Hydrogel for repairing volumetric muscle defects and preparation method thereof

Abstract

The invention discloses hydrogel for repairing volumetric muscle defects and a preparation method of the hydrogel, and the hydrogel is prepared from oxidized dextran, an alcohol-polyamino acid-bilirubin polymer and 3, 3 '-dithio-bis (propionyl hydrazide) according to the weight ratio of (50-75): (4-20): 1. The alcohol-polyamino acid-bilirubin polymer is prepared from an alcohol-polyamino acid polymer and bilirubin in a weight ratio of (6-25): 1. The hydrogel prepared by the invention not only has good characteristics of pH / ROS responsiveness, relatively strong tissue adhesion, antibacterial property, oxidation resistance and relatively low cytotoxicity, but also can remarkably promote cell migration, proliferation, new angiogenesis and myoblast differentiation, promote recovery of muscle structures and motor functions after volumetric muscle defects, and improve the recovery rate of muscle tissues and motor functions after the volumetric muscle defects. The material is expected to become a novel bioactive material capable of promoting volume muscle defect repair, and has a good application prospect in clinical treatment of large-volume tissue defect regeneration.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and more specifically, relates to a hydrogel and a preparation method and application thereof. Background Art

[0002] Volumetric Muscle Loss (VML) is mainly caused by trauma, car accidents, surgery or degenerative diseases, and the muscle defect volume / mass is greater than 20%. Skeletal muscle has a strong self-repair ability. In the face of minor injuries, it can completely heal itself under the action of skeletal muscle stem cells (satellite cells). However, when VML occurs, the self-repair process of skeletal muscle will be difficult to sustain. On the one hand, adverse microenvironmental factors such as abnormal inflammatory response and excessive oxidative stress interfere with the activity of skeletal muscle stem cells and other cells involved in repair, making it difficult to repair muscle structure after VML. On the other hand, due to the complete destruction of surrounding connective tissue, vascularization and loss of nerve redistribution ability, VML will cause a large amount of fiber deposition and scar formation, resulting in limited muscle function or even permanent disability. Its treatment has always been a huge challenge in the medical field.

[0003] At present, clinical treatment methods include surgical transplantation of autologous myocutaneous flaps or artificial grafts. However, due to the limited volume of donor muscle, large muscle defect area, easy infection, and low efficiency of muscle regeneration after transplantation, the functional recovery of the affected limb is poor. In addition, steroid administration, hormone replacement therapy, stem cell therapy, and new biomedical materials have also been used in VML treatment research. Among various new biomedical materials, hydrogels have become the most promising biomedical materials because of their ability to absorb tissue permeate, continuously provide a moist environment, and efficiently load bioactive molecules. Although many wound dressings have appeared in clinical practice, such as 3M wound dressings, film dressings, hydrocolloid dressings, alginate or calcium alginate dressings, and foam dressings, their scope of application is limited and they cannot simultaneously solve key problems such as the difficulty in repairing muscle structure and function after volumetric muscle defects.

[0004] The ideal hydrogel for volumetric muscle defect repair should have the following characteristics: microenvironment responsiveness, strong antibacterial properties, suitable mechanical properties, self-healing properties, tissue adhesion ability, good biocompatibility, strong anti-inflammatory and antioxidant properties, etc. For example: microenvironment responsive hydrogels can release bioactive molecules on demand during tissue defect repair, which is conducive to promoting tissue regeneration; strong antibacterial properties can prevent the defect site from being infected by bacteria and affecting healing; suitable mechanical properties can positively affect the behavior of wound cells through mechanical stimulation and promote defect repair; self-healing properties can keep the hydrogel stable during defect repair; strong tissue adhesion can ensure that the hydrogel fits tightly with the defect site, thereby continuously providing a moist microenvironment for the wound; excellent biocompatibility can reduce the systemic toxicity of the hydrogel and reduce side effects. Strong anti-inflammatory and antioxidant properties can regulate the wound microenvironment by reducing inflammatory responses and relieving excessive oxidative stress, which is conducive to defect repair.

[0005] Therefore, it is urgent to develop new hydrogels with the above-mentioned multifunctional properties for the treatment of volumetric muscle defects. Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide a hydrogel that can achieve muscle structure and function regeneration after volumetric muscle defect.

[0007] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0008] The first aspect of the present invention provides a hydrogel, which is prepared from oxidized dextran, alcohol-polyamino acid-bilirubin polymer and 3,3'-dithiobis(propionyl hydrazide) in a weight ratio of 50 to 75:4 to 20:1; the alcohol-polyamino acid-bilirubin polymer is prepared from alcohol-polyamino acid polymer and bilirubin in a weight ratio of 6 to 25:1.

[0009] The second aspect of the present invention provides a method for preparing the above hydrogel, comprising the following steps: mixing an aqueous solution of alcohol-polyamino acid-bilirubin polymer and an aqueous solution of 3,3'-dithiobis(propionylhydrazide), adding an aqueous solution of oxidized dextran, and leaving the mixture at room temperature for 3 minutes to 5 hours to obtain the hydrogel.

[0010] The third aspect of the present invention provides use of the hydrogel in treating volumetric muscle loss.

[0011] In view of the poor water solubility and unstable structure of bilirubin, which seriously affect its performance, in the present invention, the inventors modified bilirubin with a certain weight ratio of alcohol-polyamino acid polymer. On the one hand, the water solubility and stability of bilirubin can be greatly improved. On the other hand, since the bilirubin molecular structure is modified with amino groups, it can undergo Schiff base reaction with the aldehyde group of oxidized dextran and participate in the formation of a gel network, so that it can be used to prepare hydrogels to better exert the performance of bilirubin.

[0012] The inventors reacted the prepared alcohol-polyamino acid-bilirubin polymer with oxidized dextran and 3,3'-dithiobis(propionyl hydrazide) in a suitable weight ratio to prepare a hydrogel. On the one hand, the remaining amino groups of the alcohol-polyamino acid-bilirubin polymer and the amino groups of 3,3'-dithiobis(propionyl hydrazide) reacted with the aldehyde groups of the oxidized dextran to form a Schiff base reaction, so that the formed gel network structure was more stable and had higher mechanical strength than the network structure formed by oxidized dextran and 3,3'-dithiobis(propionyl hydrazide); on the other hand, due to the addition of the alcohol-polyamino acid-bilirubin polymer and by controlling the mass ratio of the three components within a certain range, the hydrogel had the ability to scavenge hydroxyl radicals, scavenging hydrogen peroxide, anti-inflammatory activity, antibacterial properties, and the like; and On the one hand, the disulfide bonds of 3,3'-dithiobis(propionylhydrazide) (DTPH) can give the gel network ROS responsiveness, realize the ROS responsive release of alcohol-polyamino acid-bilirubin polymer, and facilitate the repair of volumetric muscle defects; under the joint action of the above components and their proportions, the hydrogel prepared by the present invention not only has the good properties of pH / ROS responsiveness, strong tissue adhesion, antibacterial property, antioxidant property and low cytotoxicity, but also can significantly promote cell migration, proliferation, angiogenesis and myoblast differentiation, promote the recovery of muscle structure and motor function after volumetric muscle defects, and is expected to become a new type of bioactive material that can promote the repair of volumetric muscle defects, and has a good application prospect in the clinical treatment of large-volume tissue defect regeneration.

[0013] The hydrogel of the invention is easy to prepare, has low cost, is easy to operate, has no organic solvent residue, and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the pentaerythritol-ε-polylysine polymer in Example 1 of the present invention 1 H-NMR nuclear magnetic spectrum.

[0015] Figure 2 is the pentaerythritol-ε-polylysine-bilirubin polymer in Example 1 of the present invention 1 H-NMR nuclear magnetic spectrum.

[0016] Figure 3This is a comparison chart of the water solubility and stability of the pentaerythritol-ε-polylysine-bilirubin polymer and bilirubin in Example 1 of the present invention.

[0017] Figure 4 This is a picture of the hydrogel DPBD1 prepared in Example 1 of the present invention.

[0018] Figure 5 This is the pH responsiveness test result of the DPBD1 hydrogel in Example 1 of the present invention.

[0019] Figure 6 This is the ROS responsiveness test result of DPBD1 hydrogel in Example 1 of the present invention.

[0020] Figure 7 This is the tissue adhesion test result of DPBD1 hydrogel in Experimental Example 1 of the present invention.

[0021] Figure 8 This is a picture of the hydrogel DPBD3 prepared in Example 3 of the present invention.

[0022] Fig. 9 The water solubility pictures of PABR1 prepared in Example 1 of the present invention and PABR3 prepared in Comparative Example 1 are shown.

[0023] Fig.10 This is a picture of the hydrogel DGBD1 prepared in Example 5 of the present invention.

[0024] Fig.11 These are the test results of OH scavenging ability of DPBD1 hydrogel and PABR1 in Experimental Example 1 of the present invention.

[0025] Fig.12 These are the test results of OH scavenging ability of BR, DPBD3 hydrogel and DGBD1 hydrogel in Experimental Example 1 of the present invention.

[0026] Fig.13 This is a statistical chart of the OH scavenging ability test results of PABR1, BR, DPBD1 hydrogel, DGBD1 hydrogel and DPBD3 hydrogel in Experimental Example 1 of the present invention.

[0027] Fig.14 These are the test results of H2O2 scavenging ability of DPBD1 hydrogel, PABR1 and BR in Experimental Example 2 of the present invention.

[0028] Fig.15 This is the test result of the anti-inflammatory ability of DPBD1 hydrogel in Experimental Example 3 of the present invention.

[0029] Fig.16 This is the antibacterial ability test result of DPBD1 hydrogel in Test Example 4 of the present invention.

[0030] Fig.17 These are the cell viability results of the DPBD1 hydrogel, DD hydrogel and each component in Experimental Example 5 of the present invention after incubation in HUVECs cells for 24 hours.

[0031] Fig.18 These are the cell viability results of the DPBD3 hydrogel, DD hydrogel and each component in Experimental Example 5 of the present invention after incubation in C2C12 cells for 24 hours.

[0032] Fig.19 These are the cell viability results of the DGBD1 hydrogel, DD hydrogel and each component in Experimental Example 5 of the present invention after incubation in RAW264.7 cells for 24 hours.

[0033] Fig. 20 These are the results of the effects of DPBD1 hydrogel and DD hydrogel on the proliferation of C2C12 cells in Experimental Example 6 of the present invention.

[0034] Fig.21 These are the results of the effects of DPBD1 hydrogel, various hydrogel components, and DD hydrogel on HUVECs cell migration in Experimental Example 6 of the present invention, wherein A is a picture of cells migrating to the scratch area at different times; B is the statistical result of cell migration rate at 24h and 36h.

[0035] Fig. 22 These are the results of the effects of DPBD1 hydrogel and DD hydrogel on the myogenic differentiation of C2C12 cells in Experimental Example 6 of the present invention.

[0036] Fig.23 These are the results of the effects of DPBD1 hydrogel, various hydrogel components, and DD hydrogel on HUVECs in vitro angiogenesis in Experimental Example 6 of the present invention; wherein A is an angiogenesis picture; and B is the statistical result of the number of nodes in each group.

[0037] Fig.24 These are pictures of the defect sites of rat tibialis anterior muscle volumetric defect treated with DPBD1 hydrogel and DD hydrogel in Experimental Example 6 of the present invention 1 week and 4 weeks later.

[0038] Fig.25 This is the stride length of the operated legs of rats in each group after the DPBD1 hydrogel and DD hydrogel in Experimental Example 6 of the present invention were used to treat the volumetric muscle defect of the tibialis anterior muscle of rats for 1 week and 4 weeks. DETAILED DESCRIPTION

[0039] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0040] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0041] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0042] In some embodiments of the present invention, a hydrogel is disclosed, which is prepared from oxidized dextran, alcohol-polyamino acid-bilirubin polymer and 3,3'-dithiobis(propionyl hydrazide) in a weight ratio of 50 to 75:4 to 20:1; the alcohol-polyamino acid-bilirubin polymer is prepared from alcohol-polyamino acid polymer and bilirubin in a weight ratio of 6 to 25:1.

[0043] In some embodiments, the weight ratio of the oxidized dextran, alcohol-polyamino acid-bilirubin polymer and 3,3'-dithiobis(propionyl hydrazide) is 56-72:4-20:1.

[0044] In some embodiments, the weight ratio of the oxidized dextran, alcohol-polyamino acid-bilirubin polymer and 3,3'-dithiobis(propionyl hydrazide) is 68-72:4-16:1.

[0045] In some embodiments, the weight ratio of the alcohol-polyamino acid polymer to bilirubin is 10-20:1.

[0046] In some embodiments, the weight ratio of the alcohol-polyamino acid polymer to bilirubin is 10-15:1.

[0047] In some embodiments, the alcohol in the alcohol-polyamino acid-bilirubin polymer is pentaerythritol or glycerol.

[0048] In some embodiments, the polyamino acid in the alcohol-polyamino acid-bilirubin polymer is ε-polylysine.

[0049] In some embodiments, the alcohol-polyamino acid-bilirubin polymer is pentaerythritol-ε-polylysine-bilirubin polymer.

[0050] In some embodiments, the alcohol-polyamino acid-bilirubin polymer is glycerol-ε-polylysine-bilirubin polymer.

[0051] The present invention selects pentaerythritol or glycerol to graft ε-polylysine (EPL), so that the alcohol-polyamino acid polymer has rich amino functional groups on the basis of good biocompatibility and strong antibacterial property, so that it can better participate in the binding of bilirubin and improve the water solubility and stability of bilirubin.

[0052] In some of the embodiments, the pentaerythritol-ε-polylysine-bilirubin polymer is prepared by the following method: pentaerythritol is reacted with acryloyl chloride to obtain pentaerythritol-acrylate; pentaerythritol-acrylate is reacted with ε-polylysine to obtain pentaerythritol-ε-polylysine polymer; pentaerythritol-ε-polylysine polymer is reacted with bilirubin to obtain pentaerythritol-polyamino acid-bilirubin polymer.

[0053] In some of the embodiments, the glycerol-ε-polylysine-bilirubin polymer is prepared by the following method: glycerol is reacted with acryloyl chloride to obtain glycerol-acrylate; glycerol-acrylate is reacted with ε-polylysine to obtain glycerol-ε-polylysine polymer; glycerol-ε-polylysine polymer is reacted with bilirubin to obtain glycerol-polyamino acid-bilirubin polymer.

[0054] In other embodiments of the present invention, a method for preparing the above-mentioned hydrogel is disclosed, comprising the following steps: mixing an aqueous solution of an alcohol-polyamino acid-bilirubin polymer with an aqueous solution of 3,3'-dithiobis(propionylhydrazide), then adding an aqueous solution of oxidized dextran, and leaving the mixture at room temperature for 3 minutes to 5 hours.

[0055] In other embodiments of the present invention, the use of the above hydrogel in treating volumetric muscle defect is disclosed.

[0056] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0057] Example 1

[0058] The hydrogel DPBD1 provided in this embodiment is prepared from 90 μL of oxidized dextran aqueous solution (DCHO, 10 wt%), 5 μL of pentaerythritol-ε-polylysine-bilirubin polymer aqueous solution (PABR1, 10 wt%) and 5 μL of DTPH aqueous solution (3,3'-dithiobis(propionylhydrazide), 2.5 wt%), and the mass ratio of the three components is 72:4:1. The preparation method thereof comprises the following steps:

[0059] 1. Preparation of oxidized dextran (DCHO)

[0060] 2 g of dextran was dissolved in 150 mL of distilled water, 1.98 g of sodium periodate (NaIO4) was added, and the mixture was reacted at room temperature in the dark for 4 h, and then 2 mL of ethylene glycol was added to quench the reaction. After the reaction, the reaction solution was dialyzed in distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 1000, and freeze-dried to obtain oxidized dextran (DCHO) (yield 76%).

[0061] 2. Preparation of pentaerythritol-ε-polylysine-bilirubin polymer

[0062] (1) Preparation of pentaerythritol-tetraacrylate

[0063] Dissolve 2g of pentaerythritol in 100mL of dichloromethane, then add 10mL of triethylamine and 13.4g of acryloyl chloride in turn, and react at room temperature for 24h. After the reaction is completed, wash the organic phase with dilute hydrochloric acid and saturated sodium bicarbonate solution respectively. Rotary evaporate the organic phase to obtain pentaerythritol-tetraacrylate (yield 70%).

[0064] (2) Preparation of pentaerythritol-ε-polylysine polymer (PAP)

[0065] 1 g of pentaerythritol-tetraacrylate and 47.6 g of ε-polylysine (EPL, molecular weight 3500 Da) were dissolved in 10 mL of DMSO and 200 mL of H2O, respectively, and then the pentaerythritol-tetraacrylate solution was added to the ε-polylysine solution, and reacted at 80°C for 72 hours. After the reaction, the solution was dialyzed in distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 10,000, and freeze-dried to obtain pentaerythritol-ε-polylysine polymer (PAP).

[0066] Figure 1 It is a pentaerythritol-ε-polylysine polymer (PAP) 1 H-NMR nuclear magnetic spectrum. It can be seen from the figure that the characteristic peaks belonging to pentaerythritol and ε-polylysine can be displayed on the spectrum, indicating that PAP was successfully synthesized.

[0067] (3) Preparation of pentaerythritol-ε-polylysine-bilirubin polymer (PABR1)

[0068] 1g of bilirubin (BR), 1.2g of N-hydroxysuccinimide and 0.2g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride were mixed and dissolved in 50mL of DMSO and stirred at room temperature for 30min. Then 10g of pentaerythritol-ε-polylysine polymer was dissolved in 50mL of water and added to the mixed solution to react at 80°C for 72h. After the reaction was completed, the mixture was dialyzed in distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 10,000 and freeze-dried to obtain pentaerythritol-ε-polylysine-bilirubin polymer (PABR1).

[0069] Figure 2 It is a polymer of pentaerythritol-ε-polylysine-bilirubin (PABR1). 1 H-NMR nuclear magnetic spectrum. It can be seen from the figure that the characteristic peaks belonging to pentaerythritol, ε-polylysine and bilirubin can be displayed on the spectrum, indicating that PABR1 was successfully synthesized.

[0070] Figure 3 This is a comparison chart of the water solubility of PABR1 and BR. The results show that the water solubility and stability of bilirubin (PABR1) modified with pentaerythritol-ε-polylysine polymer are significantly higher than those of unmodified bilirubin (BR).

[0071] 3. Preparation of hydrogel (DPBD1)

[0072] 5 μL of pentaerythritol-ε-polylysine-bilirubin polymer (PABR1) solution (10 wt%) and 5 μL of DTPH aqueous solution (2.5 wt%) were mixed; then, 90 μL of oxidized dextran (DCHO) aqueous solution (10 wt%) was added to the mixture, mixed evenly and placed at room temperature for 3 min to form a gel state, thereby obtaining the DPBD1 hydrogel of this embodiment. Figure 4 As can be seen, DPBD1 is a soft yellow transparent gel.

[0073] The following performance tests were performed on the hydrogel DPBD1 prepared in this example.

[0074] 1. pH response test

[0075] Dilute hydrochloric acid (4 mol / L) was added to the hydrogel DPBD1. After a period of time, the hydrogel sample was observed to see whether it became a solution, so as to evaluate its pH responsiveness. The results are as follows: Figure 5 The results showed that after adding dilute hydrochloric acid to DPBD1, the Schiff base bonds in the gel network were broken, causing DPBD1 to change from a gel state to a solution state, indicating that it has pH responsiveness.

[0076] 2. Reactive oxygen species (ROS) response test

[0077] Hydrogen peroxide (H2O2) was added to the hydrogel DPBD1, and the state changes of the DPBD1 hydrogel were observed to evaluate its ROS response. Figure 6 The results showed that after adding H2O2 to DPBD1 hydrogel, DPBD1 changed from a gel state to a solution, indicating that DPBD1 is ROS responsive.

[0078] 3. Tissue Adhesion Determination

[0079] Take two fresh muscles, add 100 μL of hydrogel sample between the two muscles and press for 3 minutes at room temperature. Then clamp one of the muscles with tweezers, observe and take pictures to record the adhesion of the hydrogel to the muscle and whether the other muscle falls off, so as to evaluate the tissue adhesion of each sample. The results are as follows: Figure 7 The results showed that after lifting a piece of muscle tissue, another piece of muscle tissue could be tightly adhered to the muscle tissue above under the action of hydrogel, proving that it has strong tissue adhesion.

[0080] In summary, the hydrogel DPBD1 of this embodiment has pH responsiveness, ROS responsiveness and strong tissue adhesion.

[0081] Example 2

[0082] The hydrogel DPBD2 provided in this embodiment is prepared from 85 μL of oxidized dextran aqueous solution (DCHO, 10 wt%), 10 μL of pentaerythritol-ε-polylysine-bilirubin polymer aqueous solution (PABR1, 10 wt%) and 5 μL of DTPH aqueous solution (3,3'-dithiobis(propionylhydrazide), 2.5 wt%), and the mass ratio of the three components is 68:8:1. The preparation method is the same as that in Example 1.

[0083] The hydrogel DPBD2 of this example also has pH responsiveness, ROS responsiveness and strong tissue adhesion.

[0084] Example 3

[0085] The hydrogel DPBD3 provided in this embodiment is prepared from 90 μL of oxidized dextran aqueous solution (DCHO, 10 wt%), 5 μL of pentaerythritol-ε-polylysine-bilirubin polymer aqueous solution (PABR2, 10 wt%) and 5 μL of DTPH aqueous solution (3,3'-dithiobis(propionylhydrazide), 2.5 wt%), and the mass ratio of the three components is 72:4:1. The preparation method thereof comprises the following steps:

[0086] 1. Preparation of oxidized dextran (DCHO)

[0087] Same as Example 1.

[0088] 2. Preparation of pentaerythritol-ε-polylysine-bilirubin polymer

[0089] (1) Preparation of pentaerythritol-tetraacrylate

[0090] Same as Example 1.

[0091] (2) Preparation of pentaerythritol-ε-polylysine polymer (PAP)

[0092] Same as Example 1.

[0093] (3) Preparation of pentaerythritol-ε-polylysine-bilirubin polymer (PABR2)

[0094] 0.5g bilirubin (BR), 0.6g N-hydroxysuccinimide and 0.1g 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride were mixed and dissolved in 25mL DMSO and stirred at room temperature for 30min. Then 10g pentaerythritol-ε-polylysine polymer was dissolved in 50mL water and added to the above mixed solution to react at 80°C for 72h. After the reaction was completed, it was dialyzed in distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 10,000 and freeze-dried to obtain pentaerythritol-ε-polylysine-bilirubin polymer (PABR2).

[0095] 3. Preparation of hydrogel (DPBD3)

[0096] 5 μL of pentaerythritol-ε-polylysine-bilirubin polymer (PABR2) solution (10 wt%) and 5 μL of DTPH aqueous solution (2.5 wt%) were mixed; then, 90 μL of oxidized dextran (DCHO, stained with rhodamine for easy observation) aqueous solution (10 wt%) was added to the above mixture, mixed evenly and placed at room temperature for 10 min to form a gel state, thus obtaining the DPBD3 hydrogel of this embodiment. Figure 8 It can be seen that DPBD3 is a soft yellow transparent gel.

[0097] The hydrogel DPBD3 of this example also has pH responsiveness, ROS responsiveness and strong tissue adhesion.

[0098] Example 4

[0099] The hydrogel DPBD4 provided in this embodiment is prepared from 150 μL of oxidized dextran aqueous solution (DCHO, 10 wt%), 40 μL of pentaerythritol-ε-polylysine-bilirubin polymer aqueous solution (PABR2, 10 wt%) and 10 μL of DTPH aqueous solution (3,3'-dithiobis(propionylhydrazide), 2.5 wt%), and the mass ratio of the three components is 60:16:1. The preparation method is the same as that in Example 3.

[0100] The hydrogel DPBD4 prepared in this example also has pH responsiveness, ROS responsiveness and strong tissue adhesion.

[0101] Example 5

[0102] The hydrogel DGBD1 provided in this embodiment is prepared from 180 μL of oxidized dextran aqueous solution (DCHO, 10 wt%), 10 μL of glycerol-ε-polylysine-bilirubin polymer aqueous solution (GABR, 10 wt%) and 10 μL of DTPH aqueous solution (3,3'-dithiobis(propionylhydrazide), 2.5 wt%), and the mass ratio of the three components is 72:4:1. The preparation method thereof comprises the following steps:

[0103] 1. Preparation of oxidized dextran (DCHO)

[0104] Same as Example 1.

[0105] 2. Preparation of pentaerythritol-ε-polylysine-bilirubin polymer

[0106] (1) Preparation of glycerol-triacrylate

[0107] 2 g of glycerol was dissolved in 100 mL of dichloromethane, and then 10 mL of triethylamine and 13.4 g of acryloyl chloride were added in sequence, and the mixture was reacted at room temperature for 24 h. After the reaction was completed, the organic phase was washed with dilute hydrochloric acid and saturated sodium bicarbonate solution, respectively. The organic phase was rotary evaporated to obtain glycerol triacrylate (yield 70%).

[0108] (2) Preparation of glycerol-ε-polylysine polymer (GAP)

[0109] 1 g of glycerol-triacrylate and 47.6 g of ε-polylysine (EPL, molecular weight 3500 Da) were dissolved in 10 mL of DMSO and 200 mL of H2O, respectively. Then, the glycerol-triacrylate solution was added to the EPL solution and reacted at 80°C for 72 h. After the reaction, the mixture was dialyzed in distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 1000 and freeze-dried to obtain glycerol-ε-polylysine polymer (GAP).

[0110] (3) Preparation of glycerol-ε-polylysine-bilirubin polymer (GABR)

[0111] 1g of bilirubin (BR), 1.2g of N-hydroxysuccinimide and 0.2g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride were mixed and dissolved in 50mL of DMSO and stirred at room temperature for 30min. Then 10g of glycerol-ε-polylysine was dissolved in 50mL of water and added to the mixed solution to react at 80°C for 72h. After the reaction was completed, the mixture was dialyzed in distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 10,000 and freeze-dried to obtain glycerol-ε-polylysine-bilirubin polymer (GABR).

[0112] 3. Preparation of hydrogel (DGBD1)

[0113] 10 μL of glycerol-ε-polylysine-bilirubin polymer (GABR) aqueous solution (10 wt%) and 10 μL of DTPH aqueous solution (2.5 wt%) were mixed; then, 180 μL of oxidized dextran (DCHO, stained with rhodamine for easy observation) aqueous solution (10 wt%) was added to the above mixture, mixed evenly and placed at room temperature for 3 minutes to form a gel state, thereby obtaining the DGBD1 hydrogel of this embodiment. Fig. 9 It can be seen that DGBD1 is a soft yellow transparent gel.

[0114] The hydrogel DGBD1 of this example also has pH responsiveness, ROS responsiveness and strong tissue adhesion.

[0115] Example 6

[0116] The hydrogel DGBD2 provided in this embodiment is prepared from 140 μL of oxidized dextran aqueous solution (DCHO, 10 wt%), 5 μL of glycerol-ε-polylysine-bilirubin polymer aqueous solution (GABR, 10 wt%) and 10 μL of DTPH aqueous solution (3,3'-dithiobis(propionylhydrazide), 2.5 wt%), and the mass ratio of the three components is 56:20:1. The preparation method is the same as that in Example 5.

[0117] The hydrogel DGBD2 of this example also has pH responsiveness, ROS responsiveness and strong tissue adhesion.

[0118] Comparative Example 1

[0119] In this comparative example, pentaerythritol-ε-polylysine-bilirubin polymer PABR3 was prepared and its solubility was tested.

[0120] (1) Preparation of pentaerythritol-tetraacrylate

[0121] Same as Example 1.

[0122] (2) Preparation of pentaerythritol-ε-polylysine polymer (PAP)

[0123] Same as Example 1.

[0124] (3) Preparation of pentaerythritol-ε-polylysine-bilirubin polymer (PABR3)

[0125] 2g of bilirubin (BR), 2.4g of N-hydroxysuccinimide and 0.4g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride were mixed and dissolved in 25mL of DMSO and stirred at room temperature for 30min. Then 10g of pentaerythritol-ε-polylysine polymer was dissolved in 50mL of water and added to the above mixed solution to react at 80°C for 72h. After the reaction was completed, the mixture was dialyzed in distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 10,000 and freeze-dried to obtain pentaerythritol-ε-polylysine-bilirubin polymer (PABR3).

[0126] 0.1 g of pentaerythritol-ε-polylysine-bilirubin polymer PABR1 and pentaerythritol-ε-polylysine-bilirubin polymer PABR3 were added to 1 mL of pure water, and the results were as follows: Fig.10 As shown, from Fig.10 It can be seen that the pentaerythritol-ε-polylysine-bilirubin polymer PABR3 cannot be completely dissolved and cannot be used to prepare hydrogels.

[0127] Comparative Example 2

[0128] The hydrogel provided in this comparative example was prepared by mixing 5 μL of DTPH aqueous solution (2.5 wt%) and 90 μL of DCHO aqueous solution (10 wt%) uniformly and leaving the mixture at room temperature for 3 minutes, and was named DD.

[0129] Test Example 1 Hydroxyl Radical (·OH) Scavenging Ability Test

[0130] In this test example, the ·OH scavenging abilities of the DPBD1 hydrogel of Example 1, the DPBD3 hydrogel of Example 3, the DGBD1 hydrogel of Example 5, PABR1, and BR were measured.

[0131] Aqueous solutions of hydrogels DPBD1, DPBD3, DGBD1, PABR1 (pentaerythritol-ε-polylysine-bilirubin polymer), and BR (unmodified bilirubin) were incubated with a mixed solution of methylene blue (MB, 10 μg / mL), H2O2 (1 mM), and FeCl2·4H2O (0.2 mg / mL), respectively. The supernatants were taken at different times to measure their absorbance at 500 nm-800 nm to evaluate their ·OH scavenging ability.

[0132] MB solution has a high absorbance in the wavelength range of 500-800nm.2+ It reacts with H2O2 to generate ·OH, which can react with MB to decolorize it, reducing the absorbance of the MB solution. The ability of the hydrogel to remove ·OH can be evaluated by measuring the absorbance of the mixed solution at 500-800nm ​​at different times.

[0133] The results are as follows Figure 11 to Figure 13 As shown by Fig.11 It can be seen that compared with the MB group, MB+Fe 2+ The absorbance of the +H2O2 group was significantly reduced, indicating that the generated ·OH reacted with MB to decolorize it. Pentaerythritol-ε-polylysine-bilirubin polymer PABR1 can effectively remove ·OH, and its absorbance at different times is slightly higher than that of the MB group. The absorbance of the DPBD1 group is significantly higher than that of the MB+Fe 2+ +H2O2 group, indicating that under the action of pentaerythritol-ε-polylysine-bilirubin polymer PABR1, DPBD1 hydrogel can also effectively remove ·OH( Fig.11 ). BR, DPBD3 and DGBD1 can also effectively remove ·OH( Fig.12 ). The ·OH scavenging ability of DPBD3 is slightly lower than that of DGBD1 and DPBD1 ( Fig.13 ). Therefore, the hydrogels DGBD1, DPBD3, DPBD1, pentaerythritol-ε-polylysine-bilirubin polymer and bilirubin all have ·OH scavenging ability.

[0134] Test Example 2: Hydrogen peroxide (H2O2) removal capability test

[0135] In this test example, the scavenging ability of DPBD1 hydrogel, pentaerythritol-ε-polylysine-bilirubin polymer PABR1 and bilirubin (BR) of Example 1 on H2O2 was measured to evaluate their antioxidant properties.

[0136] A certain concentration of H2O2 was added to pentaerythritol-ε-polylysine-bilirubin polymers PABR1, DPBD1 and BR, and then placed in a 37°C shaker for different incubation times. After reaching the set time point, 50 μL of the supernatant from each group was taken and added with titanium sulfate solution (100 μL) and incubated at room temperature for 30 minutes, and then the absorbance of each sample at 405 nm was measured by an ELISA instrument. Finally, the H2O2 concentration in each sample was calculated according to the H2O2 standard curve, and the antioxidant properties of pentaerythritol-ε-polylysine-bilirubin polymers PABR1, DPBD1 and bilirubin BR were investigated.

[0137] The results are as follows Fig.14 As shown. Fig.14 It can be seen that pentaerythritol-ε-polylysine-bilirubin polymers PABR1, DPBD1 and BR can effectively remove H2O2 and have strong antioxidant properties.

[0138] Test Example 3 Anti-inflammatory ability test

[0139] RAW 264.7 macrophages were first induced with lipopolysaccharide (LPS). The growth medium was then replaced with medium containing DPBD1 (20 μg / mL), bilirubin (BR, 20 μg / mL), pentaerythritol-ε-polylysine-bilirubin polymer PABR1 (20 μg / mL) and a positive control group (Dex, concentration 2 μg / mL). After 24 hours of co-culture, the supernatant was collected and the TNF-α content in the supernatant of each group was detected using an ELISA kit to evaluate the anti-inflammatory ability of the hydrogel.

[0140] The results are as follows Fig.15 As shown. Fig.15 It can be seen that compared with the LPS group, the levels of inflammatory factors (TNF-α) in the supernatant of the BR group, PABR1 group and DPBD1 group were significantly reduced. The level of inflammatory factors (TNF-α) in the supernatant of the DPBD1 group was reduced more significantly, slightly lower than the positive control dexamethasone (Dex) group, and significantly different from the BR and PABR1 groups, indicating that DPBD1 can significantly reduce the secretion of inflammatory factors in LPS-induced macrophages and show strong anti-inflammatory activity.

[0141] Test Example 4 Antibacterial Ability Test

[0142] This test example measured the killing ability of pentaerythritol-ε-polylysine-bilirubin polymer PABR1 and DPBD1 hydrogels of Example 1; pentaerythritol-ε-polylysine-bilirubin polymer PABR2 and DPBD3 hydrogels of Example 3; and glycerol-ε-polylysine-bilirubin polymer GABR and DGPD1 hydrogels of Example 5 against methicillin-resistant Staphylococcus aureus (MRSA) to evaluate their antibacterial properties.

[0143] MRSA was cultured to the logarithmic growth phase for later use. Each hydrogel sample and control group were prepared in a 24-well plate, and then the diluted bacterial suspension (10 μL, 10 6 CFU mL -1 ) was added to the well plate and incubated in a 37°C incubator for 2 hours. Finally, the bacterial solution was diluted with PBS, and 10 μL of the dilution was plated on LB agar for 18 hours. The number of colonies on each plate was counted to evaluate the antibacterial activity of each sample.

[0144] The results are as follows Fig.16 As shown. Fig.16It can be seen that compared with the blank group (NC), the number of bacteria was significantly reduced after treatment with pentaerythritol-ε-polylysine-bilirubin polymer PABR1, PABR2 or glycerol-ε-polylysine-bilirubin polymer GABR. The number of bacteria in the hydrogel DD, DPBD1, DPBD3 and DGPD1 groups was also small, indicating that PABR1, PABR2, GABR, DPBD1, DPBD3 and DGBD1 all have strong antibacterial properties.

[0145] Test Example 5 Biocompatibility Performance

[0146] This test example compares the cytotoxicity of the DPBD1 hydrogel of Example 1, the DPBD3 hydrogel of Example 3, the DGBD1 hydrogel of Example 5, and the DD hydrogel of Comparative Example 2.

[0147] (1) After incubating HUVECs cells with different concentrations of the components of DPBD1 hydrogel, DPBD1 hydrogel and DD hydrogel for 24 h, the cell viability was detected using the CCK8 kit to evaluate the cytotoxicity. The results are as follows: Fig.17 shown.

[0148] (2) C2C12 cells were used to evaluate the cytotoxicity of the components of DPBD3 hydrogel, DPBD3 hydrogel, and DD hydrogel. Fig.18 shown.

[0149] (3) RAW264.7 cells were used to evaluate the cytotoxicity of the components of DGBD1 hydrogel, DGBD1 hydrogel, and DD hydrogel. Fig.19 shown.

[0150] Depend on Figure 17 to Figure 19 The results showed that in HUVECs cells, C2C12 cells and RAW264.7 cells, the cell viability of DPBD1 hydrogel, DPBD3 hydrogel, DGBD1 hydrogel and DD hydrogel was high at different concentrations. When the concentration reached 80 μg / mL, the cell viability was above 90%, indicating that the cytotoxicity of DPBD1, DPBD3, DGBD1 hydrogel and its components, and DD hydrogel were relatively low and had high biocompatibility.

[0151] Experimental Example 6 Effect on Repair of Volumetric Muscle Defect

[0152] This test example compares the DPBD1 hydrogel of Example 1 and the DD hydrogel of Comparative Example 1 in terms of cell proliferation, migration ability, angiogenesis ability, and volume-promoting muscle defect repair ability.

[0153] 1. Cell proliferation test

[0154] The effects of DPBD1 hydrogel and DD hydrogel on cell proliferation were determined in C2C12 cells using the AlamarBlue kit. Each sample was added to the cells, and the cell viability of each group was detected using the Alamar Blue reagent after 1, 3, and 5 days of co-culture to investigate its effect on cell proliferation. The results are shown in Figure 2. Fig. 20 shown.

[0155] Depend on Fig. 20 It can be seen that as the incubation time increases, the cell viability of each group increases. Compared with the NC group, the cell viability of the DD and DPBD1 groups was higher after 3 days of culture, indicating that the proliferation rate was accelerated; after 5 days of culture, the cell viability of the DD and DPBD1 groups increased further, and compared with the DD group, the cell viability of the DPBD1 group was significantly improved, indicating that the DPBD1 hydrogel can significantly promote cell proliferation.

[0156] 2. Cell migration ability test

[0157] First, a disinfected streaker was placed on a 6-well plate, and then C2C12 cells were inoculated in the gaps of the streaker. After the cells adhered to the wall, the streaker was taken out and DPBD1 hydrogel and DD hydrogel were added. The untreated group (negative control, NC) was used as a control, and photos were taken under a microscope at different times to record the migration of cells in each group to the scratch area, in order to investigate the effect of hydrogel on cell migration.

[0158] The results are as follows Fig.21 As shown by Fig.21 As shown in A, after 24 hours of co-culture, cells in each group migrated to the scratch area, and the cell migration rate of the DPBD1 group reached about 90%; after 36 hours, the cell migration rate of each group increased significantly, and the cell migration rate of the DPBD1 group was slightly higher than that of the DD group, close to 100% ( Fig.21 B), indicating that DPBD1 hydrogel can significantly promote cell migration.

[0159] 3. Myogenic differentiation ability test

[0160] DPBD1 hydrogel and DD hydrogel were added to differentiation medium (2% horse serum + DMEM) respectively. After C2C12 cells adhered to the wall, the growth medium was replaced with the above differentiation medium every two days. After 7 days, the effect of each sample on the generation of new myotubes (myosin heavy chain, MHC) was detected by immunofluorescence staining to investigate the effect of DPBD1 on the myogenic differentiation of C2C12 cells.

[0161] The results are as follows Fig. 22 As shown by Fig. 22It can be seen that compared with each control, the number of newly differentiated myotubes (shown by green fluorescence) from C2C12 cells in the DPBD1 group increased significantly and was higher than that in the DD group without PABR1, indicating that DPBD1 can promote myoblast differentiation and that PABR1 plays an important role in the myogenic differentiation of C2C12 cells.

[0162] 4. In vitro angiogenesis test

[0163] Matrigel was added to a pre-cooled 96-well plate (50 μL / well) and placed in a cell culture incubator to solidify for about 30-60 minutes. 80 μL of HUVECs cell suspension (1% FBS) and 20 μL of DPBD1 hydrogel or DD hydrogel dilution (final concentration 20 μg / mL) were added to the plate and placed in an incubator for 4 hours. The angiogenesis of each group was recorded by taking photos to evaluate the in vitro angiogenesis ability of each sample.

[0164] The results are as follows Fig.23 As shown by Fig.23 As shown in A and B, compared with the control, the number of nodes in the DCHO, DD and DPBD1 groups was significantly greater, and the number of nodes in the DPBD1 group was the largest, indicating that DPBD1 can promote angiogenesis.

[0165] 5. Experiment on promoting the repair of volumetric muscle defects

[0166] The repair ability of hydrogel on volumetric muscle defect was determined in the SD rat tibialis anterior muscle volumetric muscle defect model. The steps are as follows:

[0167] (1) Rats were anesthetized, and the hair of the hind legs was shaved and disinfected. The skin on the outside of the hind legs was cut open with a scalpel to expose the tibialis anterior muscle, and a rectangular defect (10 mm × 7 mm × 3 mm, muscle loss greater than 20%) was made on the muscle belly to form the VML model. Rats were randomly divided into the following four groups (n = 6): control group (blank), DCHO group, DD group, and DPBD1 group.

[0168] (2) Each material was filled into the defect and the skin incision was sutured. The untreated group served as the control.

[0169] (3) All operations were performed under sterile conditions. Rats were killed 1 week and 4 weeks after surgery. The defect repair status of each group was recorded by taking photos, and the defect tissue was taken for histological analysis. At the same time, gait analysis was performed on the operated legs of the rats 1 week and 4 weeks after surgery, and the stride length of the operated legs of each group was measured to evaluate the recovery of their motor function.

[0170] Fig.24 The prepared hydrogel was used to treat the wound recovery of rat tibialis anterior muscle volume defect after 1 week and 4 weeks. Fig.24It can be seen that one week after surgery, the defect area of ​​each group was still relatively obvious, while the defect area of ​​the DPBD1 group was reduced. Four weeks after surgery, the defect area of ​​the DPBD1 group was basically repaired, while there were still obvious defect marks in the control groups, indicating that DPBD1 hydrogel can promote the recovery of muscle structure after volumetric muscle defect.

[0171] Fig.25 The stride length of the operated leg of each group of rats after 1 week and 4 weeks of treatment with the prepared hydrogel for rat tibialis anterior muscle volume defect. The stride length of the operated leg was measured by gait analysis to measure the recovery of muscle motor function. Fig.25 It can be seen that one week after surgery, the stride lengths of the Blank, DCHO and DD groups were 9.1 cm, 9.06 cm and 9.13 cm, respectively, and the stride length of the DPBD1 group was 11.5 cm, which was significantly higher than that of the control groups. After 4 weeks, the stride length of the DPBD1 group further increased to 12.8 cm, which was comparable to that of the normal group (12.43 cm), indicating that DPBD1 can promote the recovery of muscle motor function in volumetric muscle deficiency.

[0172] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A hydrogel, characterized in that: The invention is prepared from oxidized dextran, alcohol-polyamino acid-bilirubin polymer and 3,3'-dithiobis(propionyl hydrazide) in a weight ratio of 50-75:4-20:1; the alcohol-polyamino acid-bilirubin polymer is prepared from alcohol-polyamino acid polymer and bilirubin in a weight ratio of 6-25:

1.

2. The hydrogel according to claim 1, characterized in that The weight ratio of the alcohol-polyamino acid polymer to bilirubin is 10-20:

1.

3. The hydrogel according to claim 2, characterized in that The weight ratio of the alcohol-polyamino acid polymer to bilirubin is 10-15:

1.

4. The hydrogel according to claim 1, characterized in that The weight ratio of the oxidized dextran, the alcohol-polyamino acid-bilirubin polymer and 3,3'-dithiobis(propionyl hydrazide) is 56-72:4-20:

1.

5. The hydrogel according to claim 4, characterized in that The weight ratio of the oxidized dextran, the alcohol-polyamino acid-bilirubin polymer and 3,3'-dithiobis(propionyl hydrazide) is 68-72:4-16:

1.

6. The hydrogel according to any one of claims 1 to 5, characterized in that The alcohol in the alcohol-polyamino acid-bilirubin polymer is pentaerythritol or glycerol; and / or, the polyamino acid in the alcohol-polyamino acid-bilirubin polymer is ε-polylysine.

7. The hydrogel according to claim 6, characterized in that The alcohol-polyamino acid-bilirubin polymer is a pentaerythritol-ε-polylysine-bilirubin polymer or a glycerol-ε-polylysine-bilirubin polymer.

8. The hydrogel according to claim 7, characterized in that The pentaerythritol-ε-polylysine-bilirubin polymer is prepared by the following method: pentaerythritol reacts with acryloyl chloride to obtain pentaerythritol-acrylate; pentaerythritol-acrylate reacts with ε-polylysine to obtain pentaerythritol-ε-polylysine polymer; pentaerythritol-ε-polylysine polymer reacts with bilirubin to obtain pentaerythritol-polyamino acid-bilirubin polymer; or, The glycerol-ε-polylysine-bilirubin polymer is prepared by the following method: glycerol is reacted with acryloyl chloride to obtain glycerol-acrylate; glycerol-acrylate is reacted with ε-polylysine to obtain glycerol-ε-polylysine polymer; glycerol-ε-polylysine polymer is reacted with bilirubin to obtain glycerol-polyamino acid-bilirubin polymer.

9. The method for preparing the hydrogel according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: mixing the aqueous solution of the alcohol-polyamino acid-bilirubin polymer and the aqueous solution of 3,3'-dithiobis(propionyl hydrazide), adding the aqueous solution of oxidized dextran, and placing at room temperature for 3 minutes to 5 hours to obtain the product.

10. Use of the hydrogel according to any one of claims 1 to 8 in treating volumetric muscle loss.

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