A structurally stable gelma microcarrier and a method of making the same

Abstract

The application discloses a kind of structurally stable GelMA microcarrier and preparation method thereof, it is related to the technical field of biomedical materials, a kind of structurally stable GelMA microcarrier, GelMA microcarrier is formed by low-speed stirring photo-crosslinking and low-temperature physical gelation shaping of methacrylic anhydridized gelatin, the degree of substitution of methacrylic anhydridized gelatin methacryl group is 30%-80%;The temperature of low-temperature physical gelation shaping is 0-4 ℃;Provide a kind of preparation method of GelMA microcarrier;The GelMA of the application with methacryl group is raw material, using petroleum ether-sorbitan 80 oil phase system, after high-speed emulsification, under low-speed stirring, photo-crosslinking preliminary solidification, cooling induces physical gelation and stabilizes appearance;The spherical degree of the obtained product is high, breakage rate is low, solvent residue is little, can be used for drug delivery, cell embedding, tissue engineering repair and other biomedical scenes.

Description

Technical Field

[0001] This invention relates to the field of biomedical polymer materials technology, and in particular to a structurally stable GelMA microcarrier and its preparation method. Background Technology

[0002] Nerve injury is a common and difficult-to-treat clinical condition, encompassing various types such as peripheral nerve injury, spinal cord injury, and neurodegenerative diseases of the brain. It is characterized by high incidence, difficulty in repair, and poor prognosis. With the increase in traffic injuries, sports injuries, and neurodegenerative diseases related to population aging, nerve injury has become a public health problem seriously affecting patients' quality of life. Current clinical treatment methods are very limited: peripheral nerve defects often require autologous nerve transplantation, but this suffers from drawbacks such as insufficient donor sources, donor site functional impairment, and poor compatibility; central nervous system injuries lack effective regeneration methods, and conventional drug and surgical treatments are insufficient to achieve substantial recovery of nerve function.

[0003] In recent years, neural regeneration strategies based on cell transplantation and sustained drug release have become a research hotspot. Delivering seed cells such as neural stem cells and Schwann cells, combined with neurotrophic factors and small-molecule therapeutic drugs, can synergistically regulate the injury microenvironment and promote axonal regeneration and myelin formation, representing a core pathway for achieving neural structural and functional repair. However, direct local injection of cells suffers from low cell survival rates, poor retention, and uneven distribution; free drugs, on the other hand, have limitations such as short half-lives, severe burst release, and short duration of action, making it difficult to meet the long-term, stable regulatory needs during neural repair. Therefore, constructing biocompatible delivery carriers that combine cell loading and sustained drug release functions is a key requirement in the field of neural regeneration.

[0004] Microcarriers, with their advantages of large specific surface area, controllable size, and minimally invasive delivery, have become an ideal choice for neural repair delivery systems. Methacrylic anhydride-modified gelatin (GelMA) is a photosensitive natural polymer material obtained by modifying gelatin with methacrylic anhydride. It retains gelatin's excellent biocompatibility, cell adhesion, and biodegradability, while also enabling a gentle and controllable molding process through photocrosslinking. It can provide nerve cells with a three-dimensional microenvironment similar to the extracellular matrix, and can efficiently load protein-based and small-molecule neurotherapeutic drugs, making it a core material for constructing neural repair delivery microcarriers.

[0005] Currently, GelMA microcarriers are mostly prepared using emulsification-photocrosslinking processes, but existing preparation technologies still have several technical shortcomings that limit their application in high-precision delivery scenarios: The cross-linking process is prone to droplet aggregation and insufficient uniformity of microcarriers: if high-speed stirring is maintained after emulsification for photocross-linking, continuous shear force can easily damage the droplet interface structure; if stirring is stopped and cross-linking is allowed to proceed statically, droplets are prone to collision and fusion, ultimately resulting in poor sphericity, wide particle size distribution, and poor batch-to-batch stability of the microcarriers. Neural repair delivery requires extremely high uniformity of microcarriers; uneven particle size directly leads to large differences in cell loading, inconsistent drug release behavior, and uneven repair effects in the damaged area after local delivery, making precise control difficult.

[0006] High breakage rate and poor carrier structural integrity during washing: Existing processes generally use liquid paraffin as the oil phase, which has a high boiling point and poor volatility, requiring isopropanol replacement and multi-step water washing to completely remove the oil phase and emulsifier. During the transfer of microcarriers from the oil phase to the aqueous phase, the sudden change in solvent polarity can cause non-uniform swelling of the GelMA three-dimensional network, leading to deformation, adhesion, and even breakage of the microcarriers. Under conventional processes, the breakage rate of the finished product is usually higher than 20%. Damaged microcarriers can not only cause leakage and death of loaded cells and premature drug release, but may also trigger local inflammatory reactions, which is detrimental to nerve tissue regeneration.

[0007] Organic solvent residues are difficult to remove, resulting in insufficient neurobiological safety: High-boiling-point liquid paraffin and emulsifiers are still difficult to completely remove even after multiple washing steps. Residual organic solvents have potential neurotoxicity, which can reduce the survival rate of seed cells and inhibit the growth of neurites. After implantation in the body, they may also cause local inflammation and tissue rejection, posing significant biosafety risks and failing to meet the high standards required for nerve repair implantation applications.

[0008] While current research has focused on improving the performance of GelMA microcarriers through optimization of emulsification parameters, a systematic and integrated process encompassing "crosslinking and shaping - washing and purification - solvent removal" has not yet been established. This process cannot simultaneously meet the comprehensive requirements of neural repair delivery carriers for high sphericity, high uniformity, low breakage rate, and high biocompatibility. Therefore, this study aims to provide a structurally stable GelMA microcarrier and its preparation method. Summary of the Invention

[0009] In view of the above, this invention addresses the shortcomings of existing technologies by providing a structurally stable GelMA microcarrier and its preparation method. The method addresses the following technical problems: droplet aggregation during crosslinking leads to poor sphericity and wide particle size distribution in the microcarriers; sudden changes in solvent polarity during washing cause deformation and breakage of the microcarriers, resulting in low yield; and high-boiling-point oil phase residue is difficult to remove, leading to insufficient biosafety. Using self-made GelMA as raw material and petroleum ether as the oil phase, this invention employs a dual-setting strategy of "low-speed stirring photocrosslinking + cooling physical setting," combined with low-temperature petroleum ether washing and freeze-drying solvent removal processes. This solves the technical problems of easy aggregation during crosslinking, easy breakage during washing, and high solvent residue in existing GelMA microcarriers.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A structurally stable GelMA microcarrier is provided, wherein the GelMA microcarrier is formed by dual curing of methacrylic anhydride gelatin through low-speed stirring photocrosslinking and low-temperature physical gel setting. The sphericity of the GelMA microcarrier is ≥0.96, the coefficient of variation of particle size (CV) is ≤6%, and the degree of methacryloyl substitution of the methacrylic anhydride gelatin is 30%-80%. The stirring speed for low-speed stirring photocrosslinking is 200-500 rpm, and the temperature for low-temperature physical gel setting is 0-4℃.

[0011] As a preferred embodiment: the GelMA microcarrier has a through-porous structure with a pore size of 80-250 nm; the GelMA microcarrier is loaded with active drugs, cells or growth factors.

[0012] As a preferred embodiment: the swelling rate of the GelMA microcarrier is 150%-300%, the in vitro degradation period is 7-30 days; the microcarrier breakage rate after washing is ≤3%, and the petroleum ether residue is ≤1μg / g.

[0013] A method for preparing the aforementioned GelMA microcarrier includes the following steps: First, preparation of GelMA: Dissolve methacrylic anhydride gelatin in buffer solution, heat and stir, then add methacrylic anhydride dropwise to react in the dark, and the product is dialyzed and freeze-dried to obtain GelMA powder; Second, preparation of the water-oil phase: Dissolve GelMA dry powder and photoinitiator in buffer solution to obtain GelMA prepolymer as the aqueous phase; add Span 80 emulsifier to petroleum ether and mix evenly to obtain the oil phase; Third, emulsification: The aqueous phase is added dropwise to the oil phase and stirred at high speed to form a water-in-oil emulsion; Fourth, cross-linking: Reduce the stirring speed to a low speed and keep the emulsion stirring at a low speed while irradiating with ultraviolet light to initiate the initial cross-linking and curing of GelMA; after the photo-cross-linking is completed, cool the system to allow the microcarrier to undergo low-temperature physical gelation and stabilize the morphology and structure of the microcarrier. Fifth, washing and drying.

[0014] As a preferred embodiment: in step two, the mass fraction of the GelMA prepolymer solution is 5%-15%; the photoinitiator is LAP, and the amount used is 0.5%-2% of the mass of GelMA; the aqueous phase also contains active drugs, cells or growth factors.

[0015] As a preferred embodiment: in step two, the mass fraction of the GelMA prepolymer is 8%-12%; the amount of the LAP photoinitiator is 1%-1.5% of the mass of GelMA.

[0016] As a preferred embodiment: in step two, the boiling range of the petroleum ether is 60-90℃, and the mass fraction of Span 80 is 2%-6%; in step three, the water-oil volume ratio is 1:(4-10), the stirring speed is 800-1500 rpm, and the stirring time is 5-15 min.

[0017] As a preferred embodiment: the low-speed stirring rotation speed is 200-500 rpm; the ultraviolet light wavelength is 365 nm, the irradiation time is 1-5 min, and the light intensity is 5-20 mW / cm². 2 After photocrosslinking is completed, the system is cooled to 0-4℃ and allowed to stand for setting, or liquid nitrogen is used for quenching and setting; the setting time is 5-30 minutes.

[0018] As a preferred embodiment, the washing and drying process specifically involves: adding pre-cooled low-temperature petroleum ether for resuspension and washing, discarding the supernatant, and repeating the process multiple times to remove residual emulsifier; after washing, the GelMA microcarriers are pre-frozen and then freeze-dried to remove residual petroleum ether by sublimation, yielding the finished GelMA microcarriers; the temperature of the low-temperature petroleum ether is -10-4℃, and the number of washing cycles is 3-5.

[0019] As a preferred embodiment: in step one, the reaction temperature for the addition of methacrylic anhydride in the dark is 45-55℃, and the reaction time is 20h; the molecular weight cutoff for dialysis is 8000-14000Da, and the dialysis time is 5-7 days.

[0020] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, as can be seen from the above technical solution: (1) The present invention adopts a dual shaping strategy of low-speed stirring photocrosslinking + low-temperature physical gelation, which avoids high-speed shearing from damaging the droplet interface and prevents droplet collision and aggregation during static crosslinking. The final microcarrier has high sphericity, low particle size variation coefficient, and good batch repeatability.

[0021] (2) The present invention uses a petroleum ether system for washing throughout the process. The microcarrier does not need to undergo a polarity change from oil phase to water phase, thus avoiding swelling, deformation and cracking from the source. Combined with low temperature washing conditions, the breakage rate after washing is low and the yield can reach more than 95%.

[0022] (3) The present invention uses low-boiling-point petroleum ether to replace traditional liquid paraffin. The residual solvent can be completely removed during the freeze-drying sublimation process without the need for multi-step solvent replacement. The final amount of petroleum ether residue is small and there is no risk of cytotoxicity.

[0023] (4) The present invention can flexibly adjust the degradation cycle and mechanical properties of microcarriers by controlling the degree of substitution of GelMA and crosslinking parameters, and is compatible with various functional requirements such as drug delivery and cell delivery.

[0024] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0025] Figure 1 This is a scanning electron microscope (SEM) image of the GelMA microcarrier used in this invention.

[0026] Figure 2 This is a fluorescence image of seed cells loaded on GelMA microcarriers in this invention. Detailed Implementation

[0027] The present invention is as follows Figures 1 to 2 As shown, a structurally stable GelMA microcarrier is formed by dual curing of methacrylic anhydride gelatin through low-speed stirring photocrosslinking and low-temperature physical gel setting. The sphericity of the GelMA microcarrier is ≥0.96, the coefficient of variation of particle size (CV) is ≤6%, and the degree of methacryloyl substitution of the methacrylic anhydride gelatin is 30%-80%. The stirring speed for low-speed stirring photocrosslinking is 200-500 rpm, and the temperature for low-temperature physical gel setting is 0-4℃.

[0028] The GelMA microcarrier has a through-porous structure with a pore size of 80-250 nm; the GelMA microcarrier is loaded with active drugs, cells or growth factors.

[0029] The swelling rate of this GelMA microcarrier is 150%-300%, and the in vitro degradation period is 7-30 days; after washing, the microcarrier breakage rate is ≤3%, and the petroleum ether residue is ≤1μg / g.

[0030] A method for preparing the GelMA microcarrier includes the following steps: First, preparation of GelMA: Dissolve methacrylic anhydride gelatin in buffer solution, heat and stir, then add methacrylic anhydride dropwise to react in the dark, and the product is dialyzed and freeze-dried to obtain GelMA powder; Second, preparation of the water-oil phase: Dissolve GelMA dry powder and photoinitiator in buffer solution to obtain GelMA prepolymer as the aqueous phase; add Span 80 emulsifier to petroleum ether and mix evenly to obtain the oil phase; Third, emulsification: The aqueous phase is added dropwise to the oil phase and stirred at high speed to form a water-in-oil emulsion; Fourth, cross-linking: Reduce the stirring speed to a low speed and keep the emulsion stirring at a low speed while irradiating with ultraviolet light to initiate the initial cross-linking and curing of GelMA; after the photo-cross-linking is completed, cool the system to allow the microcarrier to undergo low-temperature physical gelation and stabilize the morphology and structure of the microcarrier. Fifth, washing and drying.

[0031] In step two, the mass fraction of the GelMA prepolymer solution is 5%-15%; the photoinitiator is LAP, and the amount used is 0.5%-2% of the mass of GelMA; active drugs, cells or growth factors are also added to the aqueous phase.

[0032] In step two, the mass fraction of the GelMA prepolymer is 8%-12%; the amount of the LAP photoinitiator is 1%-1.5% of the mass of GelMA.

[0033] In step two, the boiling range of the petroleum ether is 60-90℃, and the mass fraction of Span 80 is 2%-6%; in step three, the water-oil volume ratio is 1:(4-10), the stirring speed is 800-1500 rpm, and the stirring time is 5-15 min.

[0034] The low-speed stirring speed is 200-500 rpm; the ultraviolet light wavelength is 365 nm, the irradiation time is 1-5 min, and the light intensity is 5-20 mW / cm²; after the photocrosslinking is completed, the system is cooled to 0-4℃ and allowed to stand for setting, or liquid nitrogen quenching is used for setting; the setting time is 5-30 min.

[0035] The washing and drying process is as follows: pre-cooled low-temperature petroleum ether is added for resuspension and washing, the supernatant is discarded, and the process is repeated several times to remove residual emulsifier; after washing, the GelMA microcarrier is pre-frozen and then freeze-dried, and the residual petroleum ether is sublimated to remove it, thus obtaining the finished GelMA microcarrier; the temperature of the low-temperature petroleum ether is -10-4℃, and the number of washing cycles is 3-5.

[0036] In step one, the reaction temperature for the addition of methacrylic anhydride in the dark is 45-55℃, and the reaction time is 20h; the molecular weight cutoff for dialysis is 8000-14000Da, and the dialysis time is 5-7 days.

[0037] Example 1: Preparation method of structurally stable GelMA microcarriers Step 1: Preparation of homemade GelMA: Weigh 10g of type A gelatin and add it to 100mL of PBS buffer at pH 7.4. Stir in a 50℃ water bath until completely dissolved. Slowly add 8mL of methacrylic anhydride at a rate of 1mL / min and stir at 50℃ in the dark for 20h. After the reaction is complete, dilute with preheated PBS to terminate the reaction. Transfer the solution to a dialysis bag (molecular weight cutoff 8000-14000Da) and dialyze with deionized water for 7 days, changing the water 3 times a day. Freeze-dry for 48h to obtain white sponge-like GelMA powder. NMR analysis showed that the degree of methacryloyl substitution was 58%.

[0038] Step 2: Fabrication of GelMA microcarriers: Aqueous phase preparation: Weigh 0.8g of self-made GelMA and 10mg of LAP photoinitiator, add to 10mL of pH7.4 PBS buffer, stir at 37℃ in the dark until completely dissolved, to obtain an 8% (w / w) GelMA prepolymer, which is used as the aqueous phase. Oil phase preparation: Add Span80 to petroleum ether with a boiling range of 60-90℃, stir to dissolve, to obtain a 3% (w / w) Span80-petroleum ether mixture, and measure 50mL as the oil phase. High-speed stirring emulsification: Slowly add the aqueous phase to the oil phase, maintaining high-speed stirring at 1000rpm during the addition process. After the addition is complete, continue stirring for 5min to form a uniform W / O type emulsion. Low-speed stirring photocrosslinking: Reduce the stirring speed to 300rpm and maintain low-speed uniform stirring; at the same time, place the emulsion under a 365nm UV lamp with a light intensity of 10mW / cm² for 3min to initiate the initial crosslinking and curing of GelMA. Temperature-stabilized microcarriers: After photocrosslinking, the centrifuge tubes containing the emulsion were immediately placed in a 4°C environment for 10 minutes to allow the microcarriers to undergo low-temperature physical gelation, further stabilizing their morphology. Low-temperature petroleum ether washing: Petroleum ether pre-cooled to 0°C was added to the system, and the microcarriers were gently resuspended by blowing, then the supernatant was discarded; this washing was repeated 4 times to thoroughly remove residual Span80 and unreacted monomers. Freeze-drying: The washed microcarriers were pre-frozen at -80°C for 4 hours, then transferred to a freeze dryer and vacuum-dried at -55°C for 36 hours. Residual petroleum ether was completely removed during the sublimation process, yielding the finished GelMA microcarrier product.

[0039] The microcarriers in this embodiment were tested and found to have an average particle size of 172 μm, a particle size variation coefficient (CV) of 3.9%, and a sphericity of 0.97. The microcarrier breakage rate after washing was 2.4%, and the yield was 96.1%. Gas chromatography analysis showed that the residual petroleum ether content was 0.58 μg / g.

[0040] Screening and verification of preparation process parameters: In order to optimize the aqueous phase formulation, parallel screening experiments were conducted on the mass fraction of GelMA prepolymer and the amount of LAP photoinitiator. The other preparation processes and parameters were kept the same as above. After preparation, the three core properties of microspheres, namely crosslinking degree, sphericity and washing breakage rate, were tested to screen the optimal dosage.

[0041] 1. Screening of GelMA prepolymer solution mass fraction: With the LAP dosage fixed at 1.25% of the GelMA mass, the mass fraction of the GelMA prepolymer solution was adjusted to 5%, 8%, 12%, and 15%, respectively. The results are shown in Table 1. Table 1: Screening Results of GelMA Prepolymer Mass Fraction

[0042] The results showed that when the GelMA concentration was too low, the microspheres had insufficient mechanical strength and were easily damaged during washing; when the concentration was too high, the viscosity of the aqueous phase was too large, and the uniformity of the emulsion droplets decreased; when the mass fraction was 8%-12%, the microspheres exhibited the best overall performance in terms of morphology, strength, and uniformity, which was the optimal concentration range.

[0043] 2. Screening of LAP photoinitiator dosage: With the GelMA prepolymer mass fraction fixed at 8%, the LAP dosage was adjusted to 0.5%, 1.0%, 1.25%, and 2.0% of the GelMA mass. The results are shown in Table 2. Table 2: Screening Results of LAP Photoinitiator Dosage

[0044] The results showed that when the LAP dosage was less than 1%, the cross-linking was insufficient, the microsphere structural strength was inadequate, and the washing breakage rate increased significantly; when the dosage was more than 2%, the cross-linking was excessive, the microsphere brittleness increased, and the risk of initiator residue increased; when the dosage was 1%-1.5%, the microspheres had moderate cross-linking degree, the highest sphericity, and the lowest washing breakage rate, with the best overall performance, which was the optimal dosage range.

[0045] Example 2: Preparation of drug-loaded GelMA microcarriers Preparation of drug-loaded GelMA microcarriers: (1) Self-made GelMA was prepared in the same manner as in Example 1, with a degree of substitution of 58%. (2) Aqueous phase preparation: 8% GelMA prepolymer solution was prepared, and 20 mg of deer antler polypeptide drug was added to it. The mixture was stirred and mixed in the dark. The rest was the same as in Example 1. (3) The oil phase preparation, high-speed emulsification, low-speed photocrosslinking, cooling stabilization, low-temperature washing, and freeze-drying steps were completely consistent with those in Example 1, and the deer antler polypeptide-loaded GelMA microcarriers were finally obtained.

[0046] The microcarriers were tested and found to have an average particle size of 176 μm, a CV value of 4.3%, and a washing breakage rate of 2.7%. The drug encapsulation rate was 87.8%, the 24-hour burst release rate was 13.2%, and the sustained release period could reach 21 days.

[0047] Comparative Example 1: GelMA microcarriers were prepared using a conventional liquid paraffin-water washing process. The GelMA raw material and aqueous phase were prepared in the same manner as in Example 1. The oil phase was prepared using a 3% Span80-liquid paraffin system, which was emulsified at high speed and then directly crosslinked under UV light after stirring was stopped. The washing process was conventional: the oil phase was discarded by centrifugation at 3000 rpm, and the product was washed twice with isopropanol and three times with deionized water, followed by freeze drying.

[0048] Testing revealed that the sphericity of the comparative microcarriers was 0.90, the breakage rate after washing was 28.1%, a large number of microcarriers adhered and deformed, and the yield was only 65.7%; residual organic solvent testing showed that the residual amount of liquid paraffin was 13.2 μg / g.

[0049] Comparative Example 2: Petroleum ether system microcarrier GelMA without cooling and stabilization step. The raw materials, emulsification, crosslinking, washing, and freeze-drying were the same as in Example 1, except that the cooling and stabilization step was omitted. Testing revealed that some microcarriers softened and deformed during the washing process, with the breakage rate increasing to 8.7% and the sphericity decreasing by 0.92.

[0050] Example 3: Process verification with different degrees of substitution. The amount of methacrylic anhydride added was adjusted to 4 mL, and the remaining GelMA synthesis steps were the same as in Example 1, yielding GelMA with a degree of substitution of 35%. Microcarriers were prepared according to the process in Example 1. The obtained microcarriers had an average particle size of 168 μm, a CV value of 4.6%, a washing breakage rate of 2.6%, and a degradation period of approximately 10 days, maintaining excellent morphological stability and a low breakage rate.

[0051] Example 4: Preparation and in vitro release verification of GelMA microspheres loaded with nerve growth factor (1) Preparation of self-made GelMA is the same as in Example 1, with a degree of substitution of 58%. (2) Preparation of aqueous phase: Weigh 0.8g of self-made GelMA and 10mg of LAP photoinitiator, add them to 10mL of PBS buffer at pH 7.4, stir at 37℃ in the dark until completely dissolved, and obtain a GelMA prepolymer solution with a mass fraction of 8%; add 10μg of nerve growth factor (NGF) to the prepolymer solution, mix gently, and use as the aqueous phase. (3) The steps of oil phase preparation, high-speed stirring emulsification, low-speed photocrosslinking, cooling stabilization, low-temperature petroleum ether washing, and freeze drying are all completely consistent with those in Example 1, and finally, GelMA microspheres loaded with NGF are obtained. (4) In vitro release assay: 10 mg of drug-loaded microspheres were placed in a centrifuge tube, 2 mL of PBS buffer was added, and the tube was placed in a 37°C constant temperature shaker and shaken at 100 rpm. At each set time point, all the released solution was collected and an equal amount of fresh PBS was added. The NGF concentration at each time point was detected using an ELISA kit, and the cumulative release rate was calculated. The results showed that the NGF encapsulation rate of the microspheres in this embodiment was 83.6%, and the drug loading was 0.098 μg / mg. The in vitro release results showed that the cumulative release rate was 14.7% in 24 hours, with no obvious burst release, and the release was stable and continuous for up to 14 days, which can meet the long-term drug administration needs during nerve repair.

[0052] Example 5: Preparation of Schwann cell-loaded GelMA microspheres and verification of cell activity (1) Preparation of self-made GelMA: Same as in Example 1, with a degree of substitution of 58%; After high-pressure steam sterilization, the GelMA prepolymer solution was placed in a sterile environment for later use. (2) Preparation of cell aqueous phase: Take 8% sterile GelMA prepolymer solution and add sterile LAP photoinitiator to a final concentration of 0.1%; Resuspend Schwann cells in logarithmic growth phase in the prepolymer solution, adjust the cell density to 5×10^6 cells / mL, and gently mix to obtain the cell aqueous phase. (3) Under sterile conditions, complete the oil phase preparation, high-speed emulsification, low-speed photocrosslinking, and cooling and shaping according to the process parameters of Example 1; Sterile reagents and consumables were used throughout the process. After washing with low-temperature petroleum ether, the microspheres were resuspended with sterile PBS, collected by centrifugation, and transferred to cell culture medium for culture. (4) Cell activity detection: After culturing for 1, 3, and 7 days, the cell proliferation activity was detected by CCK-8 method, and the cell survival status was observed by staining of live and dead cells. The results showed that the cell survival rate was 89.2% on day 1 after embedding, and the cells continued to proliferate on days 3 and 7. Live and dead cell staining showed that the cells in the microspheres had good morphology and no large-scale cell death. This proves that the microspheres can be used as cell carriers to provide a good three-dimensional growth microenvironment for nerve seed cells and are suitable for cell delivery for nerve repair.

[0053] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A structurally stable GelMA microcarrier, characterized in that: The GelMA microcarrier is formed by dual curing of methacrylic anhydride gelatin through low-speed stirring photocrosslinking and low-temperature physical gel setting. The sphericity of the GelMA microcarrier is ≥0.96, the particle size variation coefficient (CV) is ≤6%, and the degree of methacryloyl substitution of the methacrylic anhydride gelatin is 30%-80%. The stirring speed for low-speed stirring photocrosslinking is 200-500 rpm, and the temperature for low-temperature physical gel setting is 0-4℃.

2. The GelMA microcarrier according to claim 1, characterized in that: The GelMA microcarrier has a through-porous structure with a pore size of 80-250 nm; the GelMA microcarrier is loaded with active drugs, cells or growth factors.

3. The GelMA microcarrier according to claim 1, characterized in that: The swelling rate of the GelMA microcarrier is 150%-300%, and the in vitro degradation period is 7-30 days; the microcarrier breakage rate after washing is ≤3%, and the petroleum ether residue is ≤1μg / g.

4. A method for preparing GelMA microcarriers as described in any one of claims 1-3, characterized in that: Includes the following steps: First, preparation of GelMA: Dissolve methacrylic anhydride gelatin in buffer solution, heat and stir, then add methacrylic anhydride dropwise to react in the dark, and the product is dialyzed and freeze-dried to obtain GelMA powder; Second, preparation of the water-oil phase: Dissolve GelMA dry powder and photoinitiator in buffer solution to obtain GelMA prepolymer as the aqueous phase; add Span 80 emulsifier to petroleum ether and mix evenly to obtain the oil phase; Third, emulsification: The aqueous phase is added dropwise to the oil phase and stirred at high speed to form a water-in-oil emulsion; Fourth, cross-linking: Reduce the stirring speed to a low speed and keep the emulsion stirring at a low speed while irradiating with ultraviolet light to initiate the initial cross-linking and curing of GelMA; after the photo-cross-linking is completed, cool the system to allow the microcarrier to undergo low-temperature physical gelation and stabilize the morphology and structure of the microcarrier. Fifth, washing and drying.

5. The preparation method according to claim 4, characterized in that: In step two, the mass fraction of the GelMA prepolymer solution is 5%-15%; the photoinitiator is LAP, and the amount used is 0.5%-2% of the mass of GelMA; the aqueous phase also contains active drugs, cells or growth factors.

6. The preparation method according to claim 5, characterized in that: In step two, the mass fraction of the GelMA prepolymer is 8%-12%; the amount of the LAP photoinitiator is 1%-1.5% of the mass of GelMA.

7. The preparation method according to claim 4, characterized in that: In step two, the boiling range of the petroleum ether is 60-90℃, and the mass fraction of Span 80 is 2%-6%; in step three, the water-oil volume ratio is 1:(4-10), the stirring speed is 800-1500 rpm, and the stirring time is 5-15 min.

8. The preparation method according to claim 4, characterized in that: In step four, the low-speed stirring speed is 200-500 rpm; the ultraviolet light wavelength is 365 nm, the irradiation time is 1-5 min, and the light intensity is 5-20 mW / cm²; after the photocrosslinking is completed, the system is cooled to 0-4℃ and allowed to stand for setting, or liquid nitrogen is used for quenching and setting; the setting time is 5-30 min.

9. The preparation method according to claim 4, characterized in that: In step five, the washing and drying process specifically involves: adding pre-cooled low-temperature petroleum ether for resuspension and washing, discarding the supernatant, and repeating the process multiple times to remove residual emulsifier; after washing, the GelMA microcarriers are pre-frozen and then freeze-dried to remove residual petroleum ether by sublimation, resulting in the finished GelMA microcarriers; the temperature of the low-temperature petroleum ether is -10-4℃, and the number of washing cycles is 3-5.

10. The preparation method according to claim 4, characterized in that: In step one, the reaction temperature for adding methacrylic anhydride in the dark is 45-55℃, and the reaction time is 20h; the molecular weight cutoff for dialysis is 8000-14000Da, and the dialysis time is 5-7 days.

Images