Multifunctional hydrogel carrying neural stem cells and preparation method thereof

Abstract

The invention provides multifunctional hydrogel carrying neural stem cells. The multifunctional hydrogel mainly comprises the following components: chitosan, beta-sodium glycerophosphate, edaravone, nerve growth factors and the neural stem cells, the invention also provides a method for preparing the multifunctional hydrogel, which comprises the following steps: preparing temperature-sensitive hydrogel by taking a chitosan solution and a beta-sodium glycerophosphate solution as matrixes, preparing an edaravone-nerve growth factor mixed solution, and preparing the edaravone-nerve growth factor composite hydrogel. Sequentially adding a neural stem cell suspension and the mixed solution into a hydrogel system to obtain the multifunctional hydrogel carrying the neural stem cells. The multifunctional hydrogel disclosed by the invention realizes high-efficiency integration of three core functions of temperature-sensitive positioning, drug sustained-release microenvironment regulation and control and cell proliferation and differentiation support, and can remarkably promote spinal cord injury repair.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a multifunctional hydrogel carrying neural stem cells and a preparation method thereof. Background Art

[0002] Spinal cord injury is a severe trauma to the central nervous system that often leads to sensory and motor dysfunction below the level of injury, causing tremendous physical and mental suffering and socioeconomic burdens for patients. Currently, treatment for spinal cord injury primarily involves surgical decompression to restore stability and medication, hyperbaric oxygen therapy, and other treatments to inhibit or mitigate secondary damage. While this has alleviated symptoms to a certain extent, the effectiveness of treatment is limited, making it difficult to effectively restore damaged nerve function.

[0003] Neural stem cells have the ability to self-renew and multidirectionally differentiate. In theory, they can differentiate into neurons, astrocytes, oligodendrocytes, etc., providing new hope for the repair of spinal cord injury. However, the delivery and survival of neural stem cells in the body face many challenges. For example, direct injection of neural stem cells faces problems such as low cell survival rate and limited migration ability. In addition, the inflammatory response triggered by spinal cord injury will lead to a large accumulation of reactive oxygen free radicals. These free radicals will attack biological macromolecules such as cell membranes, proteins and nucleic acids, causing massive cell death. Therefore, developing an efficient neural stem cell delivery system and ensuring its function is of great significance for the treatment of spinal cord injury. Summary of the Invention

[0004] To address these issues, the present invention provides a multifunctional hydrogel that carries neural stem cells and a method for preparing the hydrogel. This multifunctional hydrogel is temperature-sensitive and can maintain neural stem cells at the injection site through in situ gelation. It also carries bioactive molecules and possesses powerful free radical scavenging capabilities, enabling regulation of the oxidative microenvironment at the site of injury.

[0005] To achieve the above object, the technical solution adopted by the present invention is: A multifunctional hydrogel carrying neural stem cells is characterized in that the multifunctional hydrogel mainly comprises the following components: chitosan, sodium β-glycerophosphate, edaravone, nerve growth factor and neural stem cells.

[0006] This invention utilizes chitosan, a biocompatible and biodegradable material, as a matrix. By innovatively manipulating the interaction parameters between chitosan and sodium β-glycerophosphate, a temperature-sensitive hydrogel system is constructed. At low temperatures, this system exhibits a low-viscosity, high-flowability sol state, facilitating precise targeting of spinal cord injury sites via minimally invasive injection. Upon exposure to human body temperature (around 37°C), it rapidly undergoes a phase transition to form a gel, effectively anchoring the loaded neural stem cells within the injured area, overcoming the vulnerability of traditional transplants to cell migration and loss. Specifically, the invention stably loads the bioactive molecule eladarbone within the hydrogel's three-dimensional network, enabling controlled sustained release of eladarbone. This sustained release of eladarbone effectively scavenges the large number of free radicals generated after spinal cord injury, continuously combating oxidative stress and significantly alleviating inflammatory damage. At the same time, the hydrogel and eladarone work synergistically to regulate the oxidative microenvironment in the injured area, providing suitable conditions for the effective proliferation and differentiation of neural stem cells under the action of nerve growth factor, promoting axon regeneration and synaptic connection reconstruction, thereby creating all-round favorable conditions for the repair and regeneration of spinal cord tissue. Compared with existing technologies, the multifunctional hydrogel of this invention achieves the multiple functional integration of "temperature-sensitive controllable injection and positioning," "drug sustained release to regulate the microenvironment," and "support for cell proliferation and differentiation." It expands the performance of chitosan-based hydrogels to new heights, provides a new and efficient solution for spinal cord injury repair, and has outstanding substantive characteristics and significant progress.

[0007] In addition, the present invention also provides a method for preparing a multifunctional hydrogel carrying neural stem cells, characterized in that the method comprises the following steps: Step 1: adding chitosan to glacial acetic acid and dissolving it completely to obtain a chitosan solution; Step 2: fully dissolving β-sodium glycerophosphate in deionized water to obtain a β-sodium glycerophosphate solution; Step 3, preparing an eladarbone solution, and mixing it with an equal volume of a nerve growth factor solution to obtain an eladarbone-nerve growth factor mixed solution; Step 4: adding the sodium β-glycerophosphate solution described in step 2 dropwise to the chitosan solution described in step 1 under ice bath conditions to obtain a hydrogel solution; Step 5: Under ice bath conditions, the neural stem cell suspension and the eladarone-nerve growth factor mixture described in step 3 are added to the hydrogel solution described in step 4 in sequence to obtain a multifunctional hydrogel carrying neural stem cells.

[0008] Preferably, the chitosan described in step 1 has a degree of deacetylation of 85% to 95%, a molecular weight range of 100 to 300 kDa, and a mass concentration range of 2% to 5%. The present invention selects chitosan with a degree of deacetylation in the above range and a moderate amino content, so that it has good biocompatibility, can reduce the body's immune rejection reaction, and can moderately degrade under the action of enzymes in the body; chitosan with a molecular weight of 100 to 300 kDa has a moderate molecular chain length. When combined with sodium β-glycerophosphate to form a temperature-sensitive hydrogel, it can quickly and stably achieve sol-gel transition within a suitable temperature range; the mass concentration of the chitosan solution is 2% to 5%. The hydrogel prepared in this concentration range has moderate elasticity and toughness, can withstand certain external forces, protect internal cells, and maintain good water absorption and swelling capacity, simulate the extracellular matrix environment, and promote the survival and growth of neural stem cells.

[0009] Preferably, the mass concentration of the sodium β-glycerophosphate solution in step 2 ranges from 10% to 20%. Sodium β-glycerophosphate is a key substance that triggers the sol-gel transition of the chitosan solution. This concentration range ensures that the hydrogel's gelation temperature precisely matches human body temperature (around 37°C). Upon contact with body temperature, it rapidly induces cross-linking of chitosan molecular chains, forming a stable gel structure within minutes. This ensures the localization of neural stem cells and bioactive molecules to the injury site, maintaining the hydrogel's stability and sustained effect in the body.

[0010] Preferably, the concentration of the eladarbone solution in step three is 1 to 5 mg / mL. After spinal cord injury, a large number of oxygen free radicals will be generated to induce oxidative stress. The hydrogel provided by the present invention carries the bioactive molecule eladarbone with strong free radical scavenging ability, which can effectively capture and remove reactive oxygen free radicals such as hydroxyl radicals and superoxide anions, reduce oxidative stress reactions, and protect neural stem cells from free radical damage. In the above concentration range, combined with the sustained-release properties of the hydrogel, eladarbone will not cause toxic side effects to the human body, and can effectively resist oxidative damage. The volume ratio of eladarbone to the nerve growth factor solution is 1:1. The selection of this volume ratio ensures that the two components are evenly distributed in the hydrogel and the proportions are coordinated. The antioxidant and anti-inflammatory microenvironment created by eladarbone provides favorable conditions for the nerve growth factor to play a role. Nerve growth factor promotes the differentiation of neural stem cells into neurons. The two work synergistically to optimize the survival and differentiation environment of neural stem cells, which helps repair and regenerate damaged tissues.

[0011] Preferably, the volume ratio of the sodium β-glycerophosphate solution to the chitosan solution in step 4 is 1:3 to 1:6. Sodium β-glycerophosphate is a key substance that triggers the sol-gel transition of chitosan. If its relative ratio is too high, the gelation temperature will decrease, and gelation may occur before injection, affecting the operation. If the ratio is too low, the gelation temperature will increase, and rapid gelation at human body temperature will not be possible, making it difficult to effectively fix neural stem cells at the injury site. The above volume ratio range ensures that the hydrogel maintains good fluidity at low temperatures, facilitating injection, and rapidly gels upon exposure to human body temperature, effectively fixing the carried neural stem cells and bioactive molecules.

[0012] Preferably, the neural stem cell suspension described in step five accounts for 5% to 15% by volume in the total system, and the eladarbone-nerve growth factor mixture accounts for 5% to 10% by volume in the total system. The present invention adopts the multifunctional hydrogel with the above ratio to have a multidimensional advantage. On the one hand, it ensures that the cell density in the hydrogel is within a reasonable range and that the neural stem cells can maintain a good survival state. On the other hand, it ensures that the concentrations of eladarbone and nerve growth factor in the hydrogel are in an effective range. This ratio makes the neural stem cells and the two bioactive molecules evenly distributed in the hydrogel network structure. Combined with the sustained-release properties of the hydrogel, its sustained release can be achieved, and the various components work together to achieve a repair effect on spinal cord injury. It should be noted that the total system refers to a system comprising a hydrogel solution, an eladarbone-nerve growth factor mixture, and a neural stem cell suspension.

[0013] The beneficial effects of the present invention are: 1. This invention utilizes highly biocompatible and biodegradable chitosan as a matrix. By innovatively controlling key parameters such as the chitosan and sodium β-glycerophosphate ratio and mixing conditions, a hydrogel system with precise temperature-responsive properties is constructed. At low temperatures, this system exhibits a low-viscosity, highly fluid sol state, making it suitable for minimally invasive injections. Upon exposure to human body temperature (37°C), it rapidly undergoes a sol-gel transition, forming a stable three-dimensional network structure that firmly anchors neural stem cells and bioactive molecules at the site of spinal cord injury. Compared to traditional cell transplantation techniques, this method effectively addresses the challenge of cell migration and loss.

[0014] 2. This invention stably loads edaravone into the pores of the hydrogel network, enabling sustained, controlled release. In the oxidative stress environment following spinal cord injury, the sustained release of edaravone from the hydrogel maintains an effective therapeutic concentration over an extended period, continuously scavenging reactive oxygen species such as hydroxyl radicals and superoxide anions, inhibiting lipid peroxidation and significantly reducing the expression of inflammatory factors, thereby creating a stable microenvironment for neural repair.

[0015] 3. The hydrogel system of this invention simultaneously carries nerve growth factor, leveraging the hydrogel's sustained-release properties to achieve a gradient release of the growth factor. The synergistic effect of nerve growth factor and edaravone promotes the differentiation of neural stem cells into neurons, while also inducing axonal regeneration and promoting the reconstruction of synaptic connections, significantly enhancing spinal cord function repair.

[0016] 4. This invention transcends the limitations of traditional single-functional materials, achieving the efficient integration of three core functions: temperature-sensitive targeting, sustained drug release to regulate the microenvironment, and support for cell proliferation and differentiation. Through innovative material design and preparation processes, the hydrogel possesses optimal mechanical strength, swelling properties, and biosafety. Compared to existing spinal cord injury repair materials, this multifunctional hydrogel offers a novel technological path for spinal cord injury treatment, demonstrating outstanding innovation and significant advancement. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the rheological property test results of the multifunctional hydrogel.

[0018] Figure 2 This is a graph showing the concentration changes of eladarone over time.

[0019] Figure 3 This is a graph showing the concentration changes of nerve growth factor over time.

[0020] Figure 4 This is a graph showing the hindlimb motor score of rats with spinal cord injury treated with multifunctional hydrogel. DETAILED DESCRIPTION

[0021] Example 1 Preparation of multifunctional hydrogel The multifunctional hydrogel of this embodiment mainly includes the following components: chitosan, sodium β-glycerophosphate, edaravone, nerve growth factor and neural stem cells.

[0022] This embodiment also provides a method for preparing a multifunctional hydrogel carrying neural stem cells, the specific steps of which are as follows: Step 1: Weigh 2 g of chitosan with a degree of deacetylation of 85% and a molecular weight of 100 kDa, slowly add 100 mL of 1% glacial acetic acid solution, and stir continuously at 200 r / min at a constant temperature of 25°C for 4 h until the solution is clear and transparent with no undissolved particles, thereby obtaining a uniform and stable chitosan solution; Step 2: Weigh analytical grade sodium β-glycerophosphate, add deionized water at a mass concentration of 10%, and stir at 200 r / min at a low temperature of 4°C for 30 min until completely dissolved to obtain a sodium β-glycerophosphate solution. After the solution is sterilized by filtering through a 0.22 μm filter membrane, store it in a refrigerator at 4°C away from light until it is clear and free of impurities. Step 3: Weigh pharmaceutical-grade eladarbone, add a small amount of cell culture-grade dimethyl sulfoxide, and sonicate at 25°C for 10 min until completely dissolved. Slowly add deionized water to dilute the solution to prepare a 1 mg / mL eladarbone solution, controlling the final volume of dimethyl sulfoxide to be ≤5%. Mix the eladarbone solution and the nerve growth factor solution in a 1:1 volume ratio, and stir at 200 r / min at room temperature for 10 min to obtain a uniform and stable eladarbone-nerve growth factor mixture. Step 4: Take 20 mL of the sodium β-glycerophosphate solution described in step 2 and slowly add it dropwise to 60 mL of the chitosan solution described in step 1, maintaining a stirring rate of 200 r / min during the addition process. After the addition is completed, continue stirring for 30 min to fully mix the solution to obtain a temperature-sensitive hydrogel solution; Step 5: Take neural stem cells in the logarithmic growth phase, wash them three times with serum-free medium, and adjust the cell concentration to 1×10 6 Prepare a stem cell suspension of cells / mL. In an ice bath, take 2.5 mL of the cell suspension and add it to 45 mL of the hydrogel solution obtained in step 4. Stir for 10 minutes to evenly distribute the cells in the hydrogel system. Then add 2.5 mL of the eladarone-nerve growth factor mixture obtained in step 3 and continue stirring for 10 minutes to obtain a multifunctional hydrogel carrying neural stem cells. The hydrogel is stored at 4°C and can complete the sol-gel transition at 37°C body temperature when used.

[0023] Example 2 Determination of rheological properties of multifunctional hydrogel The rheological properties of the multifunctional hydrogel were measured by rheometer. The multifunctional hydrogel was placed on a rotational rheometer and a parallel plate geometry sensor was used. The temperature was raised from 25°C to 45°C within 20 minutes in an oscillating temperature continuous sweep mode with a fixed frequency of 1 Hz and a constant stress of 0.5 Pa. The storage modulus (G') and loss modulus (G'') were measured. Figure 1 ) shows that when the temperature is below 35°C, the G' value of the multifunctional hydrogel is greater than the G' value, and it is in a liquid state. When the temperature rises to 35°C, the G' and G' values ​​are equal, indicating that the strength of the hydrogel increases. This is the sol-gel transition temperature of the multifunctional hydrogel. When the temperature rises further, the G' value is much greater than the G' value, forming a stable gel. From the above results, it can be seen that the multifunctional hydrogel of the present invention has precise temperature response characteristics, presents a low-viscosity, high-fluidity sol state in a low-temperature environment, and is suitable for minimally invasive injection needs. When exposed to human body temperature, it can quickly undergo a sol-gel transition in a short period of time, forming a stable three-dimensional network structure, effectively anchoring neural stem cells and bioactive molecules firmly to the injection site.

[0024] Example 3 Sustained-release performance test of multifunctional hydrogel PBS buffer was used to simulate the physiological environment of the human body. The multifunctional hydrogel was gelled in PBS buffer at a temperature close to that of the human body. The sampling time points were set and the concentration of eladarone in the buffer was determined by high performance liquid chromatography ( Figure 2 ), and the concentration of nerve growth factor was detected by ELISA kit ( Figure 3 The results showed that the concentrations of eladarone and nerve growth factor in the PBS buffer solution increased slowly over time, indicating that the multifunctional hydrogel has good sustained-release properties and can continuously and stably release the carried bioactive substances over a long period of time, helping to maintain their effective concentrations in the body and avoid drastic fluctuations in the concentrations of eladarone and nerve growth factor, thereby reducing toxic side effects and improving therapeutic effects.

[0025] Example 4 Evaluation of the effect of multifunctional hydrogel in promoting spinal cord injury repair A rat spinal cord injury model was established, and the multifunctional hydrogel carrying neural stem cells prepared according to the method provided in Example 1 was locally administered to the injury site for treatment. The BBB score of the rats' hind limb motor function was performed weekly for 6 weeks after surgery, and the hind limb motor function of the blank control group and the multifunctional hydrogel group was compared. Figure 4 As shown in the figure, the BBB score of the blank control group was 3.40±0.99, and the BBB score of the multifunctional hydrogel group was 7.90±0.87, that is, the hind limb joints of the multifunctional hydrogel group could achieve a larger range of motion, proving that the multifunctional hydrogel carrying neural stem cells can significantly improve the hind limb motor ability of rats with spinal cord injury.

[0026] In summary, the multifunctional hydrogel carrying neural stem cells provided by the present invention has precise temperature response characteristics, can quickly undergo sol-gel transition in a short period of time, and achieves firm anchoring of neural stem cells and bioactive molecules; the sustained-release properties of the hydrogel enable the sustained release of the bioactive molecules edaravone and nerve growth factor, which work synergistically to promote the proliferation and differentiation of neural stem cells, creating favorable conditions for spinal cord injury repair. The scope of protection of the present invention is not limited to the above-mentioned embodiments, but also includes various variations and improvements made based on the technical solutions of the present invention.

Claims

1. A multifunctional hydrogel carrying neural stem cells, characterized in that: The multifunctional hydrogel mainly comprises the following components: chitosan, sodium beta-glycerophosphate, edaravone, nerve growth factor and neural stem cells.

2. A method for preparing the multifunctional hydrogel according to claim 1, characterized in that: The method comprises the following steps: Step 1: adding chitosan to glacial acetic acid and dissolving it completely to obtain a chitosan solution; Step 2: fully dissolving β-sodium glycerophosphate in deionized water to obtain a β-sodium glycerophosphate solution; Step 3, preparing an eladarbone solution, and mixing it with an equal volume of a nerve growth factor solution to obtain an eladarbone-nerve growth factor mixed solution; Step 4: adding the sodium β-glycerophosphate solution described in step 2 dropwise to the chitosan solution described in step 1 under ice bath conditions to obtain a hydrogel solution; Step 5: Under ice bath conditions, the neural stem cell suspension and the eladarone-nerve growth factor mixture described in step 3 are added to the hydrogel solution described in step 4 in sequence to obtain a multifunctional hydrogel carrying neural stem cells.

3. The method according to claim 2, characterized in that The chitosan in step 1 has a deacetylation degree of 85% to 95%, a molecular weight range of 100 to 300 kDa, and a mass concentration range of the chitosan solution of 2% to 5%.

4. The method according to claim 2, characterized in that The mass concentration range of the β-sodium glycerophosphate solution described in step 2 is 10% to 20%.

5. The method according to claim 2, characterized in that The concentration of the eladarone solution in step 3 is 1-5 mg / mL.

6. The method according to claim 2, characterized in that The volume ratio of the β-sodium glycerophosphate solution to the chitosan solution in step 4 is 1:3 to 1:

6.

7. The method according to claim 2, characterized in that The volume of the neural stem cell suspension in step 5 accounts for 5% to 15% of the total system, and the volume of the eladarbone-nerve growth factor mixture accounts for 5% to 10% of the total system.

Images