Hydrogel for treating ischemic brain injury and preparation method thereof

Abstract

The invention relates to hydrogel for treating ischemic brain injury and a preparation method thereof. The hydrogel is prepared from the following raw materials: methylacryloylated hyaluronic acid, methylacryloylated gelatin, curcumin loaded HA-PPS (Hydroxypropyl-Polyphenylene Sulfite) nano-micelles and dendrobium polysaccharide loaded sodium alginate microspheres. Through combination of a nanotechnology and an intelligent response material, HA-PPS is adopted to load curcumin and sodium alginate microspheres are adopted to encapsulate dendrobium polysaccharide, so that the drug activity can be more effectively protected, the drug is protected to penetrate through a blood brain barrier and be accurately delivered to a brain injury area, and targeted release of the drug is realized; the anti-oxidation and anti-inflammatory effects are better under the combined action of the curcumin and the dendrobium polysaccharides, and meanwhile, a slow release function is also achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and particularly relates to a hydrogel for treating ischemic brain injury and a preparation method thereof. Background Art

[0002] Ischemic brain injury is a serious neurological disease caused by the interruption or insufficiency of blood supply to the brain, which leads to the death of brain cells due to lack of oxygen. The most common cause of this disease is cerebral apoplexy, which has a very high incidence and disability rate, and there is currently no completely effective treatment. Early intervention for ischemic brain injury usually focuses on restoring blood supply to the brain and reducing further damage to neurons. However, during the ischemia-reperfusion process (i.e., when blood flow is restored), oxidative stress will be generated, leading to functional destruction of the blood-brain barrier (BBB), damaging endothelial cells in the blood-brain barrier, increasing its permeability, and causing harmful substances such as inflammatory cells and proteins to enter the brain tissue, further aggravating brain damage. Existing clinical treatments include thrombolytic therapy, mechanical thrombectomy, and the use of antiplatelet and anticoagulant drugs, aiming to restore cerebral blood flow as quickly as possible. However, these treatments have certain limitations and risks.

[0003] The drugs currently used to treat ischemic brain injury, such as recombinant tissue plasminogen activator (rtPA), can effectively dissolve blood clots, but due to their poor targeting, they cannot act specifically on the damaged area of ​​the brain, often leading to systemic side effects, especially the risk of cerebral hemorrhage. It is difficult to control drug release, and some thrombolytic drugs and neuroprotective drugs have a short half-life in the body and are difficult to exert their effects continuously. Therefore, patients often need to take medication frequently, which affects treatment compliance and increases the incidence of drug toxicity and side effects.

[0004] The blood-brain barrier (BBB) ​​is a key barrier that prevents most drugs from entering brain tissue. Many potential therapeutic drugs cannot effectively pass through the blood-brain barrier, resulting in the inability of the drugs to reach the effective concentration required for treatment. This greatly limits the range of therapeutic drugs and also affects their therapeutic effects.

[0005] Curcumin is a natural polyphenol compound extracted from turmeric, which has multiple pharmacological activities such as anti-inflammatory, antioxidant, anti-apoptotic and neuroprotective. It can effectively scavenge free radicals, reduce the damage of oxidative stress to neurons, and thus protect brain tissue. At the same time, by inhibiting the nuclear factor-κB (NF-κB) signaling pathway, it reduces the release of proinflammatory cytokines (such as TNF-α, IL-1β), reduces the inflammatory response, and thus reduces the inflammatory damage after brain injury. However, due to its poor water solubility, low bioavailability, and easy rapid metabolism in the body, curcumin is difficult to maintain an effective concentration in the body, affecting the therapeutic effect, and curcumin has limited ability to pass the blood-brain barrier.

[0006] Dendrobium polysaccharide is a natural bioactive ingredient extracted from Dendrobium officinale, which has shown important therapeutic potential in the treatment of ischemic brain injury through multiple mechanisms such as anti-oxidation, anti-inflammation, promoting nerve regeneration, enhancing immune regulation and improving blood circulation. However, due to its poor water solubility and stability, its efficacy in the body lasts for a short time, and its bioavailability is low, which limits its actual therapeutic effect.

[0007] In recent years, the prognosis and treatment of brain injury have received widespread attention. However, conventional drugs cannot achieve targeted sustained release effects and have low utilization rates. Therefore, it is still a challenge to prepare a material that can scavenge reactive oxygen species, fight inflammation, promote neural repair, meet the mechanical strength of soft brain tissue, and whose degradation rate matches the brain tissue repair rate. Summary of the invention

[0008] In view of the above shortcomings of the current technology, the present invention provides a hydrogel for treating ischemic brain injury and a preparation method thereof. The specific scheme is as follows.

[0009] In a first aspect of the present invention, a hydrogel for treating ischemic brain injury is provided, wherein the hydrogel comprises the following raw materials: methacryloyl hyaluronic acid HAMA, methacryloyl gelatin GelMA, HA-PPS nanomicelles loaded with curcumin, and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0010] HAMA and GelMA can be cross-linked to form a hydrogel matrix. HA-PPS nano-micelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharides are loaded drugs, which can have antioxidant and anti-inflammatory effects. Among them, curcumin is encapsulated with HA-PPS nano-micelles, and HA can bind to cell surface receptors (such as CD44), which helps to accurately deliver the encapsulated curcumin to the site of brain injury, especially the inflammation and injury area; and PPS has excellent antioxidant properties, which can effectively protect curcumin from the damage of oxidative stress in the body, thereby maintaining its pharmacological activity. This is especially important for the high oxidative stress environment after brain injury. It overcomes the problem of low utilization of curcumin and can successfully cross the blood-brain barrier to achieve a sustained release of the drug. Encapsulating dendrobium polysaccharides with sodium alginate microspheres can protect dendrobium polysaccharides from degradation by digestive enzymes, improve their bioavailability in the body, and at the same time, achieve controlled release of drugs, making the release rate of dendrobium polysaccharides in the body more stable, which helps to prolong their efficacy.

[0011] In addition, HAMA also has certain anti-inflammatory properties, which can reduce local inflammatory reactions, provide a good microenvironment for repair after brain injury, and help reduce secondary injuries. The brain is an extremely soft organ, and the mechanical strength of the hydrogel needs to match the elastic modulus of the brain tissue. A hydrogel that is too hard may cause mechanical stimulation or damage to the surrounding tissue, while a hydrogel that is too soft may not provide sufficient support. The combination of GelMA and HAMA can adjust the ratio to obtain a hydrogel with the mechanical strength that meets the requirements of brain tissue. It can not only maintain its support and therapeutic functions in the body, but also will not damage brain tissue. At the same time, it helps promote the formation of new blood vessels, improves blood supply to the brain, and provides the necessary nutrients and oxygen for the regeneration of damaged tissue, further enhancing its potential for application in neural repair and regeneration.

[0012] Preferably, according to the mass ratio, the HAMA:GelMA=11.25-26 mg:45-60 mg, and more preferably, HAMA:GelMA=15-26 mg:45-60 mg, so that a hydrogel with better mechanical strength can be obtained.

[0013] Preferably, the method for preparing curcumin-loaded HA-PPS nanomicelles in the hydrogel of the present invention comprises the following steps:

[0014] 1) Synthetic polymer PPS;

[0015] Under ice bath conditions, anhydrous tetrahydrofuran, 3-mercaptopropionic acid, 1,8-diazabicyclo[5.4.0]undec-7-ene were reacted in N 2 Under atmosphere, the mixture was stirred, propylene sulfide was added dropwise, the reaction mixture was stirred at 60°C overnight, then the reaction was quenched, and the mixture was purified by precipitation in cold methanol, and the solvent was evaporated under reduced pressure to obtain yellow oily PPS;

[0016] 2) Synthesis of PPS-NH 2 ;

[0017] At room temperature, dry PPS, NHS and EDCI were mixed and dispersed in dichloromethane solvent, ethylenediamine was added dropwise to the mixture, and the mixture was stirred and reacted overnight at room temperature; then the reaction solution was diluted with dichloromethane and HO was added. 2 O and saturated NaCl solution; MgSO 4 Dry, filter, and concentrate under reduced pressure to obtain PPS-NH 2 ;

[0018] 3) Synthesis of HA-PPS conjugates.

[0019] At room temperature, NHS and EDCI were added to the acidic hyaluronic acid aqueous solution and mixed and dispersed, and then PPS-NH 2 / tetrahydrofuran solution, at room temperature, N2 The reaction was continued under protection with stirring for 24 h, dialyzed with water / methanol at a ratio of 1:1, and then the solvent was removed by lyophilization to obtain a HA-PPS conjugate;

[0020] 4) Preparation of HA-PPS nanomicelles loaded with curcumin

[0021] The HA-PPS conjugate and curcumin were dissolved in distilled water, and the curcumin-loaded HA-PPS nanomicelles were prepared by ultrasonic treatment.

[0022] In a preferred embodiment, in the step 1), anhydrous tetrahydrofuran: 3-mercaptopropionic acid: 1,8-diazabicyclo[5.4.0]undec-7-ene: propylene sulfide = 30 mL: 100 μL: 524 μL: 1.9 mL.

[0023] In a preferred embodiment, in the step 2), dichloromethane: PPS: NHS: EDCI: ethylenediamine = 20 mL: 158.6 mg: 23 mg: 48 mg: 133 μL.

[0024] In a preferred embodiment, in step 3), acidic HA:NHS:EDCI:PPS-NH 2 =100mg:7mg:14.5mg:40mg, the concentration of the acidic hyaluronic acid aqueous solution is 10mg / mL.

[0025] In a preferred embodiment, in step 4), HA-PPS conjugate: curcumin: water = 40-45 mg: 0.9 mg: 1 mL.

[0026] Preferably, the sodium alginate microspheres loaded with dendrobium polysaccharide in the hydrogel of the present invention are prepared by a microfluidic method with a mixture of dendrobium polysaccharide solution, sodium alginate solution, and Ca-EDTA solution as the aqueous phase and droplet-generated oil as the oil phase.

[0027] In a preferred embodiment, the concentration of dendrobium polysaccharide in the aqueous phase mixture is 0.5 mg / mL, and the concentration of sodium alginate is 1-2%; the Ca-EDTA solution is 1.2 parts by volume of 2M CaCl 2 The solution was mixed evenly with 4.8 parts by volume of 0.5 M EDTA solution, and then a 2 M NaOH solution was added to adjust the pH to 7.2, and then the volume was fixed to 10 parts by volume to obtain.

[0028] The present invention also provides a method for preparing the hydrogel, comprising the steps of:

[0029] Methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA are dissolved in deionized water to prepare a solution, and then a photoinitiator, HA-PPS nanomicelles loaded with curcumin, and sodium alginate microspheres loaded with dendrobium polysaccharide are added and mixed evenly to obtain a mixed solution, which is irradiated with ultraviolet light to obtain a hydrogel.

[0030] In one preferred embodiment, in the mixed solution, HAMA: GelMA: deionized water: HA-PPS nanomicelles loaded with curcumin: sodium alginate microspheres loaded with dendrobium polysaccharide = 11.25-26 mg: 45-60 mg: 0.6 mL: 200 μL: 200 μL.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention combines nanotechnology and intelligent response materials, uses HA-PPS loaded with curcumin and sodium alginate microspheres to encapsulate dendrobium polysaccharides, which can more effectively protect the activity of drugs, protect drugs from passing through the blood-brain barrier, accurately deliver them to the damaged area of ​​the brain, and achieve targeted release of drugs under specific stimulation conditions (such as inflammation or oxidative stress environment), which can effectively overcome the shortcomings of existing treatment methods.

[0033] 2. Curcumin and dendrobium polysaccharide in the hydrogel of the present invention can effectively work together to better enhance the anti-oxidation and anti-inflammatory effects of the gel, which is better than the effects of a single component.

[0034] 3. The hydrogel of the present invention has a sustained release function, which can achieve continuous drug release in the brain injury area, prolong the effective action time of the drug, reduce the frequency of administration, reduce the risk of side effects, and improve treatment efficiency.

[0035] 4. The present invention optimizes the HAMA / GelMA ratio so that the mechanical properties of the mixed material hydrogel can match those of brain tissue. It will not be too hard to further damage the soft brain tissue, but it also has a certain supporting ability, promotes the repair of brain tissue, and has a certain anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the SEM image of the hydrogel;

[0037] Figure 2 is the drug release diagram of the hydrogel;

[0038] Figure 3 For antioxidant capacity analysis;

[0039] Figure 4 Evaluation of anti-inflammatory effects. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0041] The methacryloyl hyaluronic acid HAMA, methacryloyl gelatin GelMA, HA-PPS nanomicelles doped with loaded curcumin, and sodium alginate microspheres loaded with dendrobium polysaccharide used in the hydrogels of the following examples are prepared by the following method.

[0042] (1) Preparation of methacryloyl hyaluronic acid (HAMA)

[0043] 5.0 g of hyaluronic acid was weighed and dissolved in 400 mL of deionized water, and mechanically stirred until completely dissolved. 12 mL of methacrylic anhydride was added, and the pH of the reaction solution was adjusted to 8.5 with 5 M sodium hydroxide, and stirred at room temperature for 24 hours. The reaction was then dialyzed using a cellulose dialysis bag with a molecular weight cutoff of about 1.2 kDa, and lyophilized to obtain HAMA.

[0044] (2) Preparation of methacryloyl gelatin (GelMA)

[0045] First, weigh 5.0g gelatin, add it to 50mL PBS buffer, and dissolve it in a 50℃ water bath under magnetic stirring until it is completely transparent. Then slowly add 3mL methacrylic anhydride to the gelatin solution, stir it magnetically at 700rpm for 1h, maintain the reaction temperature at 50℃, and the reaction solution contains a large number of oily droplets. After the reaction is completed, the reaction solution is transferred to a cellulose dialysis bag with a molecular weight cutoff of 3500Da and dialyzed in constant temperature deionized water at 40℃ for 3 days to remove by-products. Collect the dialyzed reaction solution, centrifuge at 5000rpm for 10 minutes to remove the precipitate, collect the supernatant, and freeze-dry at -80℃ to obtain the final product GelMA.

[0046] (3) Preparation of HA-PPS nanomicelles loaded with curcumin

[0047] 3.1 Synthetic polymer PPS.

[0048] Under ice bath conditions, first add 30 mL of anhydrous tetrahydrofuran (THF), then add 100 μL (1.15 mmol) of 3-mercaptopropionic acid (3-MPA), and stir magnetically to mix. Then add 524 μL (3.45 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene, and 2Stir for 30 min under atmosphere. Add 1.9 mL (21.15 mmol) of propylene sulfide dropwise, and stir the reaction mixture at 60°C overnight. Add 5 mL of water to quench the reaction, precipitate and purify in cold methanol, evaporate the solvent under reduced pressure to obtain yellow oily PPS, and dry it for later use.

[0049] 3.2 Synthesis of PPS-NH 2 .

[0050] First add 20 mL of dichloromethane, then add 158.6 mg (100 μmol) of dried PPS, and stir magnetically to dissolve. Continue to add 23 mg (200 μmol) of N-hydroxysuccinimide (NHS) and 48 mg (250 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), and stir magnetically at room temperature for 30 min until dissolved. Drop 133 μL (2 mmol) of ethylenediamine into the mixture, and stir and react overnight at room temperature. Add 20 mL of dichloromethane to dilute the reaction solution, and use H 2 O and saturated NaCl solution. 4 Dry and filter. The solvent is concentrated under reduced pressure to obtain PPS-NH 2 , dry and set aside.

[0051] 3.3 Synthesis of HA-PPS conjugate.

[0052] The hyaluronic acid sodium salt was dialyzed against 0.01 M HCl solution overnight and then lyophilized to obtain the acid form of hyaluronic acid. 10 mL of HCl was added to a 50 mL beaker. 2 O and 100 mg of acidic HA were dissolved by magnetic stirring. Then 7 mg (60 μmol) of N-hydroxysuccinimide and 14.5 mg (75 μmol) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide were added, and the mixture was stirred at room temperature for 30 min to completely dissolve. 40 mg of PPS-NH 2 Dissolve in 1 mL of tetrahydrofuran and add dropwise to the reaction system. 2 The reaction was continued under stirring for 24 hours. The mixture was dialyzed 3 times with water / methanol at a ratio of 1:1 for 1 day, and then dialyzed 3 times with distilled water for 1 day. The solvent was removed by freeze drying to obtain a HA-PPS conjugate.

[0053] 3.4 Preparation of curcumin-loaded HA-PPS nanomicelles

[0054] The HA-PPS conjugate and curcumin were dissolved in distilled water at a certain weight ratio and ultrasonically treated for 10 minutes to prepare HA-PPS nanogel loaded with curcumin, which was recorded as curcumin@HA-PPS.

[0055] (4) Preparation of sodium alginate microspheres loaded with Dendrobium polysaccharide

[0056] 4.1 Dissolve the dendrobium polysaccharide in deionized water and mix well to obtain a dendrobium polysaccharide solution of a certain concentration.

[0057] 4.2 Take 1.2 mL of 2M calcium chloride (CaCl 2 ) was mixed evenly with 4.8 mL of 0.5 M ethylenediaminetetraacetic acid solution (EDTA), followed by adding 2 M sodium hydroxide solution to adjust the pH to about 7.2, and finally adding ultrapure water to make the volume to 10 mL to obtain a Ca-EDTA solution.

[0058] 4.3 Dissolve sodium alginate in deionized water and mix well. Place at 60°C until completely dissolved to obtain a sodium alginate solution of a certain proportion.

[0059] 4.4 Mix the above-mentioned equal volumes of Dendrobium polysaccharide solution into the sodium alginate solution, mix well, then add the Ca-EDTA solution of equal volume to the mixed solution, mix well to obtain sodium alginate-Dendrobium polysaccharide, Ca-EDTA solution.

[0060] 4.5 Sodium alginate-Dendrobium polysaccharide, Ca-EDTA solution and 2% microdroplet generation oil were respectively loaded into a 2.5mL syringe and loaded into a microinjection pump. The flow rate of the aqueous phase (sodium alginate-Dendrobium polysaccharide, Ca-EDTA solution) was set to 0.1mL / h, and the flow rate of the oil phase (2% microdroplet generation oil) was set to 1mL / h, and microspheres were extruded through a microfluidic chip to form microspheres. The generated microdroplets were collected in an oil phase centrifuge tube with 1% acetic acid, and then allowed to stand and solidify for 30-60min to form hydrogel microspheres. Subsequently, the bottom microdroplet generation oil was taken out, a demulsifier twice the volume of the microspheres was added, and the bottom demulsifier was taken out by centrifugation at 1000rpm for 30s, and the microspheres were finally washed 2-3 times with PBS, and the hydrogel microspheres were collected and recorded as Dendrobium polysaccharide @ sodium alginate microspheres.

[0061] Example 1

[0062] A hydrogel for treating ischemic brain injury is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0063] The preparation method of the hydrogel is as follows:

[0064] 45 mg of GelMA and 20 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0065] Among them, when preparing curcumin@HA-PPS, 9 mg of HA-PPS conjugate prepared in the above step 3.3 and 0.18 mg of curcumin were dissolved in 0.2 mL of distilled water, and ultrasonic treatment was performed for 10 minutes to prepare curcumin@HA-PPS nanomicelles.

[0066] Preparation of sodium alginate microspheres loaded with dendrobium polysaccharide:

[0067] In step 4.1, 0.1 mg of Dendrobium polysaccharide was dissolved in 0.05 mL of deionized water and mixed evenly.

[0068] In step 4.3, weigh 4 mg of sodium alginate and dissolve it in 0.05 mL of deionized water and mix well.

[0069] Example 2

[0070] A hydrogel for treating ischemic brain injury is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0071] The preparation method of the hydrogel is as follows:

[0072] 45 mg of GelMA and 11.25 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0073] The preparation methods of curcumin@HA-PPS and sodium alginate microspheres loaded with dendrobium polysaccharide are the same as those in Example 1.

[0074] Example 3

[0075] A hydrogel for treating ischemic brain injury is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0076] The preparation method of the hydrogel is as follows:

[0077] 60 mg of GelMA and 26 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0078] The preparation methods of curcumin@HA-PPS and sodium alginate microspheres loaded with dendrobium polysaccharide are the same as those in Example 1.

[0079] Example 4

[0080] A hydrogel for treating ischemic brain injury is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0081] The preparation method of the hydrogel is as follows:

[0082] 60 mg of GelMA and 15 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0083] The preparation methods of curcumin@HA-PPS and sodium alginate microspheres loaded with dendrobium polysaccharide are the same as those in Example 1.

[0084] Example 5

[0085] A hydrogel for treating ischemic brain injury is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0086] The preparation method of the hydrogel is as follows:

[0087] 45 mg of GelMA and 20 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0088] The preparation method of HA-PPS nano-micelles loaded with curcumin is the same as that in Example 1. When preparing sodium alginate microspheres of dendrobium polysaccharide curcumin@HA-PPS:

[0089] In step 4.3, 2 mg of sodium alginate was weighed and dissolved in 0.05 mL of deionized water, mixed evenly, and placed at 60° C. until completely dissolved.

[0090] Example 6

[0091] A hydrogel for treating ischemic brain injury is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0092] The preparation method of the hydrogel is as follows:

[0093] 45 mg of GelMA and 20 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0094] The preparation method and formula of sodium alginate microspheres of dendrobium polysaccharide are the same as those in Example 1. When preparing curcumin@HA-PPS:

[0095] In step 3.4, 8 mg of HA-PPS conjugate prepared in step 3.3 and 0.18 mg of curcumin were dissolved in 0.2 mL of distilled water and ultrasonically treated for 10 minutes to prepare curcumin@HA-PPS nanomicelles.

[0096] Comparative Example 1

[0097] A hydrogel is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nano-micelles loaded with curcumin and sodium alginate microspheres.

[0098] The preparation method of the hydrogel is as follows:

[0099] 45 mg of GelMA and 20 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of curcumin@HA-PPS and 200 μL of sodium alginate microspheres, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0100] The preparation method and proportion of curcumin@HA-PPS are the same as those in Example 1.

[0101] The preparation method of sodium alginate microspheres is as follows:

[0102] 4.1 Take 1.2 mL of 2M calcium chloride (CaCl 2 ) was mixed evenly with 4.8 mL of 0.5 M ethylenediaminetetraacetic acid solution (EDTA), followed by adding 2 M sodium hydroxide solution to adjust the pH to about 7.2, and finally adding ultrapure water to make the volume to 10 mL to obtain a Ca-EDTA solution.

[0103] 4.2 Dissolve 2 mg of sodium alginate in 0.05 mL of deionized water and mix well. Place at 60 °C

[0104] The sodium alginate solution was obtained by adding 1% sodium alginate and stirring until the alginate was completely dissolved.

[0105] 4.3 Add an equal volume of Ca-EDTA solution to the above sodium alginate solution and mix well to obtain a sodium alginate and Ca-EDTA solution.

[0106] 4.4 Sodium alginate, Ca-EDTA solution and 2% droplet generation oil were respectively loaded into 2.5mL syringes and loaded into microinjection pumps. The flow rate of the aqueous phase (sodium alginate, Ca-EDTA solution) was set to 0.1mL / h, and the flow rate of the oil phase (2% droplet generation oil) was set to 1mL / h, and microspheres were extruded through a microfluidic chip. The generated microdroplets were collected in an oil phase centrifuge tube with 1% acetic acid, and then allowed to stand and solidify for 30-60min to form hydrogel microspheres. Subsequently, the bottom droplet generation oil was removed, and a demulsifier twice the volume of the microspheres was added, and the bottom demulsifier was removed by centrifugation at 1000rpm for 30s. Finally, the microspheres were washed 2-3 times with PBS to collect the sodium alginate microspheres.

[0107] Comparative Example 2

[0108] A hydrogel is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nano-micelles and sodium alginate microspheres carrying dendrobium polysaccharide.

[0109] The preparation method of the hydrogel is as follows:

[0110] 45 mg of GelMA and 20 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of HA-PPS and 200 μL of sodium alginate microspheres loaded with dendrobium polysaccharide, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0111] The preparation method of sodium alginate microspheres loaded with Dendrobium polysaccharide is the same as that in Example 1.

[0112] The preparation method of HA-PPS nanomicelles is as follows:

[0113] Referring to Example 1, in step 3.4, 9 mg of the HA-PPS conjugate prepared in step 3.3 was dissolved in 0.2 mL of distilled water and ultrasonically treated for 10 minutes to prepare HA-PPS nanomicelles.

[0114] Comparative Example 3

[0115] A hydrogel is cross-linked by methacryloyl hyaluronic acid HAMA and methacryloyl gelatin GelMA, and is doped with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0116] The preparation method of the hydrogel is as follows:

[0117] (1) 45 mg of GelMA and 20 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt % of LAP photoinitiator was added.

[0118] (2) Dissolve 0.18 mg of curcumin in 0.2 mL of distilled water and mix well to obtain a curcumin solution.

[0119] (3) adding the obtained curcumin solution and 200 μL of sodium alginate microspheres loaded with dendrobium polysaccharide to the mixed solution obtained in step (1), mixing them evenly, and irradiating them under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0120] The preparation method of sodium alginate microspheres loaded with Dendrobium polysaccharide is the same as that in Example 1.

[0121] Comparative Example 4

[0122] A hydrogel is formed by cross-linking methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nano-micelles loaded with curcumin and dendrobium polysaccharide.

[0123] The preparation method of the hydrogel is as follows:

[0124] (1) 45 mg of GelMA and 20 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt % of LAP photoinitiator was added.

[0125] (2) Dissolve 0.1 mg of dendrobium polysaccharide in 0.2 mL of deionized water and mix well to obtain a dendrobium polysaccharide solution.

[0126] (3) Add the obtained Dendrobium polysaccharide solution and 200 μL of curcumin@HA-PPS to the mixed solution obtained in step (1), mix them evenly, and irradiate them under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0127] The preparation method and proportion of curcumin@HA-PPS are the same as those in Example 1.

[0128] Comparative Example 5

[0129] A hydrogel is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nano-micelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0130] The preparation method of the hydrogel is as follows:

[0131] 45 mg of GelMA and 20 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of curcumin@HA-PPS and 200 μL of sodium alginate microspheres loaded with dendrobium polysaccharide, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0132] Wherein, the preparation method of curcumin@HA-PPS is the same as that in Example 1.

[0133] Preparation of sodium alginate microspheres loaded with dendrobium polysaccharide:

[0134] In step 4.1, 0.4 mg of Dendrobium polysaccharide was dissolved in 0.05 mL of deionized water and mixed evenly.

[0135] Comparative Example 6

[0136] A hydrogel is cross-linked by methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA, and is doped with HA-PPS nano-micelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

[0137] The preparation method of the hydrogel is as follows:

[0138] 45 mg of GelMA and 20 mg of HAMA were added to 0.6 mL of deionized water to prepare a solution, and then 0.2 wt% of LAP photoinitiator was added. The obtained solution was evenly mixed with 200 μL of curcumin@HA-PPS and 200 μL of sodium alginate microspheres loaded with dendrobium polysaccharide, and irradiated under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0139] Among them, when preparing curcumin@HA-PPS, 9 mg HA-PPS conjugate prepared in step 3.3 and 0.1 mg curcumin were dissolved in 0.2 mL distilled water and ultrasonically treated for 10 minutes to prepare curcumin@HA-PPS nanomicelles.

[0140] The preparation method of sodium alginate microspheres loaded with Dendrobium polysaccharide is the same as that in Example 1.

[0141] The material compositions in each embodiment and comparative example are shown in Table 1.

[0142] Table 1 Material composition of different embodiments and comparative examples

[0143]

[0144]

[0145] Performance Testing

[0146] 1. SEM image of hydrogel

[0147] Figure 1 This is a SEM cross-sectional view of the hydrogel obtained in Example 1 of the present invention.

[0148] 2. Mechanical properties of hydrogels

[0149] Test method: Place the hydrogel on the sample stage and adjust the height of the sample stage so that both the upper and lower surfaces are in contact with the fixture. Compress at a constant rate of 0.05 mm / s, and record the compression displacement (L) and load (P) until the set displacement endpoint. The mechanical strength of the hydrogel is the ratio of the load to the bottom area of ​​the hydrogel.

[0150] The test results are shown in Table 2. It can be seen that the hydrogel of the embodiment of the present invention has good strength, especially when HAMA:GelMA=15-26mg:45-60mg. In addition, it can be seen from Example 5 that sodium alginate also has a certain effect on the strength of the gel.

[0151] Table 2 Mechanical strength test results of various embodiments and comparative examples

[0152]

[0153]

[0154] 3. Drug release test of hydrogel

[0155] Test method: Curcumin solution and dendrobium polysaccharide solution were prepared according to a certain concentration gradient, and the absorbance was measured at 420nm and 490nm using a UV spectrophotometer to prepare a standard curve. Then the corresponding example and comparative example samples were placed in PBS and incubated at 37°C. The supernatant was taken on the 1st, 3rd, 5th, 7th, 14th, 21st and 28th days, and the absorbance was measured using a UV spectrophotometer. The drug release was calculated according to the standard curve.

[0156] Test results such as Figure 2 As shown, Figure 2 The upper middle figure shows the sustained release curve of curcumin through Example 1 and Comparative Example 3. After curcumin is loaded with HA-PPS, the release of curcumin can be sustained; Figure 2The lower middle figure shows, through the comparison between Example 1 and Comparative Example 4, that after wrapping Dendrobium polysaccharide with sodium alginate microspheres, the release of Dendrobium polysaccharide can be sustained-released; the drug release rate of the gel in the embodiment of the present invention can better match the rate of brain tissue repair, which is beneficial to the treatment of brain damage.

[0157] 4. Antioxidant capacity test of hydrogel

[0158] Test method: Test method: The antioxidant performance of different embodiments and comparative examples was evaluated by scavenging 1,1-diphenyl-2-pyridylhydrazide (DPPH) free radicals. The mixture of different embodiments and comparative examples and DPPH reagent was stirred and incubated in the dark for 30 minutes, and the remaining DPPH was analyzed by UV-visible spectroscopy. The scavenging rate of DPPH was determined as follows:

[0159] DVC (%) = [(A blank - A positive control) / A blank] × 100%

[0160] D (%) = [[A blank - (A measurement - A control)] / A blank] × 100%

[0161] Test results such as Figure 3 As shown, curcumin and dendrobium polysaccharide alone have the ability to scavenge free radicals and have good antioxidant effects. The hydrogel to which appropriate amounts of curcumin and dendrobium polysaccharide were added at the same time has a stronger antioxidant effect, proving that at a certain concentration, curcumin and dendrobium polysaccharide have a certain synergistic effect in antioxidant effects. However, when curcumin exceeds a certain concentration or dendrobium polysaccharide exceeds a certain concentration, its antioxidant effect will be inhibited. The two groups that did not use HA-PPS to load curcumin and did not use sodium alginate microspheres to wrap dendrobium polysaccharide had poor antioxidant effects in the later stage due to the degradation of the drug.

[0162] 5. Anti-inflammatory ability test of hydrogel

[0163] Test method: Mice were anesthetized with 0.3% sodium pentobarbital (22 mL / kg) by intraperitoneal injection and divided into sham operation group, IS (ischemic brain injury) group, Example 1 group, Comparative Example 1 group and Comparative Example 2 group. The model was generated by thread embolism middle carotid artery occlusion. An incision was made at the midline of the mouse neck to separate the common carotid artery, internal carotid artery and external carotid artery. A polyethylene-coated nylon thread (0.2-0.3 mm diameter) was inserted into the internal carotid artery and slowly advanced to the middle cerebral artery to block it. After maintaining the ischemic time for one hour, the thread embolism was removed to restore cerebral blood flow and simulate reperfusion (I / R) injury. The hydrogel was then implanted into the ischemic lesion area, and the neck opening was completely sutured with 5-0 sutures, and each layer of muscle and skin was sutured layer by layer from the inside to the outside. The neck of the sham operation group was opened, but no ischemic treatment was performed. The neck of the IS group was opened and ischemic treatment was performed, but no hydrogel was implanted. The neck of Example 1, Comparative Example 1 and Comparative Example 2 was opened, treated with ischemia, and implanted with hydrogel. Mice were killed at 48 hours, brain tissue was taken from the cerebral ischemic area, placed in an appropriate amount of PBS, and protease inhibitors were added. The tissue was thoroughly homogenized using a homogenizer, then centrifuged at 12,000 rpm for 10-15 minutes at 4°C, and the supernatant was taken for detection. Inflammatory cytokines in the supernatant were analyzed by ELISA.

[0164] from Figure 4 It can be seen that compared with the IS group, the hydrogel loaded with curcumin and dendrobium polysaccharide can effectively reduce the expression levels of inflammatory factors TNF-α, IL-6, and IL-β. Moreover, the hydrogel loaded with curcumin and dendrobium polysaccharide at the same time has a better effect on reducing the expression level of inflammatory factors than the hydrogel loaded with one of the components alone, and more effectively plays an anti-inflammatory effect, providing a stable microenvironment for brain tissue repair. The two groups of curcumin without HA-PPS loading and dendrobium polysaccharide without microspheres have a certain anti-inflammatory effect, but because the drug is easily degraded, the later treatment effect is not as good as that of Example 1.

[0165] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0166] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A hydrogel for treating ischemic brain injury, characterized in that: The hydrogel comprises the following raw materials: methacrylylated hyaluronic acid HAMA, methacrylylated gelatin GelMA, HA-PPS nano micelles loaded with curcumin, and sodium alginate microspheres loaded with dendrobium polysaccharide.

2. The hydrogel according to claim 1, characterized in that According to the mass ratio, the HAMA:GelMA=11.25-26 mg:45-60 mg, preferably, HAMA:GelMA=15-26 mg:45-60 mg.

3. The hydrogel according to claim 1, characterized in that The preparation method of the curcumin-loaded HA-PPS nanomicelles comprises the following steps: 1) Synthetic polymer PPS; Under ice bath conditions, anhydrous tetrahydrofuran, 3-mercaptopropionic acid, and 1,8-diazabicyclo[5.4.0]undec-7-ene were mixed and stirred under N2 atmosphere, and propylene sulfide was added dropwise. The reaction mixture was stirred at 60°C overnight, and then the reaction was quenched, and the mixture was purified by precipitation in cold methanol. The solvent was evaporated under reduced pressure to obtain yellow oily PPS. 2) Synthesis of PPS-NH2; At room temperature, in dichloromethane solvent, dry PPS, NHS and EDCI are mixed and dispersed, ethylenediamine is added dropwise to the mixture, and the mixture is stirred and reacted overnight at room temperature; then the reaction solution is diluted with dichloromethane, washed with H2O and saturated NaCl solution in sequence; dried with MgSO4, filtered, and concentrated under reduced pressure to obtain PPS-NH2; 3) Synthesis of HA-PPS conjugate. At room temperature, NHS and EDCI were added to the acidic hyaluronic acid aqueous solution for mixing and dispersion, and then PPS-NH2 / tetrahydrofuran solution was added dropwise, and the reaction was continued under stirring for 24 hours at room temperature and N2 protection, and dialyzed with water / methanol at a ratio of 1:1, and then the solvent was removed by freeze-drying to obtain a HA-PPS conjugate; 4) Preparation of HA-PPS nanomicelles loaded with curcumin The HA-PPS conjugate and curcumin were dissolved in distilled water, and the curcumin-loaded HA-PPS nanomicelles were prepared by ultrasonic treatment.

4. The hydrogel according to claim 3, characterized in that In the step 1), anhydrous tetrahydrofuran: 3-mercaptopropionic acid: 1,8-diazabicyclo[5.4.0]undec-7-ene: propylene sulfide = 30 mL: 100 μL: 524 μL: 1.9 mL.

5. The hydrogel according to claim 3, characterized in that In the step 2), dichloromethane: PPS: NHS: EDCI: ethylenediamine = 20 mL: 158.6 mg: 23 mg: 48 mg: 133 μL.

6. The hydrogel according to claim 3, characterized in that In the step 3), acidic HA:NHS:EDCI:PPS-NH2=100mg:7mg:14.5mg:40mg, and the concentration of the acidic hyaluronic acid aqueous solution is 10mg / mL.

7. The hydrogel according to claim 3, characterized in that In the step 4), HA-PPS conjugate: curcumin: water = 40-45 mg: 0.9 mg: 1 mL.

8. The hydrogel according to claim 1, characterized in that The sodium alginate microspheres carrying dendrobium polysaccharide are prepared by a microfluidic method with a mixture of dendrobium polysaccharide solution, sodium alginate solution and Ca-EDTA solution as the water phase and microdroplet generated oil as the oil phase.

9. The hydrogel according to claim 1, characterized in that In the aqueous phase mixture, the concentration of dendrobium polysaccharide is 0.5 mg / mL, and the concentration of sodium alginate is 1-2%. The Ca-EDTA solution is prepared by uniformly mixing 1.2 parts by volume of a 2M CaCl2 solution and 4.8 parts by volume of a 0.5M EDTA solution, then adding a 2M NaOH solution to adjust the pH to 7.2, and then fixing the volume to 10 parts by volume.

10. The method for preparing the hydrogel according to any one of claims 1 to 9, characterized in that: Includes steps: Methacrylylated hyaluronic acid HAMA and methacrylylated gelatin GelMA are dissolved in deionized water to prepare a solution, and then a photoinitiator, HA-PPS nanomicelles loaded with curcumin, and sodium alginate microspheres loaded with dendrobium polysaccharide are added and mixed evenly to obtain a mixed solution, which is irradiated with ultraviolet light to obtain a hydrogel.

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