Composite hydrogel as well as preparation method and application thereof

Abstract

The invention provides composite hydrogel and a preparation method and application thereof, and belongs to the technical field of biomedical materials, the composite hydrogel provided by the invention comprises melatonin carbon dots and porcine small intestinal submucosa hydrogel, the melatonin carbon dots are dissolved in the porcine small intestinal submucosa hydrogel, the particle size of the melatonin carbon dots is 1.5-2.5 nm, and the melatonin carbon dots are dissolved in the porcine small intestinal submucosa hydrogel. The loading capacity of the melatonin carbon dots in the composite hydrogel is 150 to 450 [mu] g / mL. The composite hydrogel provided by the invention can effectively inhibit the inflammatory response of achilles tendinopathy, can promote tendon regeneration, and has the advantages of minimally invasive effect, high drug curative effect and small side effect when being used for treating achilles tendinopathy.

Description

Technical Field

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

[0002] Achilles tendinopathy (AP) is one of the most common tendon degenerative diseases clinically. It is caused by long-term high-intensity exercise and tissue aging, and has high morbidity and disability rates.

[0003] In the treatment of Achilles tendinopathy, it is necessary to focus on both anti-inflammation and tendon regeneration. In Achilles tendinopathy, the Achilles tendon is repeatedly stimulated after injury, forming an inflammatory microenvironment. Although existing non-steroidal anti-inflammatory drugs can alleviate acute inflammatory responses and provide analgesia for Achilles tendinopathy, they usually lead to adverse reactions such as tissue adhesion and delayed repair.

[0004] In terms of tendon regeneration, there is currently a precedent for anti-inflammatory and tissue repair through platelet-rich plasma, but this method is limited by the inherent limitations of autologous platelet-rich plasma acquisition and long-term maintenance of vitality; synthetic materials such as PCL scaffolds have also been used for tendon repair and regeneration, but the degradation of some synthetic materials can trigger autologous inflammatory reactions and even toxic side effects; and stem cell therapy has great challenges in acquisition and preservation. Therefore, there is an urgent need for a new drug that has both anti-inflammatory and tendon repair-promoting properties for the treatment of Achilles tendinopathy. Summary of the Invention

[0005] The purpose of the present invention is to provide a new drug for treating Achilles tendinopathy.

[0006] To achieve the above objectives, the first aspect of the present invention provides a composite hydrogel, comprising melatonin carbon dots and porcine small intestinal submucosa hydrogel, wherein the melatonin carbon dots are dissolved in the porcine small intestinal submucosa hydrogel, the particle size of the melatonin carbon dots is 1.5-2.5 nm, and the loading amount of the melatonin carbon dots in the composite hydrogel is 150-450 μg / mL.

[0007] Melatonin is a natural metabolite of plants or animals. Studies have shown that melatonin has anti-inflammatory effects. However, melatonin is highly hydrophobic and its absorption in the body is poor, resulting in unsatisfactory therapeutic effects. The present invention converts melatonin into melatonin carbon dots, which improves the water solubility and biocompatibility of melatonin, can effectively improve the anti-inflammatory effect of melatonin, break the vicious cycle of verification microenvironment, reduce the release of inflammatory factors such as interleukin-6, and avoid the inhibitory effect of traditional non-steroidal anti-inflammatory drugs on tendon repair. In the composite hydrogel provided by the present invention, compared with traditional GelMA hydrogel, porcine small intestinal submucosa hydrogel can better mimic the extracellular matrix microenvironment, and its synergistic effect with melatonin carbon dots can effectively increase the expression of hepatitis factors such as interleukin-10 and guide tendon cell migration and proliferation; in addition, melatonin carbon dots can enhance the stability of porcine small intestinal submucosa hydrogel, and porcine small intestinal submucosa hydrogel can play a sustained-release effect on melatonin carbon dots. The two can synergize to effectively prolong the action time of the composite hydrogel.

[0008] In the composite hydrogel provided by the present invention, the particle size of melatonin carbon dots is only 1.5-2.5 nm, which can penetrate the inflammatory site and enhance the anti-inflammatory effect. In addition, the melatonin carbon dots in the composite hydrogel have a Schiff base structure, which has an immunomodulatory effect and can enhance the antagonistic ability of melatonin against inflammatory factors. At the same time, the Schiff base structure can solve the problem of anti-inflammatory and regeneration synergy through the dual effects of chemical chelation and immune regulation.

[0009] A second aspect of the present invention provides a method for preparing the aforementioned composite hydrogel, comprising the following steps:

[0010] S1: Preparation of melatonin carbon dots;

[0011] S2: Preparation of porcine small intestinal submucosa hydrogel;

[0012] S3: Mixing melatonin carbon dots and porcine small intestinal submucosa hydrogel to obtain a composite hydrogel.

[0013] Preferably, step S1 includes the following steps:

[0014] S11: Dissolve melatonin in water and heat at 160-200°C for 4-8 hours;

[0015] S12: dialyzing the product obtained after heating in step S11 to obtain melatonin carbon dots.

[0016] Preferably, in step S11, the mass ratio of melatonin to water is 1:(30-50).

[0017] Preferably, in step S12, the dialysis time is 24 to 72 hours.

[0018] The preparation method provided by this invention utilizes a hydrothermal method to prepare melatonin carbon dots, which eliminates the need for HCl and significantly improves their biosafety. Furthermore, compared to melatonin, the melatonin carbon dots produced by this method exhibit novel functional groups, significantly enhancing their anti-inflammatory activity.

[0019] Preferably, step S2 includes the following steps:

[0020] S21: The pig small intestine was soaked in a methanol-chloroform mixture and then washed with PBS. After washing with PBS, it was digested with trypsin containing EDTA to obtain SIS;

[0021] S22: SIS was soaked in SDS solution, washed with ethanol and freeze-dried;

[0022] S23: The freeze-dried SIS obtained in step S22 is digested with pepsin. After digestion, the pH is adjusted to neutral using NaOH to obtain a porcine small intestinal submucosa hydrogel.

[0023] Preferably, in step S21, the volume ratio of methanol to chloroform in the methanol-chloroform mixed reagent is 1:(1-3).

[0024] A third aspect of the present invention provides a use of the aforementioned composite hydrogel in the preparation of a drug for treating Achilles tendinopathy.

[0025] This invention combines bio-based hydrogel and melatonin carbon dots for the first time. The resulting composite hydrogel has the effect of sustained-release melatonin. Since the Achilles tendon is avascular tissue, the composite hydrogel provided by this invention can be used as an injection to effectively improve the efficacy of the drug, significantly enhancing its anti-inflammatory ability and promoting tendon regeneration.

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

[0027] 1. The composite hydrogel provided by the present invention is used as a therapeutic drug for Achilles tendinopathy. Loaded with melatonin carbon dots, the composite hydrogel eliminates the inflammatory microenvironment while promoting functional tendon regeneration through the synergistic effects of the anti-inflammatory properties of the melatonin carbon dots and the regeneration-guided properties of the porcine intestinal submucosa hydrogel, resolving the contradiction between "anti-inflammatory and repair-inhibiting" effects.

[0028] 2. The composite hydrogel provided by the present invention uses porcine small intestinal submucosa hydrogel as its matrix, completely removing immunogenic substances, having good biocompatibility and no toxic residues. It has a good sustained-release effect on melatonin carbon dots, ensuring the controlled release of melatonin carbon dots.

[0029] 3. The raw material of the composite hydrogel provided by the present invention is derived from porcine small intestine, which is widely available and low-cost. The preparation method adopts a hydrothermal method combined with dialysis purification, which is simple in process and can be modularized and produced on a large scale, showing industrial prospects.

[0030] 4. When the composite hydrogel provided by the present invention is used as a tendon repair material, systemic administration is not required, thus avoiding drug waste and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an electron microscope photograph of melatonin carbon dots prepared in Example 1 of the present invention;

[0032] Figure 2 Quantitative results of the diameter dispersion of melatonin carbon dots prepared in Example 1 of the present invention;

[0033] Figure 3 The molecular structure test results of melatonin carbon dots and melatonin prepared in Example 1 of the present invention are shown;

[0034] Figure 4 The X-ray electron spectroscopy analysis and X-ray diffraction results of the melatonin carbon dots prepared in Example 1 of the present invention are shown;

[0035] Figure 5 This is an electron microscopic photograph of the small intestinal submucosa hydrogel and composite hydrogel prepared in Example 1 of the present invention after being immersed in glutaraldehyde for 2 hours and dried;

[0036] Figure 6 The results show the swelling rate of the hydrogel and composite hydrogel in the small intestinal submucosa.

[0037] Figure 7 The results of compression modulus test of composite hydrogel and small intestinal submucosa hydrogel are shown;

[0038] Figure 8 The results of the melatonin carbon dot release characteristics test of the composite hydrogel;

[0039] Figure 9 The CCK-8 test results in Example 5;

[0040] Figure 10 The results of live and dead cell staining in Example 5;

[0041] Figure 11 The results of the cytoskeleton staining experiment in Example 5;

[0042] Figure 12 These are photos of the Achilles tendon after 1 week and 4 weeks of implantation of the material in Example 6;

[0043] Figure 13 IL-6 gene expression curves of rats in each group in Example 6;

[0044] Figure 14 IL-10 gene expression curves of rats in each group in Example 6;

[0045] Figure 15 This is the H&E staining result in Example 6. DETAILED DESCRIPTION

[0046] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter ranges described in the present invention. Reasonable variations derived therefrom are still within the scope of protection of the present invention.

[0047] It should be noted that the endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0048] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventional meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments was carried out in accordance with the protocols and parameters given by the manufacturers.

[0049] As described in the background technology, since the existing Achilles tendinopathy treatment drugs have low efficacy and high incidence of adverse reactions, in view of this, a specific embodiment of the present invention provides a composite hydrogel that can sustain the release of melatonin carbon dots. The composite hydrogel includes melatonin carbon dots and porcine small intestinal submucosa hydrogel. The melatonin carbon dots are dissolved in the porcine small intestinal submucosa hydrogel, and the melatonin carbon dots have a Schiff base structure.

[0050] In some specific embodiments, the particle size of the melatonin carbon dots is 1.5-2.5 nm.

[0051] A specific embodiment of the present invention also provides a method for preparing the aforementioned composite hydrogel, which specifically includes the following steps:

[0052] S1: Preparation of melatonin carbon dots;

[0053] S2: Preparation of porcine small intestinal submucosa hydrogel;

[0054] S3: Mixing melatonin carbon dots and porcine small intestinal submucosa hydrogel to obtain a composite hydrogel.

[0055] In the above embodiment, step S1 includes the following steps:

[0056] S11: Dissolve melatonin in water and heat at 160-200°C for 4-8 hours;

[0057] S12: dialyzing the product obtained after heating in step S11 to obtain melatonin carbon dots.

[0058] In step S11 of the above embodiment, the mass ratio of melatonin to water is 1:(30-50).

[0059] In the above embodiment, step S2 includes the following steps:

[0060] S21: The pig small intestine was soaked in a methanol-chloroform mixture and then washed with PBS. After washing with PBS, it was digested with trypsin containing EDTA to obtain SIS;

[0061] S22: SIS was soaked in SDS solution, washed with ethanol and freeze-dried;

[0062] S23: The freeze-dried SIS obtained in step S22 is digested with pepsin. After digestion, the pH is adjusted to neutral using NaOH to obtain a porcine small intestinal submucosa hydrogel.

[0063] In step S21 of the above embodiment, the volume ratio of methanol to chloroform in the methanol-chloroform mixed reagent is preferably 1:(1-3), and most preferably 1:1.

[0064] In step S22 of the above embodiment, the concentration of the SDS solution is 0.5% to 1%.

[0065] In step S3 of the above embodiment, the loading amount of melatonin carbon dots in the composite hydrogel is 150-450 μg / mL.

[0066] The technical solution of the present invention will be further described below through specific embodiments.

[0067] Example 1

[0068] Preparation of composite hydrogel

[0069] S11: Dissolve 250 mg of melatonin in 10 mL of water and heat at 180°C for 6 h.

[0070] S12: The product obtained after heating in step S11 was dialyzed in ultrapure water using a dialysis membrane (SP131060, Yuanye, China) for 48 h. The dialyzed product was freeze-dried to obtain melatonin carbon dots.

[0071] S21: Fresh porcine small intestine was mechanically treated and then immersed in a 1:1 volume ratio methanol-chloroform mixture for 12 h. After immersion, it was washed with PBS. After PBS washing, it was digested with 0.05% trypsin containing 0.05% EDTA to obtain SIS.

[0072] S22: SIS was soaked in 0.5% SDS solution for 4 h, then washed with 75% ethanol and freeze-dried;

[0073] S23: The freeze-dried SIS obtained in step S22 was digested with 1 mg / mL pepsin. The digestion was considered complete when the pH stabilized at 1-2. After the digestion was complete, the pH was adjusted to neutral using NaOH to obtain the small intestinal submucosa hydrogel.

[0074] S3: Melatonin carbon dots of different masses were taken and mixed with 1 mL of small intestinal submucosa hydrogel to obtain composite hydrogels of different concentrations. In this example, the composite hydrogels with three different melatonin carbon dot concentrations were prepared, with concentrations of 150 μg / mL, 300 μg / mL, and 450 μg / mL, respectively.

[0075] Example 2

[0076] Sample characterization

[0077] The melatonin carbon dots prepared in Example 1 were observed under a transmission electron microscope. Figure 1 As shown by Figure 1 The TEM results show that the melatonin carbon dots prepared in Example 1 are spherical, with a lattice width of 0.006 nm;

[0078] The diameter dispersion of the melatonin carbon dots prepared in Example 1 was quantified using a laser particle size analyzer. Figure 2 As shown by Figure 2 It can be seen that the average diameter of the melatonin carbon dots prepared in Example 1 is 2.1 nm;

[0079] The molecular structures of melatonin and the melatonin carbon dots prepared in Example 1 were detected by Fourier transform infrared spectroscopy. Figure 3 As shown in the Fourier transform spectrum, melatonin and melatonin carbon dots have the highest peaks at 3287, 1594, 1491, 1433, and 1211 cm -1The absorption peaks appeared at 2933 cm, which revealed the existence of NH, C=C, CN and -OC. In addition, melatonin carbon dots also produced new functional groups, such as 2933 cm -1 OH at 1620~1720cm -1 C=O / C=N at 1306cm -1 CO and 926cm -1 OH at the site.

[0080] Melatonin and the melatonin carbon dots prepared in Example 1 were subjected to X-ray photoelectron spectroscopy and X-ray diffraction analysis. The XRD results are as follows: Figure 4 As shown in A in FIG, the XRD results show that melatonin and melatonin carbon dots have obvious peaks at 2θ of 10.8°, 16.4°, and 26.1°, which means that part of the crystal structure of melatonin is retained. The XPS results are shown in FIG. Figure 4 As shown in Figure B, the XPS results show that the elemental composition of melatonin carbon dots is mainly C (72.26%), N (12.22%), and O (15.53%). The C1s spectrum of melatonin carbon dots shows that melatonin carbon dots mainly have three different types of C, namely CH at 284.4 eV ( Figure 4 B1 in), CN at 285.2eV ( Figure 4 B2 in) and C=O at 286eV ( Figure 4 B3 in the figure); the N1s spectrum of melatonin carbon dots shows that melatonin carbon dots mainly have two kinds of N, namely CN at 400.1eV and NC=O at 400.8eV; the O1s spectrum of melatonin carbon dots shows that melatonin carbon dots mainly have two kinds of O, namely C=N at 531.6eV and COC at 533.1eV.

[0081] The small intestinal submucosa hydrogel and composite hydrogel prepared in step S2 were immersed in glutaraldehyde for 2 hours and dried and then observed using a scanning electron microscope. The final results are as follows: Figure 5 As shown in Figure 3, scanning electron microscopy results showed that the addition of melatonin carbon dots did not lead to morphological changes in the small intestinal submucosa hydrogel.

[0082] Example 3

[0083] Hydrogel mechanical properties testing

[0084] 1 mL of the small intestinal submucosal hydrogel and composite hydrogel prepared in Example 1 was taken and placed in a water bath at 37°C. After the hydrogel was converted into a solid state, the solid hydrogel was immersed in deionized water. The swelling rate (SR) of the hydrogel was measured regularly. The swelling rate was calculated using the following formula:

[0085] SR=(W1-W0) / W0×100%.

[0086] Where W1 is the mass after immersion, in g; W0 is the initial mass of the hydrogel, in g. The final result is as follows Figure 6 As shown, the addition of melatonin carbon dots did not lead to changes in the swelling behavior of the small intestinal submucosa hydrogel.

[0087] The composite hydrogel and small intestinal submucosa hydrogel were compressed at a speed of 0.01 mm / s (equivalent to 0.6% strain / s) using a universal material testing machine until mechanical failure. The initial strain of 20% in the stress-strain curve represents the compression modulus, and the relevant results are shown in Figure 2. Figure 7 As shown by Figure 7 It can be seen that there is no significant difference in the compression modulus between the composite hydrogel and the small intestinal submucosa hydrogel.

[0088] Example 4

[0089] Cumulative release effect determination

[0090] The release characteristics of melatonin carbon dots from the composite hydrogel with a concentration of 300 μg / mL prepared in Example 1 at different time periods were measured by UV-visible absorption spectroscopy at 320 nm, and the results were compared with the standard curve and quantified. Figure 8 As shown by Figure 8 It can be seen that the composite hydrogel released 79% of the melatonin carbon dots within 14 days, which shows that the composite hydrogel can release melatonin carbon dots in a controllable and gradual manner, which proves the melatonin carbon dots sustained-release function of the composite hydrogel.

[0091] Example 5

[0092] Biocompatibility testing of composite hydrogels

[0093] The tendon-derived stem cells used in this example were all rat TDSCs.

[0094] Rat TDSCs were seeded into the lower chamber of a 24-well Transwell plate, and composite hydrogels containing different concentrations of melatonin carbon dots (150, 300, and 450 μg / mL) were placed in the upper chamber. After culturing for 1, 3, and 5 days, cell viability was assessed using a CCK-8 assay.

[0095] CCK-8 test results ( Figure 9 ) showed that the lower intestinal mucosal hydrogel, the composite hydrogel containing 150μg / mL melatonin carbon dots, and the composite hydrogel containing 300μg / mL melatonin carbon dots all promoted cell growth, with the composite hydrogel containing 300μg / mL melatonin carbon dots showing the greatest effect. However, the composite hydrogel containing 450μg / mL melatonin carbon dots showed an inhibitory effect on cell proliferation.

[0096] Cell live-death staining results ( Figure 10 ) and cytoskeleton staining ( Figure 11 ) also confirmed that the melatonin carbon dot composite hydrogel containing 150μg / mL and 300μg / mL did not show obvious cytotoxicity to rat TDSCs and had good biocompatibility, while the melatonin carbon dot composite hydrogel containing 450μg / mL showed obvious cytotoxicity to rat TDSCs and had poor biocompatibility.

[0097] Example 6

[0098] Repair test of Achilles tendon by composite hydrogel

[0099] The feeding, care, manipulation, and treatment of the experimental animals in this example were carried out in accordance with relevant guidelines, and all experiments were approved by the Animal Research Ethics Committee of Ningbo University.

[0100] Sixty rats were divided into four groups to evaluate the therapeutic effect of composite hydrogel on Achilles tendinopathy. The groups were divided into: (1) Sham group: rats were not treated with any treatment; (2) Achilles tendinopathy group: each rat was injected with 30 μL 10 mg / mL collagenase for 1 week to establish an Achilles tendinopathy model; (3) SIS hydrogel group: each rat was injected with 30 μL 10 mg / mL collagenase for 1 week to establish an Achilles tendinopathy model and then injected with 200 μL 10 mg / mL small intestinal submucosal hydrogel; (4) MS group: each rat was injected with 30 μL 10 mg / mL collagenase for 1 week to establish an Achilles tendinopathy model and then injected with 200 μL 10 mg / mL composite hydrogel containing 300 μg / mL melatonin carbon dots. Samples were taken 1 week and 4 weeks after implantation, and gross photos of the Achilles tendons of each group were taken. The relevant photos are shown in the figure. Figure 12 As shown by Figure 12 It can be seen that the abnormal tissue on the surface of the Achilles tendon in the SIS hydrogel group and the MS group completely disappeared at the 4th week. The SIS hydrogel group and the MS group showed therapeutic effects on Achilles tendinopathy, among which the treatment effect of the MS group was the best, and the recovery effect of the Achilles tendon was close to that of the sham group.

[0101] Interleukin-6 (IL-6) and interleukin-10 (IL-10) are inflammation-related genes. The IL-6 and IL-10 gene expression curves of the above four groups of rats were drawn. The IL-6 gene expression curves of each group are shown in Figure 2. Figure 13 As shown by Figure 13 It can be seen that IL-6 was initially expressed in the rats of the Achilles tendinopathy group, SIS hydrogel group and MS group, and the expression was highest in the Achilles tendinopathy group. The expression levels of the SIS hydrogel group and the MS group were similar. After 4 weeks, the expression levels of IL-6 in the rats of each group decreased, and the decrease was most obvious in the MS group. The IL-10 gene expression curves of each group are shown in Figure 2. Figure 14 As shown by Figure 14It can be seen that the IL-10 expression level of rats in the MS group was the highest at 1 week. After 4 weeks, except for the MS group, there was no IL-10 expression in the other groups. The above results show that the small intestinal submucosal hydrogel with the addition of melatonin carbon dots can effectively reduce the expression level of IL-6 and increase the level of IL-10, thereby achieving anti-inflammatory effects.

[0102] To study the biological healing mechanism of rats in each group, H&E staining was used to evaluate the effects of small intestinal submucosa hydrogel in the SIS hydrogel group and small intestinal submucosa hydrogel containing melatonin carbon dots in the MS group on Achilles tendon healing. Figure 15 The results of H&E staining are shown in Figure 2. Figure 15 As can be seen, the collagen fibers in the Achilles tendinopathy group showed disorder at one week, while the small intestinal submucosa hydrogel and the small intestinal submucosa hydrogel containing melatonin carbon dots promoted the recovery of the injured Achilles tendon. They showed regularity of collagen fibers at one week, but there were also significant differences between the SIS hydrogel group, the MS group, and the sham group. After four weeks, the collagen fibers in the SIS hydrogel and MS groups became more regular, with the MS group showing the best recovery effect, approaching the performance level of the sham group, while the SIS hydrogel group showed a lower degree of recovery. This shows that the small intestinal submucosa hydrogel containing melatonin carbon dots can promote the repair and regeneration of the Achilles tendon, and its effect far exceeds that of the small intestinal submucosa hydrogel.

[0103] Anti-inflammation and promoting tendon regeneration are two important goals in the treatment of Achilles tendinopathy. The composite hydrogel of sustained-release melatonin carbon dots provided by the present invention avoids the use of HCl during the preparation process, greatly improving the biosafety of the product. After combining melatonin carbon dots with bio-based hydrogels, the release of melatonin carbon dots can be effectively slowed down. Compared with melatonin, the melatonin carbon dots provided by the present invention produce new anti-inflammatory groups. Moreover, compared with simple bio-based hydrogels, the composite hydrogel provided by the present invention can effectively promote Achilles tendon regeneration, which provides new ideas for the subsequent development of drugs for the treatment of Achilles tendinopathy.

[0104] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A composite hydrogel, characterized in that The composite hydrogel includes melatonin carbon dots and porcine small intestinal submucosa hydrogel, the melatonin carbon dots are dissolved in the porcine small intestinal submucosa hydrogel, the particle size of the melatonin carbon dots is 1.5-2.5 nm, and the loading amount of the melatonin carbon dots in the composite hydrogel is 150-450 μg / mL.

2. A method for preparing the composite hydrogel according to claim 1, characterized in that: The following steps are involved: S1: Preparation of melatonin carbon dots; S2: Preparation of porcine small intestinal submucosa hydrogel; S3: Mixing melatonin carbon dots and porcine small intestinal submucosa hydrogel to obtain a composite hydrogel.

3. The preparation method according to claim 2, wherein The step S1 comprises the following steps: S11: Dissolve melatonin in water and heat at 160-200°C for 4-8 hours; S12: dialyzing the product obtained after heating in step S11 to obtain melatonin carbon dots.

4. The preparation method according to claim 3, wherein In step S11, the mass ratio of melatonin to water is 1:(30-50).

5. The preparation method according to claim 3, wherein In step S12, the dialysis duration is 24 to 72 hours.

6. The preparation method according to claim 2, wherein The step S2 comprises the following steps: S21: The pig small intestine was soaked in a methanol-chloroform mixture and then washed with PBS. After washing with PBS, it was digested with trypsin containing EDTA to obtain SIS; S22: SIS was soaked in SDS solution, washed with ethanol and freeze-dried; S23: The freeze-dried SIS obtained in step S22 is digested with pepsin. After digestion, the pH is adjusted to neutral using NaOH to obtain a porcine small intestinal submucosa hydrogel.

7. The preparation method according to claim 6, wherein In step S21, the volume ratio of methanol to chloroform in the methanol-chloroform mixed reagent is 1:(1-3).

8. Use of the composite hydrogel according to claim 1 in preparing a drug for treating Achilles tendinopathy.

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