A methylcobalamin nanofunctionalized collagen polysaccharide sponge and its application

Abstract

The present invention belongs to the field of pharmaceutical research technology, and specifically relates to a methylcobalamin nanofunctionalized collagen polysaccharide sponge and its application. The methylcobalamin nanofunctionalized collagen polysaccharide sponge is prepared according to the following steps: cross-linking is performed using the carboxyl groups on the surface of collagen with the amino groups of chitosan, and then cross-linking with hyaluronic acid to obtain a bioactive material; hydrothermal reaction is performed using methylcobalamin as a raw material to obtain methylcobalamin nanodots; methylcobalamin nanodots are mixed with the bioactive material for electrostatic adsorption to obtain a collagen polysaccharide sponge. The collagen polysaccharide sponge provided by the present invention can solve the multiple challenges faced by the application of methylcobalamin molecules in the inflammatory site of spinal cord injury, such as easy diffusion degradation, delivery barriers and microenvironment interference, and achieve long-term inflammation / immune microenvironment regulation, targeted polarization of BV2 cells and efficient nerve regeneration and other key technical problems, and has good biodegradability and cell adaptability.

Description

Technical Field

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

[0002] Spinal cord injury (SCI) is commonly caused by traffic accidents, falls, and sports injuries, often leading to loss of sensory, motor, and autonomic nervous system function, and even paralysis. Following SCI, the local microenvironment rapidly deteriorates, with a dramatic increase in reactive oxygen species levels, triggering a vigorous inflammatory response. Subsequently, the injured area experiences a variety of secondary complications, severely impacting motor function recovery. Furthermore, the inflammatory immune microenvironment hinders the stable growth and development of endogenous neural stem cells (NSCs) within the injured area. Instead, they are more likely to differentiate into astrocytes, resulting in glial scarring and hindering cell migration and neural regeneration. Microglia (BV2), key players in the inflammatory response after SCI, can polarize into either M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes, playing a crucial role in regulating the entire trajectory of the SCI microenvironment. The injury microenvironment induces microglia to polarize toward the inflammatory M1 phenotype, which in turn releases a large number of inflammatory cytokines, leading to expanded neuroinflammation, increased neuronal apoptosis, and increased axonal degeneration, further exacerbating neural tissue damage. At the same time, relevant research reports that microglia permanently switch to different transcriptional states in the spinal cord immune microenvironment, leading to longer-term changes in the spinal cord immune microenvironment, and are very likely to become new targets for spinal cord injury repair. Therefore, it is crucial to resolve the persistent inflammatory response in the injury microenvironment, protect damaged neurons, and effectively achieve targeted cascade regulation of BV2 cells.

[0003] At present, the treatment methods for SCI mainly include surgical treatment and emerging stem cell treatment. Surgical treatment can maintain the stability of the spine to reduce further damage, but patients must endure postoperative physical pain and related infections, and early 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. In recent years, biomaterials have been widely used as relevant drug carriers and anti-inflammatory preparations due to their unique physical and chemical properties, providing more treatment options for SCI.

[0004] Methylcobalamin, an endogenous coenzyme B12, promotes the synthesis of nucleic acids and proteins within nerve cells, as well as the synthesis of phosphatidylcholine, a major component of myelin. These processes are crucial for the normal function and repair of the nervous system. However, methylcobalamin has a short half-life of approximately 6–8 hours and is rapidly cleared by the kidneys, making it difficult to maintain effective concentrations at the site of injury for extended periods, necessitating frequent dosing to maintain efficacy. Furthermore, it lacks active targeting and readily diffuses from the site of injury into the systemic circulation, resulting in insufficient local drug concentrations and potentially increasing systemic side effects (e.g., rash and gastrointestinal reactions). Furthermore, traditional oral and intravenous routes of administration hinder the successful crossing of the blood-brain / spinal cord barrier. These factors hinder the long-term accumulation of methylcobalamin at the site of inflammation and its sustained therapeutic effect. Therefore, the development of novel multifunctional biomaterials that combine multiple anti-inflammatory properties, modified methylcobalamin sustained release, and the promotion of neuroregeneration and motor function recovery is of great significance. Summary of the Invention

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

[0006] The present invention provides a methylcobalamin nano-functionalized collagen polysaccharide sponge, which is prepared according to the following steps:

[0007] The carboxyl groups on the surface of collagen are cross-linked with the amino groups of chitosan, and then cross-linked with hyaluronic acid to obtain a bioactive material;

[0008] Using methylcobalamin as raw material, a hydrothermal reaction is carried out to obtain methylcobalamin nanodots;

[0009] The methylcobalamin nanodots are mixed with the bioactive material for electrostatic adsorption to obtain the methylcobalamin nanofunctionalized collagen polysaccharide sponge. The methylcobalamin nanodots are positively charged, while the bioactive material as a whole is negatively charged, so electrostatic adsorption occurs between the two, resulting in a tighter bond.

[0010] 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.

[0011] As a preferred embodiment of the present invention, the mixing mass ratio of the bioactive material to the methylcobalamin nanodots is 2-5:1.

[0012] As a preferred embodiment of the present invention, the hydrothermal reaction is maintained at 80° C. to 140° C. for 2 h to 8 h.

[0013] As a preferred embodiment of the present invention, the collagen solution and the cross-linking agent are stirred at 4°C to 10°C for 2h to 6h, the chitosan solution is added and the stirring is continued for 2h to 6h, and the sodium hyaluronate solution is added and the stirring is continued at 4°C to 10°C for 2h to 6h to obtain the bioactive material.

[0014] The present invention also provides an application of the methylcobalamin nano-functionalized collagen polysaccharide sponge in the preparation of active oxygen scavenging drugs.

[0015] The present invention also provides an application of the methylcobalamin nano-functionalized collagen polysaccharide sponge in the preparation of anti-inflammatory drugs.

[0016] As a preferred embodiment of the present invention, the methylcobalamin nano-functionalized collagen polysaccharide sponge is used to prepare a drug for promoting nerve regeneration.

[0017] Further preferably, the methylcobalamin nanofunctionalized collagen polysaccharide sponge is used to prepare a drug for treating spinal cord injury.

[0018] More preferably, the drug uses the methylcobalamin nanofunctionalized collagen polysaccharide sponge as the sole active ingredient.

[0019] More preferably, the drug is prepared by compounding the methylcobalamin nano-functionalized collagen polysaccharide sponge with pharmaceutically acceptable excipients.

[0020] The drug is prepared into an in situ transplantable preparation or an injectable preparation according to a pharmaceutically acceptable method.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention utilizes carboxyl groups on the surface of collagen to crosslink with amino groups on chitosan, which are then further crosslinked with carboxyl groups on hyaluronic acid. The negatively charged surface of hyaluronic acid and the positively charged surface of chitosan create electrostatic adsorption between the two, interconnecting the collagen, chitosan, and hyaluronic acid to form a dense three-dimensional network. This provides excellent reactive oxygen species (ROS) scavenging and highly effective anti-inflammatory properties in the injury microenvironment. Furthermore, it exhibits excellent mechanical properties, high and uniform porosity (120-170 μm), and good biodegradability and cell adaptability.

[0023] The methylcobalamin nanodots prepared by the present invention have a nanoparticle size of about 7 nm. Through efficient nano-processing, they effectively solve the key problems of methylcobalamin molecules, such as short-term effects and half-life limitations, easy degradation and instability, and sustained sustained release. In combination with the collagen-chitosan-hyaluronic acid biological "bridge", they achieve continuous and efficient nerve regeneration in the spinal cord injury microenvironment and effective regulation of inflammatory responses, and have great clinical application potential.

[0024] The present invention utilizes the prepared collagen / chitosan / hyaluronic acid composite material in combination with methylcobalamin nanoparticles (MNPs) to successfully solve the multiple challenges faced by the application of methylcobalamin molecules in the inflammatory site of spinal cord injury, such as easy diffusion degradation, delivery barriers and microenvironment interference, and achieves key technical problems in many aspects, such as long-term inflammatory / immune microenvironment regulation, targeted polarization (precise regulation) of BV2 cells and efficient nerve regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 are the scanning electron microscopy images and pore size distribution histograms of the MNP functionalized composite material MNP@C-CS@HA; a and b are the scanning electron microscopy images of the composite material, and figure c is the pore size distribution diagram.

[0026] Figure 2 This is the qPCR graph of BV2 cells before and after LPS pretreatment of the MNP functionalized composite material MNP@C-CS@HA in vitro.

[0027] Figure 3 This is a diagram of cell proliferation of the MNP-functionalized composite material MNP@C-CS@HA co-cultured with sNSCs.

[0028] Figure 4 Figure 1 shows a mouse spinal cord transection (T9) model and the in vivo biodegradability of MNP@C-CS@HA. (a) and (b) show the spinal cord injury model, (c) shows the in situ implantation of the composite material, and (d) shows the in vivo degradation of the material eight weeks after implantation. The box in (a) indicates the T9 spinal cord segment, and the box in (c) indicates the in situ implantation site.

[0029] Figure 5 Figure 3 is the BMS score and body weight of mice 12 weeks after in situ implantation of the MNP-functionalized composite material MNP@C-CS@HA into the spinal cord injury site.

[0030] Figure 6 Figure 1 shows the gait analysis (a) and hindlimb photography (b) of mice 12 weeks after the MNP-functionalized composite material MNP@C-CS@HA was in situ implanted at the spinal cord injury site.

[0031] Figure 7This is the conventional magnetic resonance imaging result of mice 12 weeks after the composite material MNP@C-CS@HA was in situ implanted into the spinal cord injury site.

[0032] Figure 8 This is an immunofluorescence image of neural differentiation in mouse tissue 12 weeks after the composite material MNP@C-CS@HA was in situ implanted into the spinal cord injury site. DETAILED DESCRIPTION

[0033] Spinal cord injuries are usually caused by traffic accidents, falls, sports injuries, etc., which often lead to loss of sensory, motor and autonomic nervous system functions, and even paralysis. The current treatments for SCI mainly include surgical treatment and emerging stem cell treatments. Surgical treatment can maintain the stability of the spine to reduce further damage, but patients must endure postoperative physical pain and related infections, and early 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.

[0034] Methylcobalamin, an endogenous coenzyme B12, promotes the synthesis of nucleic acids and proteins within nerve cells, as well as the synthesis of phosphatidylcholine, a major component of myelin. These processes are crucial for the normal function and repair of the nervous system. However, methylcobalamin has a short half-life of approximately 6–8 hours and is rapidly cleared by the kidneys, making it difficult to maintain effective concentrations at the site of injury for extended periods, necessitating frequent dosing to maintain efficacy. Furthermore, it lacks active targeting and readily diffuses from the site of injury into the systemic circulation, resulting in insufficient local drug concentrations and potentially increasing systemic side effects. Furthermore, traditional oral and intravenous routes of administration hinder the successful crossing of the blood-brain / spinal cord barrier. These factors hinder the long-term accumulation of methylcobalamin at the site of inflammation and its sustained therapeutic effect.

[0035] Based on this, the present invention provides a methylcobalamin nanofunctionalized collagen polysaccharide sponge, which is prepared according to the following steps:

[0036] The carboxyl groups on the surface of collagen are cross-linked with the amino groups of chitosan, and then cross-linked with hyaluronic acid to obtain a bioactive material;

[0037] Using methylcobalamin as raw material, a hydrothermal reaction is carried out to obtain methylcobalamin nanodots;

[0038] The methylcobalamin nanodots are mixed with bioactive materials for electrostatic adsorption to obtain a methylcobalamin nanofunctionalized collagen polysaccharide sponge.

[0039] The present invention utilizes the carboxyl group on the surface of collagen to be cross-linked with the amino group of chitosan, and then further cross-linked with the carboxyl group of hyaluronic acid, and the hyaluronic acid surface is negatively charged, the chitosan surface is positively charged, and electrostatic adsorption occurs between the two, so that collagen-chitosan-hyaluronic acid is interconnected to form a dense three-dimensional network structure, therefore possesses excellent reactive oxygen species scavenging ability and efficient anti-inflammatory ability in the injury microenvironment. Simultaneously, mechanical properties are good, porosity is high and uniform, and biodegradability and cell adaptability are good. And methylcobalamin nano dots are successfully prepared by efficient hydrothermal synthesis, finally synthesized to obtain a kind of methylcobalamin nano-functionalized collagen polysaccharide sponge, successfully solved the easy diffusion degradation, delivery barrier and microenvironmental interference and other multiple challenges faced by the application of methylcobalamin molecules in spinal cord injury inflammation site, realize long-term inflammation / immune microenvironment regulation, the key technical problems of many aspects such as the targeted polarization of BV2 cells and efficient nerve regeneration.

[0040] Example 1

[0041] A methylcobalamin nanofunctionalized collagen polysaccharide sponge is prepared according to the following steps:

[0042] (1) Take the fascia-containing cattle leg tissue as the raw material, peel off the muscle tissue and visible fat, rinse with deionized water to remove impurities, and then soak the separated fascia tissue in 75% alcohol for 2 hours for preliminary disinfection and sterilization, and then rinse twice with phosphate buffered saline (pH = 7.4) for use.

[0043] (2) The fascia tissue was placed in a 10% SDS solution at 4°C for 24 hours to remove non-collagenous components (including fat and cell membranes), and then washed twice with deionized water. The above materials were then placed in a 2% glacial acetic acid solution for low-temperature treatment for 5 days, with shaking twice a day. The collected solution was then placed in a sterile centrifuge tube and centrifuged at low temperature and high speed to collect the upper liquid and remove the undissolved tissue at the bottom.

[0044] (3) The supernatant collected by extraction was adjusted to a neutral pH with Tris-HCl buffer (pH = 7.4), and the collagen precipitate was collected. Subsequently, 2% glacial acetic acid was added to dissolve the collagen precipitate, and then phosphate buffered saline (pH = 7.4) was added to wash twice. Finally, the precipitate was centrifuged and repeated twice to obtain a high-purity collagen solution. The obtained active collagen solution was placed in a sterile EP tube, pre-frozen at -40℃ for 12 hours to freeze the contents, and then placed in a freeze dryer at -40℃ for freeze-drying.

[0045] (4) 100 mg of the synthesized active collagen was placed in a round-bottom flask (50 ml), 10 ml of PBS buffer solution was added and stirred at 37 °C for 30 min, then EDC (2 ml, 4.0 M) and NHS (2 ml, 1.0 M) were added and stirred at 4 °C for 2 h. Subsequently, 5 ml of a 2% high-viscosity chitosan solution was added and stirred for another 2 h. Then, 5 mL of a 2% high-molecular-weight sodium hyaluronate solution was added and stirred again at 4 °C for 2 h. After washing twice with phosphate buffered saline (pH = 7.4), the mixture was placed in a freeze dryer and freeze-dried at -40 °C to obtain the bioactive material, labeled as C-CS@HA.

[0046] (5) Approximately 100 mg of methylcobalamin was dissolved in 10 mL of deionized water by ultrasound and transferred to a high-pressure reactor and heated to 100 °C for 2 h. The solution was then filtered through a 0.22 μm organic filter membrane to separate large particle size impurities, poured into a centrifuge tube and centrifuged (10,000 rpm) for 10 min. The obtained solution was dialyzed against ultrapure water through a dialysis bag (MWCO: 2000 Da) for 12 h. Finally, the dialyzed solution was freeze-dried into a powder in a freeze dryer at -40 °C to obtain the final MNP nanodots, which was then stored in a refrigerator at 4 °C.

[0047] (6) 100 mg of the above-mentioned MNP biomaterial was added to 5 mL of PBS buffer solution (pH = 7.4), stirred at 4 °C for 1 h, and then 50 mg of MNP nanodots was added again and mixed thoroughly (stirred for 2 h). Finally, the mixture was placed in a freeze dryer and freeze-dried at -40 °C to obtain the MNP-functionalized collagen composite biomaterial, labeled as MNP@C-CS@HA, and stored in a refrigerator at 4 °C for future use.

[0048] Example 2

[0049] A methylcobalamin nanofunctionalized collagen polysaccharide sponge is prepared according to the following steps:

[0050] (1) Take the fascia-containing cattle leg tissue as the raw material, peel off the muscle tissue and visible fat, rinse with deionized water to remove impurities, and then soak the separated fascia tissue in 75% alcohol for 3 hours for preliminary disinfection and sterilization, and then rinse with phosphate buffered saline (pH = 7.4) three times for use.

[0051] (2) The fascia tissue was placed in a 10% SDS solution at 4 °C for 32 h to remove non-collagen components (including fat and cell membrane components), and then washed with deionized water three times. The above materials were then placed in a 3% glacial acetic acid solution for low-temperature treatment for 5 days, shaken three times a day, and then the collected solution was placed in a sterile centrifuge tube, centrifuged at low temperature and high speed, the upper liquid was collected, and the undissolved tissue at the bottom was removed. The extracted supernatant was then adjusted to a neutral pH with Tris-HCl buffer (pH = 7.4), and the collagen precipitate was collected. 3% glacial acetic acid was then added to dissolve the collagen precipitate, and then phosphate buffered saline (pH = 7.4) was added to wash three times. Finally, the precipitate was centrifuged and repeated three times to obtain a high-purity collagen solution. The obtained active collagen solution was then placed in a sterile EP tube, pre-frozen at -40 °C for 12 hours to freeze the contents, and then placed in a freeze dryer at -70 °C for freeze-drying.

[0052] (3) 150 mg of the synthesized active collagen was placed in a round-bottom flask (50 ml), 10 ml of PBS buffer solution was added and stirred at 37 °C for 30 min, then EDC (2 ml, 4.0 M) and NHS (2 ml, 1.0 M) were added and stirred at 10 °C for 4 h. Subsequently, 5 ml of a 2.5% mass fraction high-viscosity chitosan solution was added and stirred for another 2 h. Then, 5 mL of a 2.5% mass fraction high-molecular-weight sodium hyaluronate solution was added and stirred again at 10 °C for 2 h. After washing three times with phosphate buffered saline (pH = 7.4), the mixture was placed in a freeze dryer and freeze-dried at -70 °C to obtain the bioactive material, labeled as C-CS@HA.

[0053] (4) Approximately 150 mg of methylcobalamin was dissolved in 10 mL of deionized water by ultrasound and transferred to a high-pressure reactor, heated to 80°C and maintained for 8 h. The solution was then filtered through a 0.22 μm organic filter membrane to separate large particle impurities, poured into a centrifuge tube and centrifuged (10,000 rpm) for 10 min. The obtained solution was dialyzed against ultrapure water through a dialysis bag (MWCO: 2000 Da) for 12 h. Finally, the dialyzed solution was freeze-dried into a powder in a freeze dryer at -70°C to obtain the final MNP nanodots, which was then stored in a refrigerator at 4°C.

[0054] (5) Add 100 mg of the above-mentioned 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 thoroughly (stir for 2 h), and finally freeze-dry in a freeze dryer at -70 °C to obtain MNP-functionalized collagen composite biomaterial, labeled as MNP@C-CS@HA, and store in a refrigerator at 4 °C for future use.

[0055] Example 3

[0056] A methylcobalamin nanofunctionalized collagen polysaccharide sponge is prepared according to the following steps:

[0057] (1) Take the fascia-containing cattle leg tissue as the raw material, peel off the muscle tissue and visible fat, rinse with deionized water to remove impurities, and then soak the separated fascia tissue in 75% alcohol for 4 hours for preliminary disinfection and sterilization, and then rinse twice with phosphate buffered saline (pH = 7.4) for use.

[0058] (2) The fascia tissue was placed in a 10% SDS solution at 4°C for 48 hours to remove non-collagen components (including fat and cell membrane components), and then washed with deionized water three times. The above materials were then placed in a 4% glacial acetic acid solution for low-temperature treatment for 5 days, shaken three times a day, and then the collected solution was placed in a sterile centrifuge tube, centrifuged at low temperature and high speed, the upper liquid was collected, and the undissolved tissue at the bottom was removed. The extracted supernatant was then adjusted to a neutral pH with Tris-HCl buffer (pH = 8), and the collagen precipitate was collected. 4% glacial acetic acid was then added to dissolve the collagen precipitate, and then phosphate buffered saline (pH = 7.4) was added to wash three times. Finally, the precipitate was centrifuged and repeated three times to obtain a high-purity collagen solution. The obtained active collagen solution was then placed in a sterile EP tube, pre-frozen at -40°C for 12 hours to freeze the contents, and then placed in a freeze dryer at -80°C for freeze-drying.

[0059] (3) 200 mg of the synthesized active collagen was placed in a round-bottom flask (50 ml), 10 ml of PBS buffer solution was added and stirred at 37 °C for 30 min, then EDC (2 ml, 4.0 M) and NHS (2 ml, 1.0 M) were added and stirred at 8 °C for 6 h. Subsequently, 5 ml of a 3% mass fraction high-viscosity chitosan solution was added and stirred for another 2 h. Then, 5 mL of a 3% mass fraction high-molecular-weight sodium hyaluronate solution was added and stirred again at 8 °C for 2 h. After washing three times with phosphate buffered saline (pH = 7.4), the mixture was placed in a freeze dryer and freeze-dried at -80 °C to obtain the bioactive material, labeled as C-CS@HA.

[0060] (4) Approximately 200 mg of methylcobalamin was dissolved in 10 mL of deionized water by ultrasound and transferred to a high-pressure reactor and heated to 120 °C for 6 h. The solution was then filtered through a 0.22 μm organic filter membrane to separate large particle size impurities, poured into a centrifuge tube and centrifuged (10,000 rpm) for 10 min. The obtained solution was dialyzed against ultrapure water through a dialysis bag (MWCO: 2000 Da) for 12 h. Finally, the dialyzed solution was freeze-dried into a powder in a freeze dryer at -80 °C to obtain the final MNP nanodots, which was then stored in a refrigerator at 4 °C.

[0061] (5) Add 100 mg of the above-mentioned 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 thoroughly (stir for 2 h), and finally freeze-dry in a freeze dryer at -80 °C to obtain MNP-functionalized collagen composite biomaterial, labeled as MNP@C-CS@HA, and store in a refrigerator at 4 °C for future use.

[0062] Example 4

[0063] A methylcobalamin nanofunctionalized collagen polysaccharide sponge is prepared according to the following steps:

[0064] (1) Take the fascia-containing cattle leg tissue as the raw material, peel off the muscle tissue and visible fat, rinse with deionized water to remove impurities, and then soak the separated fascia tissue in 75% alcohol for 5 hours for preliminary disinfection and sterilization, and then rinse with phosphate buffered saline (pH = 7.4) three times for use.

[0065] (2) The fascia tissue was placed in a 10% SDS solution at 4 °C for 72 h to remove non-collagen components (including fat and cell membrane components), and then washed with deionized water three times. The above materials were then placed in a 5% glacial acetic acid solution for low temperature treatment for 4 days, shaken twice a day, and then the collected solution was placed in a sterile centrifuge tube, centrifuged at low temperature and high speed, the upper liquid was collected, and the undissolved tissue at the bottom was removed. The extracted supernatant was then adjusted to a neutral pH with Tris-HCl buffer (pH = 8), and the collagen precipitate was collected. 5% glacial acetic acid was then added to dissolve the collagen precipitate, and then phosphate buffered saline (pH = 7.4) was added to wash twice, and finally centrifuged to obtain the precipitate. This operation was repeated three times to obtain a high-purity collagen solution. The obtained active collagen solution was then placed in a sterile EP tube, pre-frozen at -80 °C for 12 hours to freeze the contents, and then placed in a freeze dryer at -80 °C for freeze-drying.

[0066] (3) 250 mg of the synthesized active collagen was placed in a round-bottom flask (50 ml), 10 ml of PBS buffer solution was added and stirred at 37 °C for 30 min, then EDC (2 ml, 4.0 M) and NHS (2 ml, 1.0 M) were added and stirred at 6 °C for 8 h. Subsequently, 5 ml of a 3.5% mass fraction high-viscosity chitosan solution was added and stirred for another 6 h. Then, 5 mL of a 3.5% mass fraction high-molecular-weight sodium hyaluronate solution was added and stirred again at 37 °C for 6 h. After washing three times with phosphate buffered saline (pH = 7.4), the mixture was placed in a freeze dryer and freeze-dried at -80 °C to obtain the bioactive material, labeled as C-CS@HA.

[0067] (4) Approximately 250 mg of methylcobalamin was dissolved in 10 mL of deionized water by ultrasound and transferred to a high-pressure reactor and heated to 140 °C for 2 h. The solution was then filtered through a 0.22 μm organic filter membrane to separate large particle size impurities, poured into a centrifuge tube and centrifuged (10,000 rpm) for 10 min. The obtained solution was dialyzed against ultrapure water through a dialysis bag (MWCO: 2000 Da) for 12 h. Finally, the dialyzed solution was freeze-dried to a powder in a freeze dryer at -80 °C to obtain the final MNP nanodots, which was then stored in a refrigerator at 4 °C.

[0068] (5) Add 250 mg of the above-mentioned 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 thoroughly (stir for 2 h), and finally freeze-dry in a freeze dryer at -80 °C to obtain MNP-functionalized collagen composite biomaterial, labeled as MNP@C-CS@HA, and store in a refrigerator at 4 °C for future use.

[0069] Since the properties and therapeutic effects of the biomaterials MNP@C-CS@HA prepared in Examples 1 to 4 are basically the same, the present invention will be described below using only the biomaterial MNP@C-CS@HA provided in Example 1 as an example.

[0070] 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 had a unique three-dimensional porous structure ( Figure 1 At the same time, the pore size analysis found that the pore size of the material is distributed in the range of 120~170 μm ( Figure 1 b, c), which fully demonstrates the stability of its preparation process.

[0071] Experimental Example 1

[0072] In vitro effect verification test

[0073] 0. qPCR analysis

[0074] BV2 cells were seeded into confocal microplates and treated with lipopolysaccharide (LPS) at a final concentration of 500 ng / mL to induce an inflammatory response (4 hours). MNP@C-CS@HA was then added and co-cultured with the cells for an additional 24 hours. BV2 cells not stimulated with LPS served as a negative control. The cells were then harvested, and total RNA 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. This validated the anti-inflammatory effects of each material and the polarization status of microglia.

[0075] 2. CCK-8 assay

[0076] Cell proliferation of MNP@C-CS@HA was assessed using a CCK-8 kit (C0038, Beyotime, Shanghai, China). sNSCs were seeded in a 64-well plate, followed by the addition of MNP@C-CS@HA and incubation for 72 hours. Next, 10 μL of CCK-8 solution was added to each well. After an additional 4 hours of incubation, the absorbance of each well at 450 nm was measured using a microplate reader.

[0077] 3. Results

[0078] qPCR analysis results confirmed that ( Figure 2 ), overexpression of the CD44 marker and M2 polarization of BV2 cells after treatment with MNP@C-CS@HA material. The expression of pro-inflammatory factors (iNOS, TNF-α, IL-1β) was significantly downregulated, while the expression of anti-inflammatory factors (IL-10, Arg-1) was significantly upregulated).

[0079] CCK-8 experiments found that ( Figure 3 ), MNP@C-CS@HA material has good biocompatibility and can promote the proliferation of neural stem cells.

[0080] Experimental Example 2

[0081] In vivo effect verification test

[0082] 1. Method

[0083] Mice were anesthetized with an intraperitoneal injection of 0.9% sodium pentobarbital (50 mg / kg). A 1-2 cm longitudinal incision was made in the middle of the back, centered at T9, to expose the paravertebral muscles and perform blunt dissection. The T9 lamina was exposed, and the spinous process was bitten off to expose the spinal cord. A 2 mm section of the spinal cord was removed using spring shears. The wound was irrigated with saline, and a 2 × 2 × 3 mm biomaterial scaffold was implanted into the spinal cord defect. The muscle and skin were then tightly sutured layer by layer. In the sham group, only the lamina was removed without transecting the spinal cord. In the blank control group (SCI group), the spinal cord was transected without implantation. The experimental groups were implanted with C-CS@HA and MNP@C-CS@HA biomaterial scaffolds. After surgery, each mouse received an intraperitoneal injection of 40,000 units of penicillin. Manual urination was performed twice daily for 7 consecutive days until bladder function was restored.

[0084] 2. Evaluation of animal motor function:

[0085] Motor function in mice was assessed using the BMS, body weight analysis, and footprint analysis. The BMS scoring system was used to evaluate hindlimb joint movement and coordination, ranging from 0 to 9 points. Two observers performed blinded scoring for each mouse for 3 to 5 minutes at fixed time points each week. Twelve weeks after surgery, the mice's forelimbs were painted blue and their hindlimbs red. Mice were instructed to walk along a narrow track on white paper to capture their footprints. These footprints were then scanned, and the resulting digital images were analyzed.

[0086] 3. Magnetic resonance imaging (MRI) analysis of spinal cord structure

[0087] Ninety days after spinal cord injury, rats were anesthetized and fixed in the prone position. Conventional magnetic resonance imaging (MRI) was used on a United Imaging 9.4T ultra-high-field animal MRI system to analyze the structural connectivity of the spinal cord.

[0088] 2. Immunofluorescence analysis

[0089] Twelve weeks after spinal cord injury, mice were intraperitoneally injected with 0.9% sodium pentobarbital (50 mg / kg), and 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 microtome (Leica, Germany), and then subjected to immunofluorescence analysis of GFAP and Tuj1.

[0090] 5. Results

[0091] The present invention verifies the biodegradability of MNP@C-CS@HA in vivo in a C57 mouse T9 spinal cord complete transection model. The results show that MNP@C-CS@HA material has good biodegradability and biocompatibility 12 weeks after in situ implantation at the spinal cord injury site ( Figure 4 ).

[0092] Subsequently, the BMS scores and body weight of mice were analyzed after 12 weeks of treatment, and it was found that the motor function and body weight of mice in the MNP@C-CS@HA group were significantly improved, with the BMS score exceeding 4.0 points ( Figure 5 ).

[0093] After 12 weeks, the gait analysis of mice and the photo analysis of hind leg recovery showed that the movement state of mice in the MNP@C-CS@HA group recovered most significantly, with significantly less hind limb dragging, significantly enhanced coordination, and significantly improved hind limb motor function, which was comparable to the effect of adding neurotrophic factor NT3 ( Figure 6 ).

[0094] Magnetic resonance imaging results were then used to Figure 7 ), it was further found that the MNP@C-CS@HA group showed the best recovery status among all treatment groups, with the smallest tissue damage area and cavity range.

[0095] Finally, through neural tissue immunofluorescence analysis, it was found that the MNP@C-CS@HA material had a high Tuj1+ neuronal differentiation potential (Figure 8).

[0096] These results indicate that the biomaterials provided by the present invention reshape the spinal cord injury microenvironment through multiple pathways such as anti-oxidation, regulation of BV2 polarization, and promotion of endogenous neural differentiation, significantly promoting nerve regeneration and motor function recovery, and showing great clinical application potential in the precise treatment of SCI.

[0097] Finally, the technical solutions listed in the embodiments of the present invention are only preferred implementation schemes and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A methylcobalamin nanofunctionalized collagen polysaccharide sponge, characterized in that: It is prepared according to the following steps: The carboxyl groups on the surface of active collagen are cross-linked with the amino groups of chitosan, and then cross-linked with sodium hyaluronate to obtain a bioactive material; Using methylcobalamin as raw material, a hydrothermal reaction is carried out to obtain methylcobalamin nanodots; The methylcobalamin nanodots are mixed with the bioactive material for electrostatic adsorption to obtain the methylcobalamin nanofunctionalized collagen polysaccharide sponge.

2. The methylcobalamin nanofunctionalized 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 methylcobalamin nanofunctionalized collagen polysaccharide sponge according to claim 1, characterized in that The mixing mass ratio of the bioactive material to the methylcobalamin nanodots is 2-5:

1.

4. The methylcobalamin nanofunctionalized collagen polysaccharide sponge according to claim 1, characterized in that The hydrothermal reaction is maintained at 80° C. to 140° C. for 2 h to 8 h.

5. The methylcobalamin nanofunctionalized collagen polysaccharide sponge according to claim 1, characterized in that The active collagen solution is mixed with EDC and NHS at 4°C to 10°C for 2 h to 8 h, and then the chitosan solution is added and the stirring is continued for 2 h to 6 h. The sodium hyaluronate solution is further added and the stirring is continued at 4°C to 10°C for 2 h to 6 h to obtain the bioactive material.

6. Use of the methylcobalamin nanofunctionalized collagen polysaccharide sponge according to claim 1 in the preparation of a drug for treating spinal cord injury.

7. The use according to claim 6, characterized in that The medicine uses the methylcobalamin nano-functionalized collagen polysaccharide sponge as the only active ingredient.

8. The use according to claim 6, characterized in that The medicine is prepared by compounding the methylcobalamin nano-functionalized collagen polysaccharide sponge with pharmaceutically acceptable excipients.

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