A hydrogel for the treatment of ischemic brain injury and a preparation method thereof

Abstract

The present invention relates to a hydrogel for the treatment of ischemic brain injury and a preparation method thereof. The hydrogel comprises the following raw materials: methacrylated hyaluronic acid, methacrylated gelatin, HA-PPS nanomicelles loaded with curcumin, and sodium alginate microspheres loaded with dendrobium officinale polysaccharide. By combining nanotechnology and intelligent response materials, the present invention uses HA-PPS to load curcumin and sodium alginate microspheres to encapsulate dendrobium officinale polysaccharide, which can more effectively protect the drug activity, protect the drug from crossing the blood-brain barrier, accurately deliver it to the brain injury area, and achieve targeted release of the drug; moreover, the antioxidant and anti-inflammatory effects of curcumin and dendrobium officinale polysaccharide acting together are better, and at the same time, it also has a sustained release function.

Description

Technical Field

[0001] The present 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, resulting in the death of brain cells due to hypoxia. The most common cause of this disease is stroke, which has a very high incidence and disability rate, and there is currently no completely effective treatment method. 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 the destruction of the blood-brain barrier (BBB) function, damaging the 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 exacerbating brain injury. Existing clinical treatment methods include thrombolytic therapy, mechanical thrombectomy, the application of antiplatelet and anticoagulant drugs, etc., aiming to restore cerebral blood flow as soon as possible. However, these treatment methods have certain limitations and risks.

[0003] Currently, drugs used to treat ischemic brain injury, such as recombinant tissue plasminogen activator (rtPA), although can effectively dissolve thrombus, due to its poor targeting, it cannot specifically act on the damaged area of the brain, often resulting in systemic side effects, especially the risk of intracerebral hemorrhage. The control of drug release is difficult. Some thrombolytic drugs and neuroprotective drugs have a short half-life in the body and are difficult to continuously play a role. Therefore, patients often need to take drugs frequently, which affects the treatment compliance and increases the occurrence probability of drug toxicity and side effects.

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

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

[0006] Dendrobium polysaccharide is a natural bioactive ingredient extracted from Dendrobium. Through multiple mechanisms such as antioxidation, anti-inflammation, promoting nerve regeneration, enhancing immune regulation, and improving blood circulation, it has shown important therapeutic potential in the treatment of ischemic brain injury. However, due to its poor water solubility and stability, its pharmacodynamic duration in vivo is relatively short, and its bioavailability is low, which limits the actual therapeutic effect.

[0007] In recent years, the prognosis treatment of brain injury has received extensive 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 scavenges reactive oxygen species, has anti-inflammatory effects, promotes nerve repair, conforms to the mechanical strength of soft brain tissue, and has a degradation rate matching the brain tissue repair rate. Summary of the Invention

[0008] Aiming at the above current technical deficiencies, the present invention provides a hydrogel for the treatment of ischemic brain injury and its preparation method. The specific scheme is as follows.

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

[0010] HAMA and GelMA can crosslink to form a hydrogel matrix. The HA-PPS nanomicelles loaded with curcumin and the sodium alginate microspheres loaded with dendrobium polysaccharide are the loaded drugs, which can play antioxidation and anti-inflammatory effects. Among them, curcumin is encapsulated by HA-PPS nanomicelles. HA can bind to cell surface receptors (such as CD44), which helps to accurately deliver the encapsulated curcumin to the brain injury site, especially the inflammatory and damaged areas. And PPS has excellent antioxidant properties, which can effectively protect curcumin from being damaged by in vivo oxidative stress, thus maintaining its pharmacological activity. This is particularly important for the high oxidative stress environment after brain injury. It overcomes the problem of low utilization rate of curcumin and can successfully cross the blood-brain barrier to achieve drug sustained-release effect. Encapsulating dendrobium polysaccharide with sodium alginate microspheres can protect dendrobium polysaccharide from being degraded by digestive enzymes, improve its bioavailability in vivo. At the same time, it realizes the controlled release of the drug, makes the release rate of dendrobium polysaccharide in vivo more stable, and helps to extend its therapeutic effect.

[0011] In addition, HAMA also has certain anti-inflammatory properties, which can reduce local inflammatory responses, provide a good microenvironment for the 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 brain tissue. A hydrogel that is too hard may cause mechanical irritation or injury to the surrounding tissues, while a hydrogel that is too soft may not provide sufficient support. The combination of GelMA and HAMA can obtain a hydrogel with a mechanical strength that conforms to that of brain tissue by adjusting the ratio, which can not only maintain the support and treatment functions in vivo without damaging brain tissue, but also help promote the formation of new blood vessels, improve blood supply to the brain, provide the necessary nutrients and oxygen for the regeneration of damaged tissues, and further enhance its application potential in nerve repair and regeneration.

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

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

[0014] 1) Synthesize polymer PPS;

[0015] Under ice bath conditions, anhydrous tetrahydrofuran, 3-mercaptopropionic acid, and 1,8-diazabicyclo[5.4.0]undec-7-ene are mixed and stirred under a nitrogen atmosphere, and propylene sulfide is added dropwise. The reaction mixture is stirred overnight at 60 °C, then the reaction is quenched, precipitated and purified in cold methanol, and the solvent is evaporated under reduced pressure to obtain yellow oily PPS;

[0016] 2) Synthesize PPS-NH2;

[0017] At room temperature, in dichloromethane solvent, dry PPS, NHS, and EDCI are mixed and dispersed, and ethylenediamine is added dropwise to the mixture, and the reaction is stirred overnight at room temperature; then the reaction solution is diluted with dichloromethane and washed successively with H2O and saturated NaCl solution; dried over MgSO4, filtered, and concentrated under reduced pressure to obtain PPS-NH2;

[0018] 3) Synthesize the HA-PPS conjugate.

[0019] At room temperature, NHS and EDCI are added to an aqueous solution of acidic hyaluronic acid and mixed and dispersed, and then a PPS-NH2 / tetrahydrofuran solution is added dropwise. The reaction is continued to stir for 24 h at room temperature under nitrogen protection, dialyzed with water / methanol in a ratio of 1:1, and then the solvent is removed by lyophilization to obtain the HA-PPS conjugate;

[0020] 4) Preparation of Curcumin-Loaded HA-PPS Nanomicelles

[0021] Dissolve the HA-PPS conjugate and curcumin in distilled water, and prepare curcumin-loaded HA-PPS nanomicelles by ultrasonic treatment.

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

[0023] In a preferred embodiment, in 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-NH2 = 100 mg: 7 mg: 14.5 mg: 40 mg, and the concentration of the acidic hyaluronic acid aqueous solution is 10 mg / 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 alginate microspheres loaded with dendrobium polysaccharide in the hydrogel of the present invention are prepared by a microfluidic method using a mixed solution of a dendrobium polysaccharide solution, an alginate solution, and a Ca-EDTA solution as the aqueous phase and a microdroplet-forming oil as the oil phase.

[0027] In a preferred embodiment, in the aqueous phase mixture, the concentration of dendrobium polysaccharide is 0.5 mg / mL, and the concentration of alginate is 1 - 2%; the Ca-EDTA solution is prepared by mixing 1.2 parts by volume of 2 M CaCl2 solution and 4.8 parts by volume of 0.5 M EDTA solution evenly, then adding 2 M NaOH solution to adjust the pH to 7.2, and then making up the volume to 10 parts by volume.

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

[0029] Dissolve methacrylated hyaluronic acid HAMA and methacrylated gelatin GelMA in deionized water to form a solution, then add a photoinitiator, curcumin-loaded HA-PPS nanomicelles, and alginate microspheres loaded with dendrobium polysaccharide and mix evenly to obtain a mixed solution, and irradiate with ultraviolet light to form a hydrogel.

[0030] In one of the preferred embodiments, in the mixed solution, HAMA: GelMA: deionized water: HA-PPS nanomicelles loaded with curcumin: sodium alginate microspheres loaded with dendrobium officinale 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. By combining nanotechnology and intelligent responsive materials, the present invention uses HA-PPS to load curcumin and sodium alginate microspheres to encapsulate dendrobium officinale polysaccharide, which can more effectively protect the drug activity, enable the drug to cross the blood-brain barrier, be precisely delivered to the brain injury area, and achieve targeted drug release under specific stimulation conditions (such as inflammatory or oxidative stress environment), effectively overcoming the disadvantages of existing treatment methods.

[0033] 2. Curcumin and dendrobium officinale polysaccharide in the hydrogel of the present invention can effectively act together to better enhance the antioxidant and anti-inflammatory effects of the gel, which is better than the effects of individual components.

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

[0035] 4. By optimizing the HAMA / GelMA ratio, the mechanical properties of the mixed material hydrogel of the present invention can match those of brain tissue, will not be too hard to further damage the soft brain tissue, but also have a certain supporting ability to promote the repair of brain tissue and have a certain anti-inflammatory effect. Description of the Drawings

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

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

[0038] Figure 3 It is the antioxidant capacity analysis;

[0039] Figure 4 It is the anti-inflammatory effect evaluation. Detailed Embodiments

[0040] The following specific embodiments are used to further illustrate the present invention, but the embodiments 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 technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

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

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

[0043] Weigh 5.0 g of hyaluronic acid and dissolve it in 400 mL of deionized water, and mechanically stir until completely dissolved. Add 12 mL of methacrylic anhydride, adjust the pH of the reaction solution to 8.5 with 5 M sodium hydroxide, stir at room temperature for 24 hours, and then dialyze the reaction with a cellulose dialysis bag with a cut-off molecular weight of about 1.2 kDa, and freeze-dry to obtain HAMA.

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

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

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

[0047] 3.1 Synthesis of 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 evenly. Continue to add 524 μL (3.45 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene, stir in an N2 atmosphere for 30 min. Dropwise add 1.9 mL (21.15 mmol) of propylene sulfide, and stir the reaction mixture overnight at 60 °C. Add 5 mL of water to quench the reaction, precipitate and purify in cold methanol, and evaporate the solvent under reduced pressure to obtain yellow oily PPS, and dry it for later use.

[0049] 3.2 Synthesis of PPS-NH2.

[0050] First, add 20 mL of dichloromethane, and then add 158.6 mg (100 μmol) of dried PPS, and dissolve it by magnetic stirring. Continuously 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 wash it successively with H2O and saturated NaCl solution. Dry it with MgSO4 and filter. Concentrate the solvent under reduced pressure to obtain PPS-NH2, and dry it for standby.

[0051] 3.3 Synthesis of HA-PPS conjugate.

[0052] Dialyze sodium hyaluronate in 0.01 M HCl solution overnight, and then lyophilize it to obtain hyaluronic acid in acid form. Add 10 mL of H2O and 100 mg of acid-form HA to a 50 mL beaker, and dissolve it by magnetic stirring. Then add 7 mg (60 μmol) of N-hydroxysuccinimide and 14.5 mg (75 μmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and stir the mixture at room temperature for 30 min until completely dissolved. Dissolve 40 mg of PPS-NH2 in 1 mL of tetrahydrofuran, and drop it into the reaction system. Continue to stir and react for 24 h at room temperature under N2 protection. Dialyze 3 times with water / methanol in a ratio of 1:1 for 1 day, and then dialyze 3 times with distilled water for 1 day. Remove the solvent by lyophilization to obtain the HA-PPS conjugate.

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

[0054] Dissolve the HA-PPS conjugate and curcumin in distilled water according to a certain weight ratio, and ultrasonically treat for 10 minutes to prepare curcumin-loaded HA-PPS nanogel, denoted as curcumin@HA-PPS.

[0055] (4) Preparation of alginate microspheres loaded with dendrobium officinale polysaccharide

[0056] 4.1 Take dendrobium officinale polysaccharide and dissolve it in deionized water, and mix it evenly to obtain a dendrobium officinale polysaccharide solution with a certain concentration.

[0057] 4.2 Take 1.2 mL of 2 M calcium chloride (CaCl2) and mix it evenly with 4.8 mL of 0.5 M ethylenediaminetetraacetic acid solution (EDTA), then add 2 M sodium hydroxide solution to adjust the pH to about 7.2, and finally add ultrapure water to make up the volume to 10 mL to obtain the Ca-EDTA solution.

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

[0059] 4.4 Mix the above-mentioned equal-volume polysaccharide solution of Dendrobium officinale with the sodium alginate solution. After mixing evenly, add a Ca-EDTA solution with the same volume as the mixed solution, and mix evenly to obtain a sodium alginate-polysaccharide of Dendrobium officinale, Ca-EDTA solution.

[0060] 4.5 Load the sodium alginate-polysaccharide of Dendrobium officinale, Ca-EDTA solution and 2% microdroplet-forming oil into 2.5 mL syringes respectively, and load them onto a micro-injection pump. Set the flow rate of the aqueous phase (sodium alginate-polysaccharide of Dendrobium officinale, Ca-EDTA solution) to 0.1 mL / h and the flow rate of the oil phase (2% microdroplet-forming oil) to 1 mL / h, and extrude through a microfluidic chip to form microspheres. Collect the generated microdroplets with an oil-phase centrifuge tube containing 1% acetic acid, and then let it stand and solidify for 30 - 60 min to form hydrogel microspheres. Subsequently, take out the bottom microdroplet-forming oil, add a demulsifier with twice the volume of the microspheres, centrifuge at 1000 rpm for 30 s and take out the bottom demulsifier. Finally, wash the microspheres with PBS 2 - 3 times, and collect the obtained hydrogel microspheres, denoted as polysaccharide of Dendrobium officinale@ sodium alginate microspheres.

[0061] Example 1

[0062] A hydrogel for the treatment of ischemic brain injury, which is cross-linked by methacrylated hyaluronic acid HAMA and methacrylated gelatin GelMA, and doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres loaded with polysaccharide of Dendrobium officinale.

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

[0064] Add 45 mg of GelMA and 20 mg of HAMA to 0.6 mL of deionized water to prepare a solution, and then add 0.2 wt% LAP photoinitiator. Mix the obtained solution evenly with 200 μL of curcumin@HA-PPS and 200 μL of polysaccharide of Dendrobium officinale@ sodium alginate microspheres, and irradiate and mold under a 408 nm ultraviolet lamp to obtain a hydrogel.

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

[0066] When preparing sodium alginate microspheres loaded with polysaccharide of Dendrobium officinale:

[0067] In step 4.1, take 0.1 mg of polysaccharide of Dendrobium officinale and dissolve it in 0.05 mL of deionized water, and mix evenly.

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

[0069] Example 2

[0070] A hydrogel for the treatment of ischemic brain injury, which is crosslinked by methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA), and doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

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

[0072] Add 45 mg of GelMA and 11.25 mg of HAMA to 0.6 mL of deionized water to prepare a solution, then add 0.2 wt% LAP photoinitiator. Mix the resulting solution evenly with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiate and mold it under a 408 nm ultraviolet lamp to obtain the hydrogel.

[0073] Among them, 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 the treatment of ischemic brain injury, which is crosslinked by methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA), and doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres loaded with dendrobium polysaccharide.

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

[0077] Add 60 mg of GelMA and 26 mg of HAMA to 0.6 mL of deionized water to prepare a solution, then add 0.2 wt% LAP photoinitiator. Mix the resulting solution evenly with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiate and mold it under a 408 nm ultraviolet lamp to obtain the hydrogel.

[0078] Among them, 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 the treatment of ischemic brain injury, which is crosslinked by methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA), and 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% LAP photoinitiator was added. The resulting solution was mixed evenly with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiated and molded under a 408 nm ultraviolet lamp to obtain the hydrogel.

[0083] Among them, 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 the treatment of ischemic brain injury, which is crosslinked by methacrylated hyaluronic acid HAMA and methacrylated gelatin GelMA, and 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% LAP photoinitiator was added. The resulting solution was mixed evenly with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiated and molded under a 408 nm ultraviolet lamp to obtain the hydrogel.

[0088] Among them, the preparation method of HA-PPS nanomicelles 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 and mixed evenly, and placed at 60 °C until completely dissolved.

[0090] Example 6

[0091] A hydrogel for the treatment of ischemic brain injury, which is crosslinked by methacrylated hyaluronic acid HAMA and methacrylated gelatin GelMA, and 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% LAP photoinitiator was added. The resulting solution was mixed evenly with 200 μL of curcumin@HA-PPS and 200 μL of dendrobium polysaccharide@sodium alginate microspheres, and irradiated and formed under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0094] Among them, the preparation method and formula of the sodium alginate microspheres of dendrobium polysaccharide were 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 ultrasonic treatment was performed for 10 minutes to prepare curcumin@HA-PPS nanomicelles.

[0096] Comparative Example 1

[0097] A hydrogel is crosslinked by methacrylated hyaluronic acid HAMA and methacrylated gelatin GelMA, and doped with HA-PPS nanomicelles loaded with curcumin and sodium alginate microspheres.

[0098] The preparation method of this 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% LAP photoinitiator was added. The resulting solution was mixed evenly with 200 μL of curcumin@HA-PPS and 200 μL of sodium alginate microspheres, and irradiated and formed under a 408 nm ultraviolet lamp to obtain a hydrogel.

[0100] Among them, the preparation method and ratio of curcumin@HA-PPS were 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 2 M calcium chloride (CaCl2) and mix it evenly with 4.8 mL of 0.5 M ethylenediaminetetraacetic acid solution (EDTA). Subsequently, 2 M sodium hydroxide solution was added to adjust the pH to about 7.2, and finally ultrapure water was added to make up the volume to 10 mL to obtain a Ca-EDTA solution.

[0103] 4.2 Take 2 mg of sodium alginate and dissolve it in 0.05 mL of deionized water and mix evenly, and place it at 60 °C

[0104] until it is completely dissolved to obtain a sodium alginate solution.

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

[0106] 4.4 Load sodium alginate, Ca-EDTA solution and 2% microdroplet-forming oil into 2.5 mL syringes respectively, and load them onto a micro-injection pump. Set the flow rate of the aqueous phase (sodium alginate, Ca-EDTA solution) to 0.1 mL / h and the flow rate of the oil phase (2% microdroplet-forming oil) to 1 mL / h, and extrude through a microfluidic chip to form microspheres. Collect the generated microdroplets with an oil-phase centrifuge tube containing 1% acetic acid, and then let them stand and solidify for 30 - 60 min to form hydrogel microspheres. Subsequently, take out the bottom microdroplet-forming oil, add a demulsifier twice the volume of the microspheres, centrifuge at 1000 rpm for 30 s and take out the bottom demulsifier. Finally, wash the microspheres with PBS 2 - 3 times to collect sodium alginate microspheres.

[0107] Comparative Example 2

[0108] A hydrogel is crosslinked by methacrylated hyaluronic acid HAMA and methacrylated gelatin GelMA, and is doped with HA-PPS nanomicelles and sodium alginate microspheres loaded with dendrobium polysaccharide.

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

[0110] Add 45 mg of GelMA and 20 mg of HAMA to 0.6 mL of deionized water to prepare a solution, then add 0.2 wt% LAP photoinitiator. Mix the obtained solution evenly with 200 μL of HA-PPS and 200 μL of sodium alginate microspheres loaded with dendrobium polysaccharide, and irradiate and mold under ultraviolet light at 408 nm to obtain a hydrogel.

[0111] The preparation method of the 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, dissolve 9 mg of the HA-PPS conjugate prepared in step 3.3 in 0.2 mL of distilled water, and ultrasonically treat for 10 minutes to prepare HA-PPS nanomicelles.

[0114] Comparative Example 3

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

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

[0117] (1) Dissolve 45 mg of GelMA and 20 mg of HAMA in 0.6 mL of deionized water to form a solution, and then add 0.2 wt% LAP photoinitiator.

[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) Add the obtained curcumin solution and 200 μL of alginate microspheres loaded with dendrobium polysaccharide to the mixture obtained in step (1), mix well, and irradiate and form under ultraviolet light at 408 nm to obtain a hydrogel.

[0120] Among them, the preparation method of the alginate microspheres loaded with dendrobium polysaccharide is the same as that in Example 1.

[0121] Comparative Example 4

[0122] A hydrogel is crosslinked by methacrylated hyaluronic acid HAMA and methacrylated gelatin GelMA, and is doped with HA-PPS nanomicelles loaded with curcumin and dendrobium polysaccharide.

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

[0124] (1) Dissolve 45 mg of GelMA and 20 mg of HAMA in 0.6 mL of deionized water to form a solution, and then add 0.2 wt% LAP photoinitiator.

[0125] (2) Dissolve 0.1 mg of dendrobium polysaccharide in 0.2 mL of deionized water, 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 mixture obtained in step (1), mix well, and irradiate and form under ultraviolet light at 408 nm to obtain a hydrogel.

[0127] Among them, the preparation method and ratio of curcumin@HA-PPS are the same as those in Example 1.

[0128] Comparative Example 5

[0129] A hydrogel is crosslinked by methacrylated hyaluronic acid HAMA and methacrylated gelatin GelMA, and is doped with HA-PPS nanomicelles loaded with curcumin and alginate microspheres loaded with dendrobium polysaccharide.

[0130] The preparation method of this 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% LAP photoinitiator was added. The resulting solution was mixed evenly with 200 μL of curcumin@HA-PPS and 200 μL of alginate microspheres loaded with dendrobium officinale polysaccharide, and irradiated and formed under a 408 nm ultraviolet lamp to obtain a hydrogel.

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

[0133] When preparing the alginate microspheres loaded with dendrobium officinale polysaccharide:

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

[0135] Comparative Example 6

[0136] A hydrogel is crosslinked by methacrylated hyaluronic acid HAMA and methacrylated gelatin GelMA, and doped with HA-PPS nanomicelles loaded with curcumin and alginate microspheres loaded with dendrobium officinale polysaccharide.

[0137] The preparation method of this 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% LAP photoinitiator was added. The resulting solution was mixed evenly with 200 μL of curcumin@HA-PPS and 200 μL of alginate microspheres loaded with dendrobium officinale polysaccharide, and irradiated and formed under a 408 nm ultraviolet lamp to obtain a hydrogel.

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

[0140] The preparation method of the alginate microspheres loaded with dendrobium officinale polysaccharide was the same as that in Example 1.

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

[0142] Table 1 Material Compositions of Different Examples and Comparative Examples

[0143]

[0144]

[0145] Performance Test

[0146] 1. SEM Images of the Hydrogel

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

[0148] 2. Mechanical properties of the hydrogel

[0149] Test method: Place the hydrogel on the sample stage, adjust the height of the sample stage so that both the upper and lower surfaces are in contact with the fixture. Compress it at a constant rate of 0.05 mm / s, record the compression displacement (L) and load (P) until the set displacement end point. 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 hydrogels of the examples of the present invention have good strength, especially when HAMA: GelMA = 15 - 26 mg: 45 - 60 mg. In addition, it can also be seen from Example 5 that sodium alginate also has a certain influence on the strength of the gel.

[0151] Table 2 Test results of the mechanical strength values of each example and comparative example

[0152]

[0153]

[0154] 3. Drug release test of the hydrogel

[0155] Test method: Prepare curcumin solution and dendrobium polysaccharide solution according to a certain concentration gradient, measure the absorbance using an ultraviolet spectrophotometer at 420 nm and 490 nm respectively, and make a standard curve. Then put the corresponding example and comparative example samples into PBS and incubate at 37°C. Take the supernatant on the 1st, 3rd, 5th, 7th, 14th, 21st, and 28th days respectively, measure the absorption value using an ultraviolet spectrophotometer, and calculate the drug release amount according to the standard curve.

[0156] The test results are as Figure 2 shown, Figure 2 In the upper figure above, through Example 1 and Comparative Example 3, the sustained-release curve of curcumin is reflected. After loading curcumin with HA-PPS, it can play a sustained-release effect on the release of curcumin; Figure 2 In the lower figure below, through the comparison of Example 1 and Comparative Example 4, it is reflected that after encapsulating dendrobium polysaccharide with sodium alginate microspheres, it can play a sustained-release effect on the release of dendrobium polysaccharide; The drug release rate of the hydrogel of the examples of the present invention can better fit the rate of brain tissue repair, which is beneficial to the treatment of brain injury.

[0157] 4. Antioxidant capacity test of the hydrogel

[0158] Test method: The method of scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals was adopted to evaluate the antioxidant properties of different examples and comparative examples. The mixtures of different examples and comparative example samples with DPPH reagent were stirred and incubated in the dark for 30 minutes, and the remaining DPPH was analyzed by ultraviolet-visible spectroscopy. The formula for determining the scavenging rate of DPPH is:

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

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

[0161] The test results are as Figure 3 shown that curcumin and dendrobium polysaccharide both have the ability to scavenge free radicals and have good antioxidant effects. The hydrogel with appropriate amounts of curcumin and dendrobium polysaccharide added has a stronger antioxidant effect, proving that curcumin and dendrobium polysaccharide have a certain synergistic effect in antioxidant action at a certain concentration. However, when the concentration of curcumin or dendrobium polysaccharide exceeds a certain level, it will have an inhibitory effect on its antioxidant effect. For the two groups without loading curcumin with HA-PPS and without encapsulating dendrobium polysaccharide with sodium alginate microspheres, due to the degradation of the drug, the antioxidant effect becomes poor in the later stage.

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

[0163] Test method: Mice were anesthetized by intraperitoneal injection with 0.3% sodium pentobarbital (22 mL / kg) and divided into a sham operation group, an IS (ischemic brain injury) group, an example 1 group, a comparative example 1 group, and a comparative example 2 group. The model was generated by the middle cerebral artery occlusion method of the suture method. An incision was made at the midline of the mouse neck, and the common carotid artery, internal carotid artery, and external carotid artery were isolated. A nylon wire with a polyethylene coating (0.2 - 0.3 mm in diameter) was inserted into the internal carotid artery and slowly advanced to the middle cerebral artery to block it. After maintaining an ischemic time of one hour, the suture was removed to restore cerebral blood flow, simulating reperfusion (I / R) injury. Then the hydrogel was 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 inside to outside. The sham operation group had a neck opening but no ischemic treatment. The IS group had a neck opening and ischemic treatment but no hydrogel implantation. The example 1 group, the comparative example 1 group, and the comparative example 2 group had neck openings, ischemic treatment, and hydrogel implantation. The mice were sacrificed at the 48th hour, and brain tissues were taken from the ischemic area of the brain, placed in an appropriate amount of PBS, and protease inhibitors were added. The tissues were thoroughly homogenized using a homogenizer, and then centrifuged at 12,000 rpm for 10 - 15 minutes at 4°C, and the supernatant was taken for detection. The inflammatory cytokines in the supernatant were analyzed by ELISA.

[0164] It can be seen from Figure 4 that compared with the IS group, the hydrogels loaded with curcumin and polysaccharide of Dendrobium officinale can effectively reduce the expression levels of inflammatory factors TNF-α, IL-6, and IL-β. Moreover, the hydrogel loaded with both curcumin and polysaccharide of Dendrobium officinale has a better effect on reducing the expression levels of inflammatory factors than the hydrogel loaded with only one of the components, and can more effectively play an anti-inflammatory effect, providing a stable microenvironment for brain tissue repair. For the two groups of curcumin not loaded with HA-PPS and polysaccharide of Dendrobium officinale not encapsulated with microspheres, although they have a certain anti-inflammatory effect, due to the easy degradation of the drugs, the later treatment effect is inferior to that of Example 1 group.

[0165] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0166] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A hydrogel for the treatment of ischemic brain injury, characterized in that, The hydrogel comprises the following raw materials: methacrylated hyaluronic acid (HAMA), methacrylated gelatin (GelMA), HA-PPS nanomicelles loaded with curcumin, and sodium alginate microspheres loaded with dendrobium officinale polysaccharide.

2. The hydrogel according to claim 1, wherein According to the mass ratio, HAMA: GelMA = 11.25 - 26 mg: 45 - 60 mg.

3. The hydrogel according to claim 2, wherein According to the mass ratio, HAMA: GelMA = 15 - 26 mg: 45 - 60 mg.

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

5. The hydrogel according to claim 4, 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.

6. The hydrogel according to claim 4, wherein In the step 2), dichloromethane: PPS: NHS: EDCI: ethylenediamine = 20 mL: 158.6 mg: 23 mg: 48 mg: 133 μL.

7. The hydrogel according to claim 4, wherein In the step 3), acidic HA: NHS: EDCI: PPS-NH2 = 100 mg: 7 mg: 14.5 mg: 40 mg, and the concentration of the aqueous solution of acidic hyaluronic acid is 10 mg / mL.

8. The hydrogel according to claim 4, wherein In the step 4), HA-PPS conjugate: curcumin: water = 40 - 45 mg: 0.9 mg: 1 mL.

9. The hydrogel according to claim 1, characterized in that, The sodium alginate microspheres loaded with dendrobium officinale polysaccharide are prepared by a microfluidic method using a mixed solution of a dendrobium officinale polysaccharide solution, a sodium alginate solution, and a Ca-EDTA solution as the aqueous phase and a microdroplet-forming oil as the oil phase.

10. The hydrogel according to claim 9, wherein, In the aqueous phase, the concentration of dendrobium officinale polysaccharide is 0.5 mg / mL, and the concentration of sodium alginate is 1 - 2%. The Ca-EDTA solution is obtained by uniformly mixing 1.2 parts by volume of 2M CaCl2 solution and 4.8 parts by volume of 0.5M EDTA solution, then adding 2M NaOH solution to adjust the pH to 7.2, and then making up the volume to 10 parts by volume.

11. The preparation method of the hydrogel according to any one of claims 1-10, characterized in that, It includes the steps of: Dissolving methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) in deionized water to form a solution, then adding a photoinitiator, HA-PPS nanomicelles loaded with curcumin, and sodium alginate microspheres loaded with dendrobium officinale polysaccharide and mixing uniformly to obtain a mixed solution, and irradiating with ultraviolet light to form a hydrogel.

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