mL of triple-distilled water to obtain a mixed solution. Place the mixed solution in an 80°C oven to allow it to fully react for 8 hours to form an IPA-loaded hydrogel. Soak the hydrogel in distilled water for 24 hours to fully remove unreacted small molecules. A drug-loaded hydrogel with a total molar concentration of PEGDA+Lys of 0.7 M was obtained. The surface structure scanning electron microscopy image is shown below. Figure 1 As shown in b of part B.

[0079] Example 3

[0080] Add 3 mg of indole-3-propionic acid (IPA) to 1 mL of triple-distilled water, and use sodium hydroxide to adjust the pH until it is completely dissolved. Dissolve 1.12 g of polyethylene glycol diacrylate (PEGDA) and 0.1160 g of lysine (Lys) in 1 mL of triple-distilled water to obtain a mixed solution. Place the mixed solution in an 80°C oven to allow it to fully react for 8 hours to form an IPA-loaded hydrogel. Soak the hydrogel in distilled water for 24 hours to fully remove unreacted small molecules. A drug-loaded hydrogel with a total molar concentration of PEGDA+Lys of 0.8 M was obtained. The surface structure scanning electron microscopy image is shown below. Figure 21 As shown in c of part B.

[0081] Example 4

[0082] Add 3 mg of indole-3-propionic acid (IPA) to 1 mL of triple-distilled water, and use sodium hydroxide to adjust the pH until it is completely dissolved. Dissolve 1.26 g of polyethylene glycol diacrylate (PEGDA) and 0.1314 g of lysine (Lys) in 1 mL of triple-distilled water to obtain a mixed solution. Place the mixed solution in an 80°C oven to allow it to fully react for 8 hours to form an IPA-loaded hydrogel. Soak the hydrogel in distilled water for 24 hours to fully remove unreacted small molecules. A drug-loaded hydrogel with a total molar concentration of PEGDA+Lys of 0.9 M was obtained. The surface structure scanning electron microscopy image is shown below. Figure 1 As shown in d of part B.

[0083] Comparative Example 1

[0084] 98 mg of polyethylene glycol diacrylate (PEGDA) and 10.22 mg of lysine were dissolved in triple-distilled water to obtain a mixed solution, which was then placed in an oven at 80° C. to allow it to fully react for 8 h, thereby successfully preparing a blank hydrogel (Gel).

[0085] Comparative Example 2

[0086] 0.6 mg of indole-3-propionic acid was added to triple-distilled water and the pH was adjusted with sodium hydroxide until it was completely dissolved. This successfully prepared the drug group (IPA).

[0087] Test Example 1: Surface Topography Analysis

[0088] The present invention uses lysine and polyethylene glycol diacrylate as raw materials to prepare a hydrogel loaded with indole-3-propionic acid through Michael addition reaction. After the lysine / polyethylene glycol diacrylate solution containing indole-3-propionic acid is fully mixed, it is heated in an oven at 80°C for 4 hours. When the solution changes from liquid to gel state, the polyethylene glycol diacrylate / lysine hydrogel loaded with indole-3-propionic acid (IPA@Gel) is successfully synthesized. By changing the concentrations of polyethylene glycol diacrylate and lysine, four hydrogels with different raw material ratios were successfully prepared, and the states before and after the formation of the hydrogel were photographed with a camera. Figure 1 As shown in part A, the yellow color of the hydrogel changes from light to dark as the concentration increases.

[0089] Test Example 2: H NMR and IR analysis

[0090] 10 mg of PEGDA and 10 mg of lysine were weighed and dissolved in deuterated heavy water. The deuterated heavy water was used to synthesize the drug-loaded hydrogel in an 80°C oven. The molecular structures of PEGDA, lysine and drug-loaded hydrogel were detected and analyzed using a nuclear magnetic resonance hydrogen spectrometer. The results are shown in Figure 2. Figure 2 As shown in Part A, the PEGDA proton spectrum shows acrylate peaks at δ6.35, 6.12, and 5.90 ppm. The signals at δ2.8 and 3.3 ppm originate from the amino groups in Lys. The poly(β-amino ester) hydrogel is formed by an addition reaction between PEGDA monomer and Lys in a 2:1 ratio. The proton spectrum reveals that when the two monomers are uniformly mixed, a Michael addition reaction occurs at 80°C. Upon heating, the peak area of ​​the amino groups decreases significantly, indicating that the hydrogel prepared via the Michael addition reaction was successfully synthesized.

[0091] The structures of lysine, polyethylene glycol diacrylate and indole-3-propionic acid were characterized by Fourier transform infrared spectroscopy. Figure 2 As shown in part B, 1637nm -1 and 1716nm -1 The characteristic absorption peaks at 3400nm are the stretching vibrations of C=C and C=O in the acrylate group of polyethylene glycol diacrylate. After the reactants are fully mixed and reacted, the obtained drug-loaded hydrogel has no amino characteristic peaks, indicating that lysine and polyethylene glycol diacrylate are successfully cross-linked. -1 The absorption peak at is the stretching vibration of carboxylic acid in indole-3-propionic acid, indicating that indole-3-propionic acid is successfully loaded.

[0092] Test Example 3: Analysis of rheological properties of gelation time

[0093] The rheological properties of the material were tested using parallel plates on a HAAKE MARS rotational rheometer. At a test frequency of 1 Hz and a test distance of 1 mm, the test temperature was set to 80°C, and a time oscillation test was performed on the hydrogel sample (V = 1 mL); after gelation, the time oscillation test was continued for 30 minutes to detect the gelation stability. Then, a cyclic strain sweep test was performed at 37°C. 3 cycle tests were performed with 1% strain and 500% strain, and each strain condition was kept for five minutes. The shear thinning properties of the hydrogel material were measured at 37°C using parallel plates, and the shear rate range was set to 0.01-10s. -1 The experiment was performed in triplicate (n=3).

[0094] like Figure 3 As shown in the figure, the results show that at 80℃, the hydrogels all gelled stably in 40 to 50 minutes, and there was no significant difference in the gelation time of each component. The similar change trend of the hydrogels indicated that they had similar rheological characteristics. The rheometer was then used to detect the changes in the storage modulus and loss modulus 30 minutes after gelation, and no significant fluctuations were found ( Figure 4 (E) in the middle proves that the hydrogel can maintain stability after gelation and can provide a stable drug delivery system.

[0095] Test Example 4: Mechanical properties test

[0096] The stress-strain curve, Young's modulus, and maximum compressive strength of IPA@Gel medical hydrogel were tested using a universal electronic mechanical tensile machine. Similarly, cyclic compression was performed with a maximum strain of 60%. Each sample was subjected to 5 cycles to obtain a multiple cyclic stress-strain curve. The experiment was repeated five times (n=5). The results are shown in Figure 2. Figure 4 As shown in Figure A, the hydrogel surface first undergoes elastic deformation as the tensile testing machine's compression head compresses. The curve at this stage is characterized by a stress-strain relationship that is essentially proportional. The hydrogel then enters the yield phase. As the compressive force continues to increase, the strain also increases, but the fluctuation range decreases significantly.

[0097] The results show that if Figure 4 As shown in Figure B, as the hydrogel concentration increases, the Young's modulus also increases. Indole-3-propionic acid-loaded hydrogels with Young's moduli ranging from 1.9 to 6.1 kPa were successfully synthesized via Michael addition reaction. The Young's moduli of the 0.6M, 0.7M, 0.8M, and 0.9M indole-3-propionic acid-loaded hydrogels were 1.9 ± 1.1 kPa, 3.4 ± 0.9 kPa, 4.0 ± 1.2 kPa, and 6.1 ± 1.1 kPa, respectively. These are similar to the Young's modulus of the normal spinal cord, thus avoiding mechanical mismatch between the two.

[0098] Test Example 5: Thixotropy Analysis

[0099] The mechanical modulus of the drug-loaded hydrogel under cyclic changes of strain 1% and 500% was analyzed using a rheometer. After three high and low strain shear rate changes, Figure 5 As shown in the figure, the hydrogel has good recovery ability and the sample shows rapid thixotropic recovery. Figure 5 The G' and G" of the 0.9 M drug-loaded hydrogel shown in D are closest to those at the initial shearing stage.

[0100] Test Example 6: Cyclic Compression Test Analysis

[0101] When hydrogel materials are implanted in the body, they will be subjected to friction or mechanical pressure from the surrounding tissues. Therefore, we conducted a cyclic compression test on the hydrogel. Using a universal tensile testing machine, the deformation was controlled at 60%, and five consecutive cyclic compressions were performed, and the stress-strain data of the hydrogel were recorded. Figure 7 As shown, the tested hydrogels all have good elastic deformation ability and no significant strength degradation or plastic deformation occurs. Figure 6 The cyclic compression curves of the medium C 0.8M hydrogel have the best coincidence, indicating that this hydrogel has the best elastic deformation performance and quickly recovers to its original shape after stress is eliminated.

[0102] Test Example 7: Contact Angle Test

[0103] In order to evaluate the hydrophilicity of the hydrogel material, a drop of distilled water was dropped on the surface of the hydrogel material on a contact angle analyzer, the moment of contact was recorded, and the contact angle was analyzed on a contact angle analyzer. Figure 7 As shown in Figure 3B, the contact angles of 0.6M, 0.7M, 0.8M and 0.9M drug-loaded hydrogels were 52.38±9.26, 49.10±6.73, 44.02±4.71 and 53.26±3.94°, respectively. There was no significant difference between the groups. The contact angles of the hydrogels were all less than 90 degrees, indicating good hydrophilicity, which is conducive to cell adhesion, proliferation and differentiation on their surfaces.

[0104] Test Example 8: Swelling Test

[0105] The surface moisture of the newly prepared hydrogel was blotted dry and weighed as W0. The weighed hydrogel was immersed in PBS (pH = 7.4) at room temperature for 10, 20, 30, 60, 120, and 240 minutes. The swollen hydrogel was taken out and the surface moisture was also blotted dry, and the weight was W1. The experiment was repeated three times (n = 3). The results are shown in Figure 2. Figure 8As shown in the figure, the mass change of the hydrogel in PBS for 4 hours shows that the hydrogel swells rapidly in the first 30 minutes, and then swells slowly. Furthermore, the results show that the swelling rate decreases with increasing hydrogel concentration. The swelling rates of 0.6M, 0.7M, 0.8M, and 0.9M drug-loaded hydrogels are 90.14±53.01%, 69.37±46.96%, 43.05±15.81%, and 21.04±8.23%, respectively.

[0106] Test Example 9: Degradation Test

[0107] The prepared hydrogel was immersed in 3 mL PBS (pH = 7.4) at 37°C for 28 days. At the predetermined time points, the hydrogel was collected, dried, and weighed (n = 3). Figure 9 As shown in the figure, the hydrogel degraded significantly in the first 3 days, and then the rate slowed down in the next four weeks. At 4 weeks, the degraded mass of the hydrogel reached 74.88±5.56%, 71.22±9.35%, 66.46±6.11% and 62.26±8.55% of the original mass, respectively, indicating that the drug-loaded hydrogel has the potential for long-term implantation.

[0108] Test Example 10: In vitro H2O2 removal performance test

[0109] The Gris Hydrogen Peroxide Kit was used to test the H2O2 scavenging performance of indole-3-propionic acid. The principle of the kit is that hydrogen peroxide, as one of the important reactive oxygen species, is indispensable in biotic and abiotic stress, growth and development regulation, and programmed cell death. Hydrogen peroxide reacts with titanium salts to form a peroxide-titanium complex. This yellow precipitate can be dissolved by concentrated sulfuric acid and has a maximum absorption peak at a wavelength of 415nm. The color depth is linearly related to the concentration of hydrogen peroxide. 0mg, 0.75mg, 1.5mg, and 3mg of indole-3-propionic acid were set, 0.02mM H2O2 was added, and the mixture was shaken for 2h to mix thoroughly. The absorbance was measured at a wavelength of 415nm using the Gris Hydrogen Peroxide Kit.

[0110] like Figure 10 As shown, IPA has a significant ability to scavenge hydrogen peroxide compared to the control group, with no significant difference between 0.75mg, 1.5mg, and 3mg. To prolong the release and duration of IPA, this study selected a 3mg hydrogel-loaded IPA content. Subsequent in vitro cell and in vivo experiments demonstrated that this content was non-physiologically toxic.

[0111] Test Example 11: Injectability Analysis

[0112] The shear thinning of hydrogels is a phenomenon in which their viscosity decreases with increasing shear stress, which determines the extrudability of hydrogels during application. A full understanding of the shear thinning properties of hydrogels is helpful in evaluating the printability and extrudability of hydrogels. Figure 11 As shown in the figure, the viscosity trend as a function of shear rate was measured using a rheometer. Since the 0.8 M indole-3-propionic acid-loaded hydrogel exhibited the highest degree of overlap in its cyclic compression curves and rapidly recovered to its initial shape after stress relief, it was selected as a representative viscosity-shear rate graph. The results showed that as the shear rate increased, the viscosity decreased, indicating that the hydrogel exhibited shear-thinning properties and could be injected in situ into the injury site via a syringe, reducing secondary damage to the wound.

[0113] Test Example 12: In vitro indole-3-propionic acid release test

[0114] The ultraviolet absorption peak of indole-3-propionic acid is at 270nm, and ultraviolet absorbance is selected to study the release of indole-3-propionic acid in vitro. The hydrogel loaded with indole-3-propionic acid (0.5g) was incubated with 3mL PBS (pH=7.4) at 37°C. At a specific time point, the supernatant of the liquid was recovered, and then 3mL of fresh PBS was added and incubated. The test wavelength was set to 270nm, and the absorbance of the supernatant was measured and recorded using an enzyme marker. At the same time, the absorbance values ​​of indole-3-propionic acid at different concentrations were measured to draw a standard curve (the linear correlation between the absorbance value and the concentration was R 2 =0.9863), including concentrations of 0.0625 mg / mL, 0.125 mg / mL, 0.1875 mg / mL, 0.25 mg / mL, and 0.375 mg / mL. The in vitro release of indole-3-propionic acid at corresponding time points was calculated by reference to the standard curve, and an in vitro release curve of indole-3-propionic acid was plotted.

[0115] from Figure 12 As shown in Figure B, IPA release is a continuous process, with rapid release within the first 24 hours followed by a slow release, reaching 33.51 ± 0.52% at 72 hours. These release kinetics indicate that indole-3-propionic acid can be slowly released after implantation into the injury site, reducing the stress on the injured area caused by rapid, high-volume release.

[0116] Test Example 13: Cytotoxicity and Hemolysis Assay

[0117] We used hemolysis analysis to evaluate the blood compatibility of the hydrogel. The hydrogel was incubated with red blood cells for 60 minutes, and the mixture was centrifuged. Figure 13As shown in B, there was no obvious hemolysis in the hydrogel group and the saline group, and there was obvious red hemolysis in the positive group (H2O). The hemolysis rate in the supernatant culture medium was measured by an enzyme-linked microplate reader. The hemolysis rates of 0.6M, 0.7M, 0.8M and 0.9M hydrogels were 3.77±0.94%, 3.15±0.80%, 3.82±0.67% and 3.29±0.58%, respectively. The results of erythrocyte morphology observation were consistent with the measurement results. After incubation with saline and hydrogel, the erythrocytes maintained their structural integrity and roundness, while there were basically no normal cells in the positive control group ( Figure 14 A).

[0118] After the hydrogel extract was co-cultured with rat fibroblast L929 cells and the extract was co-cultured with rat dorsal root ganglion neuroblastoma ND7 / 23 cells for 24 hours and 72 hours, the biocompatibility of the hydrogel was evaluated by cytotoxicity test. Figure 13 DE assays using a CCK-8 cell proliferation assay revealed that cell viability in the drug-loaded hydrogel extract group was greater than 85% compared to the normal culture group, with no significant differences between the groups. Furthermore, the cell number did not decrease over time. Both hemocompatibility and cytocompatibility testing demonstrated that the hydrogel possessed excellent biocompatibility and potential for in vivo implantation.

[0119] Test Example 14: Live / Dead Staining

[0120] This study used a live / dead staining method to evaluate the biological behavior of ND7 / 23 cells grown on hydrogels. The cells were directly seeded onto the hydrogels and stained using the live / dead staining method. Based on the differences in plasma membrane integrity and intracellular esterase activity between live and dead cells, live cells were labeled with AM fluorescence, emitting strong green fluorescence. Correspondingly, dead cells were labeled with PI, producing red fluorescence.

[0121] The results are as follows Figure 14 As shown in the figure, the cell viability of all hydrogels is above 85%, and most cells emit green fluorescence. This shows that the cells on the hydrogel can maintain good vitality and proliferation ability, and also shows that the drug-loaded hydrogel has good cell compatibility. At the same time, we counted the living cells on the hydrogel, and the 0.7M hydrogel unit image (unit area is 5.76mm) 2 )The number of live cells of ND7 / 23 cells was 110.00±26.55, which was higher than that of 0.6M (100.00±24.49), 0.8M (84.67±30.58) and 0.9M (100.67±21.75) hydrogels. This may be because the mechanical properties of 0.7M hydrogel are more in line with the tissue mechanical properties required for nerve cell growth. Therefore, in subsequent in vivo studies, the present invention selected 0.7M drug-loaded hydrogel for experiments.

[0122] Test Example 15: Oxidative stress cell model test

[0123] The UV-irradiated hydrogel was cut into regular pieces and placed in a 24-well plate. ND7 / 23 rat dorsal root ganglion neuroblastoma cells were inoculated at a density of 2×10 4 / well seeded on the hydrogel (n=4). Five groups were set up: ND7 / 23 cells cultured normally without any intervention (negative control), cells subsequently stimulated with H2O2 (positive control), blank hydrogel group, drug-loaded hydrogel group, and drug group. The negative control was normal cultured cells without any treatment. After 24 hours of cell adhesion and growth, the remaining three groups were replaced with a medium containing 0.2mM H2O2 and cultured for another 24 hours. The CCK-8 cell viability assay kit was then used to detect cell viability after H2O2 stimulation to verify the cell's vitality in response to oxidative stress.

[0124] The results are as follows Figure 15 As shown in Figure 2, compared with the control group, the IPA-loaded hydrogel can significantly reduce cell apoptosis caused by oxidative stress, and the cell viability reaches 103.89±7.55% ( Figure 16 B) It is clear that IPA plays an important role in inhibiting oxidative stress and can effectively address the phenomenon of uncontrolled oxidative stress at the damaged site.

[0125] Test Example 16: Behavioral evaluation of the right hemisection animal model of spinal cord injury

[0126] An animal model of right hemisection of spinal cord injury in SD rats was established. Four groups were set up, namely PBS group, Gel group, IPA@Gel group and IPA group, with 5 rats in each group. After the rats were anesthetized with 10% concentration of chloral hydrate, the surgical area on the back was shaved and the surface was disinfected. The rat's back was cut open along the midline to expose the T9-T10 segment of the rat spinal cord, and the right spinal cord was contused with a length of 2mm in the injured area to establish a right spinal cord hemisection model in rats. The BBB score and thermal pain test were used to evaluate the recovery effect of the injectable drug-loaded hydrogel in repairing the motor and sensory functions of spinal cord injury.

[0127] The Basso, Beattie & Bresnahan (BBB) ​​score is a scoring method in which three people who are not familiar with the experiment evaluate the hind limb behavior of the rats and score them according to the scoring standard without interfering with each other. The average value is calculated. According to the scoring standard, if the hind limbs are completely paralyzed, it is scored as 0 points, and if the motor function of the hind limbs is completely normal, it is scored as 21 points. That is, the higher the score, the more normal the motor function of the hind limbs. Figure 16As shown in A, the BBB score continued to improve over time, indicating that the condition of the rat's right lower limb continued to recover and improve. At 28 days after surgery, the scores of the indole-3-propionic acid loaded hydrogel group and the drug group were 13.07±5.10 and 15.73±2.02, respectively, which were higher than those of the spinal cord injury control group (5.53±4.98) and the blank hydrogel (10.53±7.53). Figure 16 A).

[0128] On days 14, 21, and 28 after surgery, a thermal pain meter was used to test and record the time the rats felt pain at 40°C on the injured side of their right hind limb. The results before surgery (day -1) were used as the baseline. The results of the thermal pain test represent the time the rats felt pain on the injured side of the spinal cord. Figure 16 As shown in Figure B, as the postoperative injury repair time progressed, pain perception became significantly faster than in the early stages. The thermal pain perception times of the drug-loaded hydrogel group and the drug group at the fourth week were 3.818±0.871 seconds and 3.356±0.881 seconds, respectively. These reaction times were faster than those of the spinal cord injury control group (5.106±2.277 seconds) and the blank hydrogel group (4.200±2.169 seconds), and recovery was close to preoperative levels. The continuous increase in BBB scores and the continuous reduction in thermal pain time indicate that the drug-loaded hydrogel group significantly promoted the functional recovery of the rat spinal cord nerves.

[0129] Subsequently, in order to further analyze the behavioral recovery of rats with spinal cord injury, the rats' gait was recorded and analyzed at two and four weeks. Each rat walked a distance of 90 cm in a specific direction on the instrument, and the relevant gait data was recorded. Figure 17 As shown, compared with the spinal cord injury control group, the injured SD rats in the drug-loaded hydrogel group showed significant gait recovery. The maximum contact area of ​​the rats' hind limbs at four weeks post-surgery was greater than that at two weeks post-surgery. The contact area in the drug-loaded hydrogel group was significantly higher than that in the spinal cord injury control group and the blank hydrogel group, but not significantly different from the drug group. The rats in the drug-loaded hydrogel group also recovered toe splay function, while the corresponding function in the spinal cord injury control group was not fully restored.

[0130] Finally, at two and four weeks after surgery, the rats with spinal cord injury were euthanized by carbon dioxide asphyxiation. After perfusion with paraformaldehyde solution, the spinal cord tissue was removed and the area of ​​the injured area was analyzed. Figure 18 As shown in the figure, at two and four weeks after surgery, the spinal cord injury area in the drug-loaded hydrogel group was the smallest (2 weeks, 0.078±0.010cm 2 ; 4 weeks, 0.047±0.14cm 2 ), drug group (2 weeks, 0.080±0.010cm 2 ; 4 weeks, 0.061±0.008cm 2) and blank hydrogel group (2 weeks, 0.095±0.014cm 2 ; 4 weeks, 0.076±0.014cm 2 ), spinal cord injury control group (2 weeks, 0.104±0.016cm 2 ; 4 weeks, 0.095±0.017cm 2 ) had the largest injury area. However, there was no significant difference between the drug-loaded hydrogel group and the drug group. Over time, the spinal cord injury area decreased significantly, indicating significant recovery from spinal cord injury.

[0131] Test Example 17: Histological Examination

[0132] (1) Immunofluorescence staining

[0133] In order to further explore the repair mechanism, the present invention performed immunofluorescence staining of neurofilaments and glial fibrillary acid protein on tissue sections of spinal cord injury sites. Figure 19 As shown in AB, after treatment with the drug-loaded hydrogel, the number of NF-200-positive neurons increased and the area of ​​GFAP-positive regions decreased, indicating that the hydrogel loaded with indole-3-propionic acid has the effect of promoting the growth of nerve fibers and inhibiting the proliferation of early astrocytes, further inhibiting the formation of glial scars. Quantitative results showed that the changes from the second to the fourth week showed that over time, the number of NF-200-positive neurons in the drug-loaded hydrogel group was the highest, exceeding that of the spinal cord injury control group, the blank hydrogel group, and the drug group; the number of GFAP-positive astrocytes in the drug-loaded hydrogel group was less than that in the blank hydrogel and drug groups, but there was no significant statistical difference ( Figure 19 CD). Spinal cord injury leads to a significant decrease in NF-200-positive neurons, while the drug-loaded hydrogel group reverses this decrease. In summary, the drug-loaded hydrogel group inhibits the proliferation of early astrocytes and promotes the expression of positive neurons.

[0134] (2) Immunofluorescence staining

[0135] β-Tubulin III (Tuj-1) is a widely documented neuronal marker whose expression increases during the early stages of neural differentiation and decreases in aged spinal cord tissue. Neuron-specific class III β-tubulin (Tuj-1) is present in newly formed immature postmitotic neurons, differentiated neurons, and some mitotically active neural precursors.

[0136] Two and four weeks after surgery, the rats were perfused and the tissues around the injured area were sampled. Figure 20As shown, at two and four weeks after surgery, there was no significant change in the fluorescence grayscale value, but the drug-loaded hydrogel group had the highest expression of Tuj-1 at the injury site, followed by the drug group, but the expression was greater than that of the blank hydrogel group and the spinal cord injury control group. These results indicate that the drug-loaded hydrogel group promoted the growth and differentiation of newborn neurons in the spinal cord injury site.

[0137] (3) H&E staining of visceral tissue

[0138] The biocompatibility of the drug-loaded hydrogel was evaluated by hematoxylin & eosin staining. Four weeks after surgery, the rats with spinal cord injury were perfused with paraformaldehyde, and the major metabolic organs (heart, liver, spleen, lung, and kidney) were stained with hematoxylin & eosin, and pathological sections were observed under a microscope.

[0139] like Figure 21 As shown, compared with the spinal cord injury control group, the heart, liver, spleen, lung, and kidney of the blank hydrogel, drug-loaded hydrogel, and drug-loaded groups showed similar morphology, with no significant differences in cell nuclei and cytoplasm. All metabolic organs showed no obvious necrosis, and no structural atrophy, damage, fibrosis, or other pathological structural changes were observed. This suggests that the indole-3-propionic acid-loaded hydrogel has no toxic side effects, good in vivo biocompatibility, and great application potential.

[0140] The injectable hydrogel provided by the embodiments of the present invention and its preparation method and application have the following advantages:

[0141] (1) The medical hydrogel of poly-β-amino ester loaded with indole-3-propionic acid (IPA@Gel) of the embodiment of the present invention is obtained by cross-linking a polymer composed of polyethylene glycol diacrylate, lysine / arginine / cystamine / ethylenediamine and other molecules containing diprimary amino groups and indole-3-propionic acid through Michael addition reaction. The polymers are highly cross-linked, and the hydrogel surface has no porous structure. It has good swelling, degradability, hydrophilicity and biocompatibility, and has good mechanical properties with a Young's modulus of 1.9KPa-6.1KPa. It is applied to spinal cord injury and can provide suitable scaffold support for the cavity formed at the injury site, promote the regeneration of cells at the injury site, and enhance the regeneration of spinal cord injury.

[0142] 2) The IPA@Gel medical hydrogel provided in the embodiment of the present invention can reduce the ROS level in the injured area to promote the regeneration and repair of the damaged spinal cord, and can significantly reduce the cell apoptosis caused by oxidative stress. In vitro cell experiments show that the 3.4KPa drug-loaded hydrogel is most suitable for nerve cell adhesion and proliferation, and can reduce the cell apoptosis caused by oxidative stress. The IPA@Gel medical hydrogel provided in the embodiment of the present invention was applied to the spinal cord injury model. Through behavioral evaluation, it was proved that the IPA@Gel medical hydrogel can effectively promote the behavioral recovery of rats with spinal cord injury. At the same time, it was verified that the IPA@Gel hydrogel has a promoting effect on the growth of rat neurons and has no obvious biological toxicity.

[0143] (3) The degradation performance of the hydrogel provided by the embodiment of the present invention affects the efficiency of tissue regeneration at the implant site. If the hydrogel degrades too quickly, the new tissue growth will lose its attachment and cannot be fully repaired; if the degradation is too slow, the growth of new tissue will be hindered. The introduction of disulfide bonds into the IPA@Gel medical hydrogel provided by the embodiment of the present invention can, on the one hand, enhance cell adhesion and facilitate the regeneration of cells at the site of loss; on the other hand, it makes the degradation of the IPA@Gel medical hydrogel reduction-sensitive. Under reducing conditions, the degradation rate will be accelerated, and the degradation of the hydrogel can be regulated by changes in the microenvironment.

[0144] (4) The IPA@Gel medical hydrogel provided in the embodiment of the present invention has shear thinning properties and is therefore injectable, which is beneficial for the delivery of the hydrogel through minimally invasive methods.

[0145] (5) The preparation method of the IPA@Gel medical hydrogel provided in the embodiment of the present invention is simple and controllable. The degradation rate of the polyethylene glycol diacrylate / lysine / indole-3-propionic acid medical hydrogel and the release rate of indole-3-propionic acid can be regulated by changing the temperature of the Michael addition reaction, the dosage ratio of the three raw materials or the concentration of the reaction system. The swelling properties, hydrophilicity, mechanical properties, etc. of the IPA@Gel medical hydrogel can also be regulated by changing the concentration of the reaction system, so that the PCA medical hydrogel is more conducive to the repair and functional recovery of the nerve injury site, providing a promising biochemical material for in-depth research in the field of nerve regeneration.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An injectable hydrogel, characterized in that: The hydrogel is a medical hydrogel of poly-β-amino ester loaded with indole-3-propionic acid, and its raw materials include: polyethylene glycol diacrylate as scaffold material; a molecule containing a diprimary amine group as a crosslinking agent, the crosslinking agent being crosslinked with polyethylene glycol diacrylate via an acrylate bond; Indole-3-propionic acid as the active ingredient.

2. The injectable hydrogel according to claim 1, wherein: The molecule containing a diprimary amine group is selected from at least one of lysine, arginine, cystamine or ethylenediamine.

3. The injectable hydrogel according to claim 1, wherein: The swelling ratio of the hydrogel after immersion in phosphate buffered saline (PBS) with a pH of 7.4 at 37° C. for 4 hours ranged from 21.04±8.23% to 90.14±53.01%, and the residual mass after immersion for 28 days ranged from 62.26±8.55% to 74.88±5.56% of the original mass.

4. The injectable hydrogel according to claim 1, wherein: The Young's modulus of the hydrogel is 1.9-6.1 KPa, which is achieved by controlling the total molar concentration of polyethylene glycol diacrylate and the cross-linking agent in the reaction system within the range of 0.6-0.9 M.

5. The injectable hydrogel according to claim 1, wherein: The hydrogel has a porous structure, and the cumulative release amount of indole-3-propionic acid in a phosphate buffer solution with a pH of 7.4 at 37° C. in vitro for 72 hours is 33.51±0.52%.

6. The injectable hydrogel according to claim 1, wherein: The hydrogel has shear-thinning properties, with viscosity decreasing as shear rate increases, and can be delivered to the injury site via a syringe.

7. A method for preparing the injectable hydrogel according to claim 1, characterized in that: The following steps are involved: (a) adding indole-3-propionic acid to triple-distilled water to obtain an indole-3-propionic acid solution; (b) polyethylene glycol diacrylate and a cross-linking agent are dissolved in triple-distilled water, mixed, and then added with indole-3-propionic acid solution; (c) the mixed solution obtained in step (b) is subjected to a Michael addition reaction to form a hydrogel loaded with indole-3-propionic acid; (d) soaking the hydrogel obtained in step (c) in distilled water to fully remove unreacted small molecules.

8. The preparation method according to claim 7, characterized in that: In step (a), the pH value is adjusted to 5.0-7.4 using sodium hydroxide until the indole-3-propionic acid is completely dissolved; In step (b), the total molar concentration of polyethylene glycol diacrylate and the cross-linking agent is controlled to be 0.6-0.9 M to regulate the Young's modulus, swelling ratio and degradation rate of the hydrogel; In step (c), the reaction is carried out at 50-80° C. for 8-12 hours; In step (d), the soaking time is 24-48 hours.

9. Use of the injectable hydrogel according to claim 1 in preparing biomaterials for repairing spinal cord injuries.

10. The use according to claim 9, characterized in that: The hydrogel is injected into the spinal cord injury area to inhibit oxidative stress and cell apoptosis; promote nerve axon regeneration and motor function recovery; and reduce the formation of glial scars.

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