A multifunctional injectable hydrogel for treating ischemic stroke, its preparation method and uses
A multi-functional hydrogel with NO-releasing colloid and drug-loaded nanoparticles addresses the lack of effective treatments for ischemic stroke by promoting neural repair and vascular regeneration, overcoming the limitations of the blood-brain barrier.
Patent Information
- Application Number
- CN202411228796.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-03
- Publication Date
- 2025-07-15
- Estimated Expiration
- 2044-09-03
AI Technical Summary
The existing drugs for the treatment of ischemic stroke have problems such as narrow time window, blood-brain barrier obstruction, limited effects and complexity of multiple targets, resulting in poor treatment effects and lack of effective neuroprotective drugs, and a high mortality and disability rate.
A multifunctional injectable hydrogel containing colloids and drug-loaded nanoparticles that can sustainably release NO, and load MIF inhibitors to achieve nerve damage repair, anti-inflammatory and pro-angiogenesis.
In the cerebral ischemic microenvironment, it exerts the functions of nerve damage repair, anti-inflammatory, anti-blood-brain barrier destruction and pro-angiogenesis, realizes the recovery of nerve function in ischemic stroke and improves the quality of life.
Smart Images
Figure CN119055583B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a multifunctional injectable hydrogel for treating ischemic stroke, a preparation method thereof, and uses thereof. Background Art
[0002] Stroke is a global disease that seriously threatens human life and health and is also one of the main causes of global population death. Among them, ischemic stroke (cerebral infarction) is the most common type of stroke, accounting for about 80%. In conventional treatment regimens, the time window for acute mechanical thrombectomy and intravenous thrombolysis is narrow. Most patients with cerebral infarction do not benefit and may also be accompanied by related complications. In addition, due to the very complex process of ischemic injury and chronic recovery, the treatment mechanism has the characteristics of multiple targets and multiple pathways.
[0003] At present, there are few clinically effective neuroprotective drugs during the recovery period after cerebral ischemia, and the effects of the approved neuroprotective agents are also relatively limited. Therefore, the current mortality and disability rates of cerebral infarction are extremely high, seriously endangering human health, bringing a huge burden to families and society, and being one of the important reasons for poverty due to illness. After cerebral infarction, the brain undergoes a series of pathophysiological processes due to ischemia and hypoxia, leading to cell metabolic disorders. For example, excitotoxicity, oxidative stress, and mitochondrial dysfunction can induce a variety of cellular cascades, ultimately resulting in neuronal apoptosis, necrosis, and autophagy. The pathophysiological mechanism after cerebral infarction is complex and has multiple targets, which is also one of the reasons for the poor effect of targeting a single treatment target after cerebral infarction.
[0004] In addition, another obstacle is the protective effect of the blood-brain barrier. As a biological barrier that protects the brain from toxins and infections, the blood-brain barrier also prevents most therapeutic drugs from effectively entering the brain, and most therapeutic drugs have problems such as short circulation time, poor stability, and their own toxicity. Therefore, finding a comprehensive treatment method that can continuously repair nerve damage, reasonably control neuroinflammation, and promote angiogenesis, and using new technical means to enable these key molecules to play a role in the core area of ischemic brain injury to improve the prognosis of ischemic brain injury and improve the quality of life of patients with cerebral infarction is a difficult problem that needs to be solved urgently in clinical practice. Summary of the Invention
[0005] In view of the above deficiencies in the prior art, the present invention provides a multifunctional injectable hydrogel for treating ischemic stroke, a preparation method thereof, and uses thereof, which can play the functions of nerve injury repair, anti-inflammatory, anti-blood-brain barrier damage, and promoting angiogenesis in the cerebral ischemic microenvironment, and ultimately achieve the recovery of nerve function after ischemic stroke.
[0006] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is:
[0007] A multifunctional injectable hydrogel for treating ischemic stroke, comprising a colloid that can sustainably release NO and drug-loaded nanoparticles crosslinked therewith; the drug-loaded nanoparticles are loaded with an MIF inhibitor.
[0008] Furthermore, the colloid that can sustainably release NO is gelatin grafted with an NO donor, which is crosslinked with the drug-loaded nanoparticles through oxidized dextran.
[0009] Furthermore, the preparation method of the gelatin grafted with an NO donor is as follows:
[0010] (1) Bromoacetic acid and silver nitrate are placed in anhydrous acetonitrile, stirred at 70-90 °C for 15-18 h, filtered, the filtrate is concentrated under vacuum, then dichloromethane is added, incubated at room temperature for 2-5 h and filtered again to remove dichloromethane to obtain 2-(nitrooxy)acetic acid;
[0011] (2) The carboxyl group of 2-(nitrooxy)acetic acid is activated in a buffer solution, then gelatin is added and stirred for reaction for 42-50 h to prepare gelatin G grafted with an NO donor. NO 。
[0012] Furthermore, the weight ratio of bromoacetic acid to silver nitrate is 1:1.5-2.
[0013] Furthermore, NHS and EDC are used to activate the carboxyl group of 2-(nitrooxy)acetic acid.
[0014] Furthermore, the buffer solution is 2-morpholinoethanesulfonic acid with a pH value of 6.
[0015] Furthermore, the preparation method of the drug-loaded nanoparticles is as follows:
[0016] (1) 4-Carboxyphenylboronic acid, EDC and a catalyst are added to a PF127 solution, and the reaction is carried out at 40-50 °C for 20-30 h in a protective gas atmosphere, filtered and concentrated, precipitated with pre-cooled ether, and then dialyzed and freeze-dried for standby;
[0017] (2) The product obtained in step (1) and the MIF inhibitor are dissolved and sonicated evenly, the organic solvent is removed by rotary vacuum evaporation to obtain a uniform thin film, then water at 40-50 °C is added, and sonication hydration is carried out at 40-50 °C. After the thin film peels off, it is filtered through a filter membrane to obtain the drug-loaded nanoparticles.
[0018] Furthermore, the mass ratio of PF127, 4-carboxyphenylboronic acid, EDC and the catalyst is 10-20:0.5-2:1-2:0.1-0.8.
[0019] Furthermore, the catalyst is DMAP.
[0020] Furthermore, the MIF inhibitor is ISO-1.
[0021] For the preparation method of the above multifunctional injectable hydrogel, G NO , oxidized dextran and drug-loaded nanoparticles are mixed and stirred evenly, and then G NO and the drug-loaded nanoparticles can be crosslinked through oxidized dextran.
[0022] Furthermore, the mass ratio of G NO to oxidized dextran is 1:1.
[0023] Furthermore, the preparation method of oxidized dextran is as follows:
[0024] Dissolve dextran and sodium periodate in deionized water (DW), and stir at 37 °C for 4 h; then, add ethylene glycol and continue to stir for 2 h to mix evenly, and finally dialyze with DW and freeze-dry to obtain.
[0025] Use of the above multifunctional injectable hydrogel in the preparation of drugs for nerve injury repair, promoting angiogenesis, anti-blood-brain barrier disruption or treating ischemic stroke.
[0026] Advantages of the present invention:
[0027] The sustained-release NO multifunctional hydrogel loaded with PF127B@ISO-1 nanoparticles prepared by the present invention can respond to the microenvironment and can play functions of nerve injury repair, anti-inflammatory, anti-blood-brain barrier disruption and promoting angiogenesis in the cerebral ischemia microenvironment, and finally achieve the recovery of nerve function after ischemic stroke. Description of the Drawings
[0028] Figure 1 For the performance detection of the multifunctional injectable hydrogel prepared by the present invention;
[0029] Figure 2 For the expression of inflammation, angiogenesis and neuroprotection-related proteins in the brains of experimental mice after drug administration;
[0030] Figure 3 For the changes in nerve function and behavior of experimental mice over time after drug administration;
[0031] Figure 4 For the degree of motor activity of experimental mice after drug administration. Detailed Embodiments
[0032] The following describes the specific embodiments of the present invention to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the concept of the present invention are within the scope of protection.
[0033] Example 1 Preparation of a multifunctional injectable hydrogel
[0034] 1. Preparation of NO-grafted gelatin (G NO )
[0035] Bromoacetic acid (2.00 g, 14.3 mmol) and silver nitrate (3.70 g, 21.8 mmol) were stirred in anhydrous acetonitrile (80 mL) at 70 °C for 18 h. After filtration, the product was concentrated in vacuo and dichloromethane (100 mL) was added. The mixture was incubated at room temperature for 2 h and then filtered again. Subsequently, dichloromethane was removed to obtain 2-(nitrooxy)acetic acid as a yellow oil. 2-(Nitrooxy)acetic acid (3.5 mL), N-hydroxysuccinimide (NHS) (6.3 g), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (10.5 g) were dissolved in 100 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.0) to activate the carboxyl group. After 2 h, gelatin (5.5 g) was added and the reaction was stirred for 48 h, then dialyzed against deionized water and freeze-dried to obtain G NO .
[0036] 2. Synthesis of PF127-BA
[0037] (1) 20 g of PF127 was dissolved in dichloromethane, and then 1.5 g of 4-carboxyphenylboronic acid, 1.92 g of EDC, and 611 mg of 4-dimethylaminopyridine (DMAP) were added respectively. Under nitrogen protection, the reaction was carried out at 40 °C for 24 h. After filtration and concentration of the reactants, they were added dropwise to pre-cooled diethyl ether for precipitation, and then dialyzed and freeze-dried in a methanol / water solution.
[0038] (2) 100 mg of PF127B and 10 mg of ISO-1 were respectively dissolved in 5 mL of tetrahydrofuran, and the mixture was sonicated in a water bath for 10 minutes to fully dissolve it; at 40 °C, the organic solvent was removed by rotary evaporation under reduced pressure, and at this time, a uniform film was left on the bottle; 20 mL of pre-heated 40 °C aqueous solution was added to the rotary evaporation flask, and ultrasonic hydration was carried out in a 40 °C water bath until the film on the bottle wall peeled off. After filtration through a 0.22 μm filter membrane, it was stored at 4 °C to obtain PF127-BA.
[0039] 3. Preparation of oxidized dextran
[0040] Dissolve 10.0 g of dextran and 8.0 g of sodium periodate in deionized water (DW), and stir at 37 °C for 4 h. Then, add 1.2 mL of ethylene glycol to the above solution and stir for 2 h to mix evenly. Finally, dialyze with deionized water (DW) for 48 h, and obtain oxidized dextran (OD) after freeze-drying.
[0041] 4. Preparation of hydrogel
[0042] React the synthesized gelatin grafted with NO, oxidized dextran, and PF127 nanoparticles loaded with ISO-1. The gelatin grafted with NO and the PF127 nanoparticles loaded with ISO-1 can be cross-linked by oxidized dextran, and then the hydrogel is prepared.
[0043] Example 2
[0044] 1. Detect the performance of the hydrogel prepared in Example 1, and the results are shown in Figure 1 . Figure 1 Among them, (A) is the detection of the typical peaks of each component by nuclear magnetic resonance hydrogen spectrum; (B) is the injectability and gel-forming performance of the synthesized hydrogel; (C) is the rheological property detection of three hydrogels, where: Gel1 is Gel-NO; Gel2 is Gel-NO-PF127BA; Gel3 is Gel-NO-PF127B@ISO-1.
[0045] As Figure 1 shown, the present invention has successfully grafted the NO donor onto gelatin, and both oxidized dextran (OD) and PF127-BA have been successfully synthesized, and typical peaks can be seen in the nuclear magnetic resonance hydrogen spectrum (* marks the typical peaks of each substance, Figure 1 A); at the same time, the hydrogel synthesized by the method of the present invention can have a fixed shape and has injectability ( Figure 1 B).
[0046] Furthermore, measure the mechanical properties of the hydrogel obtained by oscillatory shear rheology. The results show that the storage modulus (G’) significantly exceeds the loss modulus (G”), and is similar to the modulus of brain tissue, and the mechanical properties of the hydrogel match those of brain tissue. Moreover, when the strain increases, the storage modulus is lower than the loss modulus, indicating that all kinds of hydrogels prepared by the present invention have injectability ( Figure 1 C).
[0047] 2. Comparison of the model group (PT group): The mouse model of photochemically induced ischemic cerebral infarction was divided into the Gel-NO hydrogel injection group (PT + Gel-NO), the Gel-NO + empty PF127BA nanoparticle hydrogel injection group (PT + Gel-NO-PF127BA), and the Gel-NO + PF127B@MIF inhibitor nanoparticle hydrogel injection group (PT + Gel-NO-PF127B@ISO-1). The expression levels of proteins related to inflammation, angiogenesis, and neuroprotection in the model mice after treatment were evaluated. The results are shown in Figure 2 , Figure 2 . Immunofluorescence staining was performed on the brain tissues of the mice in the above groups respectively. It was found that compared with the sham operation group, the expressions of TUNEL ( Figure 2 A), 4-HNE ( Figure 2 B), MIF ( Figure 2 C), MMP-9 ( Figure 2 D), CD31 ( Figure 2 E), and α-SMA ( Figure 2 F) in the brain tissues of the treatment groups all increased.
[0048] 2. Comparison of the model group (PT group): The mouse model of photochemically induced ischemic cerebral infarction was divided into the Gel-NO hydrogel injection group (PT + Gel-NO), the Gel-NO + empty PF127BA nanoparticle hydrogel injection group (PT + Gel-NO-PF127BA), and the Gel-NO + PF127B@MIF inhibitor nanoparticle hydrogel injection group (PT + Gel-NO-PF127B@ISO-1). The dosage was 10 μL. The treatment of the model mice 21 days after medication was evaluated. The results are shown in Figure 3 , Figure 3 . (A) is the neurological function score (mNSS); (B) is the duration of each group of mice on the rotarod in the rotarod test; (C) is the distance of the mice's movement in the open field test; (D) is the speed of the mice's movement in the open field test.
[0049] As Figure 3 shown, after treatment, the neurological function scores (mNSS) of the ischemic cerebral infarction mice all decreased ( Figure 3 A), the duration on the rotarod in the rotarod test prolonged ( Figure 3 B), the distance of movement in the open field test increased ( Figure 3 C), and the speed became faster ( Figure 3 D), indicating that the voluntary motor function recovered well. Moreover, with the prolongation of the treatment time, the effect was better.
[0050] In addition, in the open field test, compared with the model group, the motor activity of the treatment group increased significantly, especially in the Gel-NO-PF127B@ISO-1 group. With the extension of the treatment time, both the motor activity and the curiosity about the environment increased ( Figure 4 ).
[0051] According to the above test results, it can be seen that the multifunctional injectable hydrogel prepared by the present invention can be used to treat ischemic cerebral infarction, indicating that it has good clinical significance and application value for the treatment of cerebral infarction.
[0052] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.
Claims
1. A multifunctional injectable hydrogel for treating ischemic stroke, characterized in that, The multifunctional injectable hydrogel is Gel-NO-PF127B@ISO-1, including a colloid G that can sustainably release NO. NO ; The colloid G NO is gelatin grafted with an NO donor, which is crosslinked with a drug-loaded nanoparticle PF127-BA through oxidized dextran; the drug-loaded nanoparticle is loaded with an MIF inhibitor ISO-1.
2. The multifunctional injectable hydrogel according to claim 1, wherein, The preparation method of the grafted NO donor gelatin is as follows: (1) Bromoacetic acid and silver nitrate are placed in anhydrous acetonitrile, stirred at 70-90 °C for 15-18 h, filtered, the filtrate is concentrated under vacuum, then dichloromethane is added, incubated at room temperature for 2-5 h and filtered again to remove dichloromethane to obtain 2-(nitrooxy)acetic acid; (2) Carboxyl activation of 2-(nitrooxy)acetic acid is carried out in a buffer solution, and then gelatin is added, followed by stirring and reacting for 42 to 50 h to obtain gelatin G grafted with NO donors. NO .
3. The multifunctional injectable hydrogel according to claim 2, wherein The weight ratio of the bromoacetic acid to the silver nitrate is 1:1.5-2.
4. The multifunctional injectable hydrogel according to claim 2, wherein NHS and EDC are used to activate the carboxyl group of 2-(nitrooxy)acetic acid.
5. The multifunctional injectable hydrogel according to claim 1, wherein The preparation method of the drug-loaded nanoparticles is as follows: (1) 4-Carboxyphenylboronic acid, EDC and a catalyst are added to the PF127 solution, and the reaction is carried out at 40-50 °C for 20-30 h in a protective gas atmosphere, filtered and concentrated, then precipitated with pre-cooled ether, and then dialyzed and freeze-dried for standby; (2) The product obtained in step (1) and the MIF inhibitor ISO-1 are dissolved and sonicated uniformly, the organic solvent is removed by rotary vacuum evaporation to obtain a uniform thin film, then water at 40-50 °C is added, and ultrasonic hydration is carried out at 40-50 °C. After the thin film peels off, it is filtered through a filter membrane to obtain the drug-loaded nanoparticles.
6. The multifunctional injectable hydrogel according to claim 5, wherein The mass ratio of the PF127, 4-carboxyphenylboronic acid, EDC and the catalyst is 10-20:0.5-2:1-2:0.1-0.
8.
7. A method for preparing the multifunctional injectable hydrogel according to any one of claims 1 to 6, characterized in that, Mix G NO , oxidized dextran, and drug-loaded nanoparticles evenly by stirring, and then G NO and the drug-loaded nanoparticles can be crosslinked by oxidized dextran.
8. Use of the multifunctional injectable hydrogel according to any one of claims 1-6 in the preparation of a drug for nerve injury repair, promoting angiogenesis, anti-blood-brain barrier disruption or treating ischemic stroke.