Mecobalamin nano-functionalized collagen polysaccharide sponge and application thereof

Abstract

The invention belongs to the technical field of medicine research, and particularly relates to a mecobalamin nano-functionalized collagen polysaccharide sponge and application thereof. The mecobalamin nano-functionalized collagen polysaccharide sponge is prepared according to the following steps: cross-linking carboxyl on the surface of collagen with amino of chitosan, and cross-linking with hyaluronic acid to obtain a bioactive material; the preparation method comprises the following steps: carrying out hydrothermal reaction by taking mecobalamine as a raw material to obtain mecobalamine nanodots; and mixing the mecobalamin nanodots with a bioactive material, and carrying out electrostatic adsorption to obtain the collagen polysaccharide sponge. The collagen polysaccharide sponge provided by the invention can solve multiple challenges of easy diffusion and degradation, delivery barrier, microenvironment interference and the like faced by application of mecobalamine molecules in spinal cord injury inflammation parts, and realizes key technical problems in multiple aspects of long-term inflammation / immune microenvironment regulation, targeted polarization of BV2 cells, efficient nerve regeneration and the like; the biodegradability and the cell adaptability are relatively good.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug research, and particularly relates to a methylcobalamin-functionalized collagen polysaccharide sponge and its application. Background Art

[0002] Spinal cord injury (SCI) is usually caused by traffic accidents, falls, sports injuries, etc., and often leads to the loss of sensory, motor and autonomic nerve functions, and even paralysis. After spinal cord injury, the local microenvironment will deteriorate rapidly, and the levels of a large number of reactive oxygen species will increase sharply, triggering a strong inflammatory response. Subsequently, the injured area will experience a variety of secondary complications, seriously affecting the recovery of motor nerve function. In addition, the inflammatory immune microenvironment makes it difficult for endogenous NSCs to stably grow and develop in the injured area, and is more likely to differentiate into astrocytes, producing glial scars, which hinder cell migration and nerve regeneration. Microglia (BV2), as key participants in the inflammatory response after spinal cord injury, can polarize into two phenotypes, M1 (pro-inflammatory) and M2 (anti-inflammatory), and play a key role in regulating the entire trajectory of the spinal cord injury microenvironment. The injury microenvironment will induce microglia to polarize into the inflammatory M1 phenotype, and then cause the expansion of neuroinflammation, neuronal apoptosis and increased axonal degeneration by releasing a large number of inflammatory factors, further aggravating nerve tissue damage. At the same time, according to relevant research reports, microglia will permanently switch to different transcriptional states in the spinal cord immune microenvironment, resulting in more long-term changes in the spinal cord immune microenvironment, and most likely becoming a new target for spinal cord injury repair. Therefore, it is crucial to solve the persistent inflammatory response in the injury microenvironment, protect the damaged neurons and effectively achieve the targeted cascade regulation of BV2 cells.

[0003] Currently, the treatment methods for SCI mainly include surgical treatment and emerging stem cell treatment methods. Surgical treatment can maintain the stability of the spine to reduce further damage, but patients must bear the postoperative physical pain and related infections, and the efficacy of early decompression surgery for complete SCI is limited. Stem cell transplantation is a potential repair method that may change traditional clinical treatment. However, stem cell transplantation may also block blood vessels, induce tumors, and cause a series of rejection reactions in the body, and the mechanism of action of stem cell transplantation is not yet fully understood. In recent years, biomaterials have been widely used as related drug carriers and anti-inflammatory agents due to their unique physical and chemical properties, providing more treatment options for SCI.

[0004] Methylcobalamin is an endogenous coenzyme B12 that can promote the synthesis of nucleic acids and proteins in nerve cells, as well as the synthesis of lecithin, the main component of myelin sheaths. These processes are crucial for the normal function and repair of the nervous system. However, methylcobalamin has a short half-life of about 6-8 hours and is easily cleared rapidly by the kidneys, making it difficult to maintain an effective concentration at the injury site for a long time and requiring frequent administration to maintain efficacy. Moreover, it lacks active targeting ability and easily diffuses from the injury site to the systemic circulation, resulting in insufficient local drug concentration and potentially increasing systemic side effects (such as rashes and gastrointestinal reactions). In addition, the traditional routes of oral and intravenous administration will prevent these drugs from smoothly crossing the blood-brain / spinal cord barrier. These factors make it difficult for methylcobalamin to accumulate at the inflammatory site for a long time and exert a continuous therapeutic effect. Therefore, it is of great significance to develop new multifunctional biomaterials with multiple anti-inflammatory properties, modified methylcobalamin sustained-release function, promotion of nerve regeneration, and restoration of motor function. Summary of the Invention

[0005] In view of the multiple challenges currently faced by methylcobalamin in the application at the spinal cord injury inflammatory site, such as easy diffusion and degradation, delivery barriers, and microenvironment interference, as well as the key technical issues of how to efficiently achieve the long-term regulation of the inflammatory / immune microenvironment, targeted polarization of BV2 cells, and nerve regeneration of biomaterials, the present invention has developed a multifunctional composite biological "bridge" through a synthesis method of low-temperature extraction, dilute acid purification, and multiple chemical crosslinking, and successfully prepared methylcobalamin nanodots by an efficient hydrothermal synthesis method, and finally synthesized a methylcobalamin nanofunctionalized collagen polysaccharide sponge.

[0006] The present invention provides a methylcobalamin nanofunctionalized collagen polysaccharide sponge, which is prepared according to the following steps: Crosslink the carboxyl groups on the surface of collagen with the amino groups of chitosan, and then crosslink with hyaluronic acid to obtain a bioactive material; Use methylcobalamin as a raw material for hydrothermal reaction to obtain methylcobalamin nanodots; Mix the methylcobalamin nanodots with the bioactive material for electrostatic adsorption to obtain the methylcobalamin nanofunctionalized collagen polysaccharide sponge. Methylcobalamin nanodots carry a positive charge, and the bioactive material as a whole carries a negative charge, so electrostatic adsorption will occur between the two, making the combination tighter.

[0007] As a preferred embodiment of the present invention, the mass ratio of the active collagen, chitosan, and sodium hyaluronate is 1-2.5:1:1.

[0008] As a preferred embodiment of the present invention, the mixing mass ratio of the bioactive material to the methylcobalamin nanodots is 2-5:1.

[0009] As a preferred embodiment of the present invention, the hydrothermal reaction is carried out at 80°C to 140°C for 2 h to 8 h.

[0010] As a preferred embodiment of the present invention, the solution of collagen and the crosslinking agent are stirred and mixed at 4°C to 10°C for 2 h to 6 h, then the solution of chitosan is added and stirring continues for 2 h to 6 h, and then the solution of sodium hyaluronate is added and stirred at 4°C to 10°C for 2 h to 6 h to obtain the bioactive material The present invention also provides an application of the methylcobalamin nano-functionalized collagen polysaccharide sponge in the preparation of active oxygen scavenging drugs.

[0011] The present invention also provides an application of the methylcobalamin nano-functionalized collagen polysaccharide sponge in the preparation of anti-inflammatory drugs.

[0012] As a preferred embodiment of the present invention, the methylcobalamin nano-functionalized collagen polysaccharide sponge is used for the preparation of drugs for promoting nerve regeneration.

[0013] Further preferably, the methylcobalamin nano-functionalized collagen polysaccharide sponge is used for the preparation of drugs for treating spinal cord injury.

[0014] More preferably, the drug uses the methylcobalamin nano-functionalized collagen polysaccharide sponge as the sole active ingredient.

[0015] More preferably, the drug is prepared by compounding the methylcobalamin nano-functionalized collagen polysaccharide sponge with pharmaceutically acceptable excipients.

[0016] The drug is prepared into an in-situ transplantable preparation or an injection preparation according to a pharmaceutically acceptable method.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention crosslinks the carboxyl groups on the surface of collagen with the amino groups of chitosan, and then further crosslinks with the carboxyl groups of hyaluronic acid. Moreover, the surface of hyaluronic acid is negatively charged and the surface of chitosan is positively charged, and there will be an electrostatic adsorption effect between the two, so as to connect collagen-chitosan-hyaluronic acid to form a dense three-dimensional network structure. Therefore, it has excellent reactive oxygen species (ROS) scavenging ability and high anti-inflammatory ability in the injury microenvironment. At the same time, it has good mechanical properties, high and uniform porosity (120 - 170 μm), good biodegradability and cell adaptability.

[0018] The methylcobalamin nanodots prepared by the present invention have a nanosize of about 7 nm. Through efficient nanosizing treatment, the short-term effect and half-life limitation of methylcobalamin molecules, as well as key problems such as easy degradation, instability, and sustained release, are effectively solved. Combined with the collagen-chitosan-hyaluronic acid biological "bridge", continuous and efficient nerve regeneration in the spinal cord injury microenvironment and effective regulation of inflammatory responses are achieved, showing great potential for clinical application.

[0019] The present invention uses the prepared collagen / chitosan / hyaluronic acid composite material, combined with methylcobalamin nanosizing (MNP), to successfully solve multiple challenges faced by the application of methylcobalamin molecules in the inflammatory site of spinal cord injury, such as easy diffusion and degradation, delivery barriers, and microenvironment interference, and realizes key technical problems in multiple aspects such as long-term regulation of the inflammatory / immune microenvironment, targeted polarization (precision regulation) of BV2 cells, and efficient nerve regeneration. Brief Description of the Drawings

[0020] Figure 1 It is the scanning electron microscope image and pore size distribution histogram of the MNP-functionalized composite material MNP@C-CS@HA; a and b are the scanning electron microscope images of the composite material, and c is the pore size distribution diagram.

[0021] Figure 2 It is the qPCR graph of BV2 cells before and after LPS pretreatment in vitro of the MNP-functionalized composite material MNP@C-CS@HA.

[0022] Figure 3 It is the cell proliferation graph of the co-culture of the MNP-functionalized composite material MNP@C-CS@HA and sNSCs.

[0023] Figure 4 It is the animal model of complete spinal cord transection (T9) in mice and the in vivo biodegradability experiment of MNP@C-CS@HA. a and b are the spinal cord injury modeling diagrams, c is the in-situ implantation diagram of the composite material, and d is the in vivo degradation diagram 8 weeks after the material is implanted at the injury site. The area within the frame in a refers to the spinal cord at the T9 segment, and the area within the frame in c refers to the in-situ transplantation position of the material.

[0024] Figure 5 It is the BMS score and body weight graph of mice 12 weeks after the in-situ implantation of the MNP-functionalized composite material MNP@C-CS@HA at the spinal cord injury site.

[0025] Figure 6 It is the gait analysis graph (a) and hind limb photograph (b) of mice 12 weeks after the in-situ implantation of the MNP-functionalized composite material MNP@C-CS@HA at the spinal cord injury site.

[0026] Figure 7These are the conventional magnetic resonance imaging results of mice after the in-situ implantation of the composite material MNP@C-CS@HA at the spinal cord injury site for 12 weeks.

[0027] Figure 8 These are the immunofluorescence images of nerve differentiation in mouse tissues after the in-situ implantation of the composite material MNP@C-CS@HA at the spinal cord injury site for 12 weeks. Specific embodiments

[0028] Spinal cord injury is usually caused by traffic accidents, falls, sports injuries, etc., and often leads to the loss of sensory, motor, and autonomic nerve functions, and even paralysis. Currently, the treatment methods for SCI mainly include surgical treatment and emerging stem cell treatment methods. Surgical treatment can maintain the stability of the spine to reduce further damage, but patients must endure the postoperative physical pain and related infections, and the early implementation of decompression surgery has limited efficacy for complete SCI. Stem cell transplantation is a potential repair method that may change traditional clinical treatment. However, stem cell transplantation may also block blood vessels, induce tumors, and cause a series of rejection reactions in the body, and the mechanism of action of stem cell transplantation is not yet fully understood.

[0029] Methylcobalamin is an endogenous coenzyme B12, which can promote the synthesis of nucleic acids and proteins in nerve cells, as well as the synthesis of lecithin, the main component of myelin sheath. These processes are crucial for the normal function and repair of the nervous system. However, the half-life of methylcobalamin is relatively short, about 6-8 hours, and it is easily cleared by the kidneys quickly, making it difficult to maintain an effective concentration at the injury site for a long time, and frequent administration is required to maintain the efficacy. Moreover, it lacks active targeting ability and is easily diffused from the injury site to the systemic circulation, resulting in insufficient local drug concentration, and may increase systemic side effects. In addition, the traditional routes of oral and intravenous administration will hinder these drugs from smoothly crossing the blood-brain / spinal cord barrier. These factors make it difficult for methylcobalamin to accumulate at the inflammatory site for a long time and play a continuous therapeutic role.

[0030] Based on this, the present invention provides a methylcobalamin nano-functionalized collagen polysaccharide sponge, which is prepared according to the following steps: Crosslink the carboxyl groups on the surface of collagen with the amino groups of chitosan, and then crosslink with hyaluronic acid to obtain a bioactive material; Use methylcobalamin as a raw material for hydrothermal reaction to obtain methylcobalamin nanodots; Mix the methylcobalamin nanodots with the bioactive material for electrostatic adsorption to obtain a methylcobalamin nano-functionalized collagen polysaccharide sponge.

[0031] The present invention crosslinks the carboxyl groups on the surface of collagen with the amino groups of chitosan, and then further crosslinks with the carboxyl groups of hyaluronic acid. Since the surface of hyaluronic acid is negatively charged and the surface of chitosan is positively charged, electrostatic adsorption occurs between the two, thereby connecting collagen-chitosan-hyaluronic acid to form a dense three-dimensional network structure. Therefore, it has excellent reactive oxygen species scavenging ability and high anti-inflammatory ability in the damaged microenvironment. At the same time, it has good mechanical properties, high and uniform porosity, good biodegradability and cell adaptability. Methylcobalamin nanodots were successfully prepared by an efficient hydrothermal synthesis method, and finally a methylcobalamin nanofunctionalized collagen polysaccharide sponge was synthesized, which successfully solved multiple challenges faced by the application of methylcobalamin molecules in the inflammatory site of spinal cord injury, such as easy diffusion and degradation, delivery barriers and microenvironment interference, and achieved key technical problems in long-term regulation of the inflammatory / immune microenvironment, targeted polarization of BV2 cells and efficient nerve regeneration.

[0032] Example 1 A methylcobalamin nanofunctionalized collagen polysaccharide sponge is prepared according to the following steps: (1) Take the beef leg tissue containing fascia as the raw material, strip the muscle tissue and visible fat, rinse with deionized water to remove impurities, and then immerse the separated fascia tissue in 75% alcohol for 2 h for preliminary disinfection and sterilization, and then rinse 2 times with phosphate buffer solution (pH = 7.4) for standby.

[0033] (2) Immerse the fascia tissue in 10% SDS solution at 4 °C for 24 h to remove non-collagen components (including components such as fat and cell membranes), and wash 2 times with deionized water. Subsequently, the above materials are placed in a 2% glacial acetic acid solution for low-temperature treatment for 5 days, shaken 2 times a day. Then, the collected solution is placed in a sterile centrifuge tube, centrifuged at a low temperature and high speed, and the upper layer liquid is collected to remove the undissolved tissue at the bottom.

[0034] (3) Adjust the pH of the extracted and collected supernatant to neutral with Tris-HCl buffer solution (pH = 7.4), collect the obtained collagen precipitate, then add 2% glacial acetic acid to dissolve the collagen precipitate, and then add phosphate buffer solution (pH = 7.4) to wash 2 times. Finally, centrifuge to obtain the precipitate, and repeat this operation 2 times to obtain a high-purity collagen solution. Place the obtained active collagen solution in a sterile EP tube, pre-freeze at -40 °C for 12 h to freeze the content, and then place it in a freeze dryer for freeze-drying at -40 °C for standby.

[0035] (4) Place the synthesized 100 mg of active collagen in a round-bottom flask (50 ml), add 10 ml of PBS buffer solution, stir at 37 °C for 30 min, then add EDC (2 ml, 4.0 M) and NHS (2 ml, 1.0 M), and stir at 4 °C for 2 h. Subsequently, add 5 ml of 2% high-viscosity chitosan solution and continue stirring for 2 h. Then add 5 mL of 2% high-molecular-weight sodium hyaluronate solution and stir again at 4 °C for 2 h. After washing twice with phosphate buffer solution (pH = 7.4), place it in a freeze dryer and freeze-dry at -40 °C to obtain the bioactive material, labeled as C-CS@HA.

[0036] (5) Dissolve approximately 100 mg of mecobalamin in 10 mL of deionized water by ultrasound, transfer it to a high-pressure reactor, heat to 100 °C and maintain for 2 h. Then filter the solution through a 0.22 μm organic filter membrane to separate large-particle impurities, pour it into a centrifuge tube and centrifuge (10,000 rpm) for 10 minutes. Dialyze the obtained solution against ultrapure water through a dialysis bag (MWCO: 2000 Da) for 12 h. Finally, freeze-dry the dialyzed solution in a freeze dryer at -40 °C into a powder to obtain the final MNP nanodots, and store them in a refrigerator at 4 °C.

[0037] (6) Add 100 mg of the above MNP biomaterial to 5 mL of PBS buffer solution (pH = 7.4), stir at 4 °C for 1 h, then add another 50 mg of MNP nanodots and mix well (stir for 2 h). Finally, place it in a freeze dryer and freeze-dry at -40 °C to obtain the MNP-functionalized collagen composite biomaterial, labeled as MNP@C-CS@HA, and store it in a refrigerator at 4 °C for standby.

[0038] Example 2 A mecobalamin nano-functionalized collagen polysaccharide sponge is prepared according to the following steps: (1) Take bovine leg tissue containing fascia as raw material, strip the muscle tissue and visible fat, rinse with deionized water to remove impurities, then soak the separated fascia tissue in 75% alcohol for 3 h for preliminary disinfection and sterilization, and then rinse 3 times with phosphate buffer solution (pH = 7.4) for standby.

[0039] (2) Place the fascia tissue in 10% SDS solution and soak it at 4 °C for 32 h to remove non-collagen components (including fat and cell membrane components, etc.), and wash it 3 times with deionized water. Subsequently, place the above materials in 3% glacial acetic acid solution and treat them at low temperature for 5 days, shaking 3 times a day. Then place the collected solution in a sterile centrifuge tube, centrifuge at high speed at low temperature, collect the upper liquid, and remove the undissolved tissue at the bottom. Then adjust the pH of the extracted and collected supernatant to neutral with Tris-HCl buffer (pH = 7.4), collect the obtained collagen precipitate, then add 3% glacial acetic acid to dissolve the collagen precipitate, and then wash it 3 times with phosphate buffered saline (pH = 7.4). Finally, centrifuge to obtain the precipitate, and repeat this operation 3 times to obtain a high-purity collagen solution. Subsequently, place the obtained active collagen solution in a sterile EP tube, pre-freeze it at -40 °C for 12 hours to freeze the contents, and then place it in a freeze dryer and freeze-dry it at -70 °C for standby.

[0040] (3) Place 150 mg of the synthesized active collagen in a round-bottom flask (50 ml), add 10 ml of PBS buffer solution and stir at 37 °C for 30 min, then add EDC (2 ml, 4.0 M) and NHS (2 ml, 1.0 M) and stir at 10 °C for 4 h. Subsequently, add 5 ml of 2.5% high-viscosity chitosan solution and continue to stir for 2 h. Then add 5 mL of 2.5% high-molecular-weight sodium hyaluronate solution and stir again at 10 °C for 2 h. After washing 3 times with phosphate buffered saline (pH = 7.4), place it in a freeze dryer and freeze-dry it at -70 °C to obtain a bioactive material, labeled as C-CS@HA.

[0041] (4) Dissolve approximately 150 mg of mecobalamin in 10 mL of deionized water by ultrasound, and transfer it to a high-pressure reactor, heat it to 80 °C and maintain it for 8 h. Then filter the solution through a 0.22 μm organic filter membrane to separate large-particle impurities, pour it into a centrifuge tube and centrifuge (10,000 rpm) for 10 minutes. Dialyze the obtained solution against ultrapure water through a dialysis bag (MWCO: 2000 Da) for 12 h. Finally, freeze-dry the dialyzed solution in a freeze dryer at -70 °C into a powder to obtain the final MNP nanodots, and store them in a refrigerator at 4 °C.

[0042] (5) Add 100 mg of the above MNP biomaterial to 5 mL of PBS buffer solution (pH = 7.4), stir at 4 °C for 1 h, then add 50 mg of MNP nanodots again and mix well (stir for 2 h). Finally, place it in a freeze dryer and freeze-dry it at -70 °C to obtain an MNP-functionalized collagen composite biomaterial, labeled as MNP@C-CS@HA, and store it in a refrigerator at 4 °C for standby.

[0043] Example 3 A mecobalamin-functionalized collagen polysaccharide sponge is prepared according to the following steps: (1) Take the beef leg tissue containing fascia as raw material, strip the muscle tissue and visible fat, rinse with deionized water to remove impurities, then soak the separated fascia tissue in 75% alcohol for 4 h for preliminary disinfection and sterilization, and then rinse 2 times with phosphate buffered saline (pH = 7.4) for standby.

[0044] (2) Immerse the fascia tissue in 10% SDS solution at 4 °C for 48 h to remove non-collagen components (including components such as fat and cell membranes), and wash 3 times with deionized water. Subsequently, place the above materials in a 4% glacial acetic acid solution for low-temperature treatment for 5 days, shake 3 times a day. Then, place the collected solution in a sterile centrifuge tube, centrifuge at low temperature and high speed, collect the upper liquid, and remove the undissolved tissue at the bottom. Then, adjust the pH of the extracted and collected supernatant to neutral with Tris-HCl buffer (pH = 8), collect the obtained collagen precipitate, then add 4% glacial acetic acid to dissolve the collagen precipitate, add phosphate buffered saline (pH = 7.4) and wash 3 times, and finally centrifuge to obtain the precipitate. Repeat the operation 3 times to obtain a high-purity collagen solution. Subsequently, place the obtained active collagen solution in a sterile EP tube, pre-freeze at -40 °C for 12 h to freeze the content, and then place it in a freeze dryer and freeze-dry at -80 °C for standby.

[0045] (3) Place 200 mg of the synthesized active collagen in a round-bottom flask (50 ml), add 10 ml of PBS buffer solution and stir at 37 °C for 30 min, then add EDC (2 ml, 4.0 M) and NHS (2 ml, 1.0 M) and stir at 8 °C for 6 h. Subsequently, add 5 ml of 3% high-viscosity chitosan solution and continue to stir for 2 h. Then add 5 mL of 3% high-molecular-weight sodium hyaluronate solution and stir again at 8 °C for 2 h. After washing 3 times with phosphate buffered saline (pH = 7.4), place it in a freeze dryer and freeze-dry at -80 °C to obtain a bioactive material, labeled as C-CS@HA.

[0046] (4)Dissolve approximately 200 mg of mecobalamin in 10 mL of deionized water by ultrasound, and transfer it to a high-pressure reactor, heat it to 120 °C and maintain for 6 h. Then filter the solution through a 0.22 μm organic filter membrane to separate large-particle impurities, pour it into a centrifuge tube and centrifuge (10,000 rpm) for 10 minutes. Dialyze the obtained solution against ultrapure water through a dialysis bag (MWCO: 2000 Da) for 12 h. Finally, lyophilize the dialyzed solution at -80 °C in a freeze dryer into a powder to obtain the final MNP nanodots, and store them in a refrigerator at 4 °C.

[0047] (5)Add 100 mg of the above MNP biomaterial to 5 mL of PBS buffer solution (pH = 7.4), stir at 4 °C for 1 h, then add another 50 mg of MNP nanodots and mix well (stir for 2 h). Finally, place it in a freeze dryer and lyophilize at -80 °C to obtain the MNP-functionalized collagen composite biomaterial, labeled as MNP@C-CS@HA, and store it in a refrigerator at 4 °C for standby.

[0048] Example 4 A mecobalamin nano-functionalized collagen polysaccharide sponge is prepared according to the following steps: (1)Take bovine leg tissue containing fascia as raw material, strip the muscle tissue and visible fat, rinse with deionized water to remove impurities, then soak the separated fascia tissue in 75% alcohol for 5 h for preliminary disinfection and sterilization, and then rinse 3 times with phosphate buffer solution (pH = 7.4) for standby.

[0049] (2)Soak the fascia tissue in 10% SDS solution at 4 °C for 72 h to remove non-collagen components (including components such as fat and cell membranes), and wash 3 times with deionized water. Subsequently, place the above material in a 5% glacial acetic acid solution for low-temperature treatment for 4 days, shake 2 times a day. Then place the collected solution in a sterile centrifuge tube, centrifuge at low temperature and high speed, collect the upper liquid, and remove the undissolved tissue at the bottom. Then adjust the pH of the extracted and collected supernatant to neutral with Tris-HCl buffer solution (pH = 8), collect the obtained collagen precipitate, then add 5% glacial acetic acid to dissolve the collagen precipitate, add phosphate buffer solution (pH = 7.4) and wash 2 times, and finally centrifuge to obtain the precipitate. Repeat this operation 3 times to obtain a high-purity collagen solution. Subsequently, place the obtained active collagen solution in a sterile EP tube, pre-freeze at -80 °C for 12 hours to freeze the content, and then place it in a freeze dryer and lyophilize at -80 °C for standby.

[0050] (3)Place the synthesized 250 mg of active collagen in a round-bottom flask (50 ml), add 10 ml of PBS buffer solution, stir at 37 °C for 30 min, then add EDC (2 ml, 4.0 M) and NHS (2 ml, 1.0 M), and stir at 6 °C for 8 h. Subsequently, add 5 ml of a 3.5% high-viscosity chitosan solution and continue stirring for 6 h. Then add 5 mL of a 3.5% high-molecular-weight sodium hyaluronate solution and stir again at 37 °C for 6 h. After washing 3 times with phosphate buffer solution (pH = 7.4), place it in a freeze dryer and freeze-dry at -80 °C to obtain the bioactive material, labeled as C-CS@HA.

[0051] (4)Dissolve approximately 250 mg of mecobalamin in 10 mL of deionized water by ultrasound, transfer it to a high-pressure reactor, heat to 140 °C and maintain for 2 h. Then filter the solution through a 0.22 μm organic filter membrane to separate large-particle impurities, pour it into a centrifuge tube and centrifuge (10,000 rpm) for 10 minutes. Dialyze the obtained solution against ultrapure water through a dialysis bag (MWCO: 2000 Da) for 12 h. Finally, freeze-dry the dialyzed solution in a freeze dryer at -80 °C into a powder to obtain the final MNP nanodots, and store them in a refrigerator at 4 °C.

[0052] (5)Add 250 mg of the above MNP biomaterial to 5 mL of PBS buffer solution (pH = 7.4), stir at 4 °C for 1 h, then add 50 mg of MNP nanodots again and mix well (stir for 2 h). Finally, place it in a freeze dryer and freeze-dry at -80 °C to obtain the MNP-functionalized collagen composite biomaterial, labeled as MNP@C-CS@HA, and store it in a refrigerator at 4 °C for standby.

[0053] Since the properties and therapeutic effects of the biomaterial MNP@C-CS@HA prepared in Examples 1 to 4 are basically the same, the following description of the present invention will only take the biomaterial MNP@C-CS@HA provided in Example 1 as an example.

[0054] The biomaterial MNP@C-CS@HA prepared by the method of Example 1 was observed and analyzed by scanning electron microscopy (SEM), and it was found that it has a unique three-dimensional porous structure ( Figure 1 as shown in a). At the same time, through pore size analysis, it was found that the pore size of this material is distributed around 120 - 170 μm ( Figure 1 as shown in b and c), which fully proves the stability of its preparation process.

[0055] Experimental Example 1 In vitro effect verification test 0, qPCR analysis BV2 cells were seeded into confocal dishes and stimulated with lipopolysaccharide at a final concentration of 500 ng / mL to induce an inflammatory response (4 h). Then, MNP@C-CS@HA was added and co-cultured with the cells for an additional 24 h. Meanwhile, BV2 cells without lipopolysaccharide stimulation were used as negative controls. Subsequently, the cells were collected, and total RNA of BV2 was extracted for qPCR analysis to measure the expression of M1 markers (iNOS, IL-6, and TNF-α), M2 markers (Arg-1 and IL-10), and CD44, and to verify the anti-inflammatory effects of the materials in each group and the polarization state of microglia.

[0056] 2, CCK-8 assay The CCK-8 kit (C0038, Beyotime, Shanghai, China) was used to evaluate the cell proliferation of MNP@C-CS@HA. First, sNSCs were seeded into 64-well plates, and then MNP@C-CS@HA materials were added for co-incubation for 72 h. Next, 10 μL of CCK-8 solution was added to the wells. After continued incubation for 4 h, the absorbance of each well at a wavelength of 450 nm was measured using a microplate reader.

[0057] 3, Results The results of qPCR analysis confirmed that Figure 2 ), after treatment with MNP@C-CS@HA materials, there was overexpression of the CD44 marker and M2 polarization of BV2 cells. The expression of pro-inflammatory related factors (iNOS, TNF-α, IL-1β) was significantly down-regulated, while the expression of anti-inflammatory related factors (IL-10, Arg-1) was significantly up-regulated).

[0058] The CCK-8 experiment found that Figure 3 ), the MNP@C-CS@HA material had good biocompatibility and could promote the proliferation of neural stem cells.

[0059] Experimental Example 2 In vivo efficacy verification test 1, Method Mice were anesthetized by intraperitoneal injection of 0.9% sodium pentobarbital (50 mg / kg). A 1-2 cm longitudinal incision was made in the middle of the back of the mice centered on T9 to expose the paravertebral muscles for blunt dissection. The T9 lamina was exposed, and the spinous process was bitten off to expose the spinal cord. 2 mm of the spinal cord was cut off using spring scissors. The wound was rinsed with saline, and a 2 × 2 × 3 mm biomaterial scaffold was implanted into the spinal cord defect site. Then the muscle and skin were sutured tightly layer by layer. In the Sham group, only the lamina was resected without cutting the spinal cord. In the blank control group (SCI group), no material was implanted after cutting the spinal cord. In the experimental group, C-CS@HA and MNP@C-CS@HA biomaterial scaffolds were implanted. After the operation, each mouse was intraperitoneally injected with 40,000 units of penicillin for 7 consecutive days, and manually assisted urination was performed 2 times a day until bladder function recovered.

[0060] 2. Evaluation of animal motor function: The motor function of mice was evaluated by BMS, body weight analysis, and footprint analysis. The BMS scoring system was used to evaluate the joint movement and coordination of the hind limbs of mice, with a range of 0 to 9 points. At a fixed time point every week, 2 observers performed a 3-5 min double-blind experiment score on each mouse. At 12 weeks after the operation, the forelimbs of the mice were painted blue and the hind limbs were painted red. The mice were allowed to walk on a narrow track on white paper to capture their footprints. Subsequently, these footprints were scanned, and the generated digital images were analyzed.

[0061] 3. Magnetic resonance imaging (MRI) - related analysis of spinal cord structure 90 days after spinal cord injury, the rats were anesthetized and fixed in the prone position. The structural connection status of the spinal cord was analyzed using conventional magnetic resonance imaging on a United Imaging 9.4T ultra-high field animal magnetic resonance imaging system.

[0062] 2. Immunofluorescence analysis 12 weeks after spinal cord injury, mice were intraperitoneally injected with 0.9% sodium pentobarbital (50 mg / kg), spinal cord tissues were collected, washed with saline, and fixed with 4% paraformaldehyde. The tissues were sectioned at a thickness of 10 μm using a cryostat (Leica, Germany), and then immunofluorescence analysis of GFAP and Tuj1 was performed.

[0063] 5. Results The biodegradability of MNP@C-CS@HA in vivo was verified in a C57 mouse T9 spinal cord complete transection model. The results showed that the MNP@C-CS@HA material had good biodegradability and biocompatibility 12 weeks after in-situ implantation into the spinal cord injury site ( Figure 4 ).

[0064] Subsequently, after 12 weeks of treatment, mouse BMS scores and body weight were analyzed, and it was found that both the motor function and body weight of the mice in the MNP@C-CS@HA group were significantly improved, with the BMS score exceeding 4.0 points ( Figure 5 ).

[0065] Through gait analysis of the mice after 12 weeks and photo analysis of the recovery of the hind legs, it was found that the motor status of the mice in the MNP@C-CS@HA group recovered most significantly. The dragging of the hind limbs of the mice was significantly reduced, the coordination was significantly enhanced, and the motor function of the hind limbs was significantly improved, which was equivalent to the effect of adding the neurotrophic factor NT3 ( Figure 6 ).

[0066] Subsequently, through magnetic resonance imaging results ( Figure 7 ), it was further found that the MNP@C-CS@HA group showed the best recovery status among the treatment groups, with the smallest tissue damage area and cavity range.

[0067] Finally, through immunofluorescence analysis of nerve tissue, it was found that the MNP@C-CS@HA material had a high potential for Tuj1+ neuron differentiation (Figure 8).

[0068] These results indicate that the biomaterial provided by the present invention remodels the spinal cord injury microenvironment through multiple pathways such as antioxidant, regulating BV2 polarization, and promoting endogenous nerve differentiation, significantly promotes nerve regeneration and motor function recovery, and shows great clinical application potential in the precise treatment of SCI.

[0069] Finally, the technical solutions listed in the embodiments of the present invention are only preferred implementation solutions and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A mecobalamin-nanofunctionalized collagen polysaccharide sponge, characterized in that, It is prepared according to the following steps: Crosslink the carboxyl groups on the surface of collagen with the amino groups of chitosan, and then crosslink with hyaluronic acid to obtain a bioactive material; Use mecobalamin as a raw material for hydrothermal reaction to obtain mecobalamin nanodots; Mix the mecobalamin nanodots with the bioactive material for electrostatic adsorption to obtain the mecobalamin nanofunctionalized collagen polysaccharide sponge.

2. The mecobalamin nano-functionalized collagen polysaccharide sponge according to claim 1, characterized in that, The mass ratio of the active collagen, chitosan and sodium hyaluronate is 1-2.5:1:

1.

3. The mecobalamin-nanofunctionalized collagen polysaccharide sponge according to claim 1, wherein The mixing mass ratio of the bioactive material and the mecobalamin nanodots is 2-5:

1.

4. The mecobalamin nano-functionalized collagen polysaccharide sponge according to claim 1, wherein The hydrothermal reaction is carried out at 80°C - 140°C for 2 h - 8 h.

5. The mecobalamin-nanofunctionalized collagen polysaccharide sponge according to claim 1, characterized in that, Mix the collagen solution and the crosslinking agent by stirring at 4°C - 10°C for 2 h - 8 h, then add the chitosan solution and continue stirring for 2 h - 6 h, and then add the sodium hyaluronate solution and stir at 4°C - 10°C for 2 h - 6 h to obtain the bioactive material.

6. Use of the mecobalamin nanofunctionalized collagen polysaccharide sponge according to claim 1 in the preparation of active oxygen scavenging drugs or anti-inflammatory drugs.

7. The application according to claim 6, characterized in that The mecobalamin nanofunctionalized collagen polysaccharide sponge is used for the preparation of drugs for promoting nerve regeneration.

8. The application according to claim 7, wherein The mecobalamin nanofunctionalized collagen polysaccharide sponge is used for the preparation of drugs for treating spinal cord injury.

9. The application according to claim 8, wherein The drug uses the mecobalamin nanofunctionalized collagen polysaccharide sponge as the only active ingredient.

10. The application according to claim 8, characterized in that, The drug is compounded from the mecobalamin nanofunctionalized collagen polysaccharide sponge and pharmaceutically acceptable excipients.

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