Multifunctional injectable hydrogel and its preparation method and application

Abstract

The application discloses a multifunctional injectable hydrogel and a preparation method and application thereof, and relates to the technical field of multifunctional hydrogel preparation and application. In the preparation, CMC-AGE and OD-SH are prepared respectively, and DSPE-se-se-PEG@AC NPs nanoparticles are prepared, then the multifunctional injectable hydrogel is prepared by forming a hydrogel through CMC-AGE and OD-SH and adding DSPE-se-se-PEG@AC NPs nanoparticles and / or beta nerve growth factor. The application also includes the multifunctional injectable hydrogel prepared by the above method and the application thereof. The application constructs an in-situ hydrogel delivery platform with good biocompatibility, is used for co-delivery of DSPE-se-se-PEG@AC NPs and NGF, is used in ischemic stroke treatment, and reshapes the pathological microenvironment of a cerebral infarction area, so that cerebral ischemic injury is reduced.

Description

Technical Field

[0001] This invention relates to the field of multifunctional hydrogel preparation and application technology, specifically to a multifunctional injectable hydrogel, its preparation method, and its application. Background Technology

[0002] The latest statistics show that stroke is the second leading cause of death worldwide, with ischemic stroke accounting for 86.8% of all stroke events. Its high mortality and long-term disability rates impose a huge health and economic burden on patients and global healthcare systems. Currently, tissue plasminogen activator (tPA) remains the only approved drug for the treatment of acute ischemic stroke. However, because the therapeutic window of tPA is only 4.5 hours, only about 6% of patients benefit, and the majority of these patients suffer from long-term disabilities such as hemiplegia, aphasia, sensory loss, and cognitive impairment. Therefore, developing new treatment strategies to mitigate ischemic brain injury is crucial.

[0003] Focal cerebral ischemia leads to insufficient cerebral perfusion, mitochondrial dysfunction, and imbalance in mitochondrial energy metabolism, resulting in excessive ROS production, triggering inflammatory responses, apoptosis, and ultimately brain cell death. Even if blood supply is restored, ischemia-reperfusion occurs, and excessive ROS-induced inflammatory responses may lead to irreversible brain damage and dysfunction. Therefore, research on regulating the pathological microenvironment of cerebral ischemia-injury has attracted much attention. Currently, intravenous administration has been proposed to improve the pathological microenvironment after stroke; however, more than 98% of small molecules with a mass <500 Da and almost all small molecules with a mass >500 Da cannot effectively penetrate the blood-brain barrier (BBB). The off-target effects and low homing efficiency caused by the BBB limit its therapeutic efficacy, while in situ injection seems to be a feasible method to overcome these shortcomings. Surprisingly, stroke provides a unique opportunity for in situ injection of hydrogels. After the initial loss of neurons in cerebral infarction, microglia recruit and clear debris, forming a compartmentalized infarct cavity that can accommodate the hydrogel without compressing the surrounding parenchyma. Currently, in-situ hydrogels carrying therapeutic molecules or cells have been reported for the treatment of ischemic stroke with satisfactory results. Furthermore, compared to intravenous administration, local hydrogel injection, by bypassing the BBB and interacting directly with the peri-infarct area where neuroplasticity is strongest, can improve drug delivery efficiency, enhance therapeutic effects, and minimize systemic toxicity.

[0004] In response to the severe pathological state following stroke, various pharmaceutical preparations with antioxidant and anti-inflammatory functions have been proposed to alleviate cerebral ischemia-reperfusion injury. Among these drugs and preparations, atorvastatin calcium (AC) has been well-established for its lipid-lowering properties in cardiovascular and cerebrovascular diseases and has been approved by the FDA for stroke prevention. Furthermore, AC has been shown to possess anti-inflammatory and antioxidant capabilities, removing superoxide by inhibiting pro-inflammatory polarization of microglia and increasing the activity of antioxidant enzymes. Meanwhile, the crucial role of microcirculation remodeling and neuroprotection in the late recovery period after stroke is widely recognized. NGF (β-nerve growth factor), as an important neurotrophic factor, possesses dual functions of angiogenesis and neurogenesis, playing a vital role in nerve repair. Moreover, NGF and its receptors are widely distributed in the brain. As an exogenous supplement for neurological diseases lacking endogenous neurotrophic factors or with impaired neurotrophic effects, NGF can maintain neuronal survival and function, thereby accelerating the repair of the nervous system after stroke, making it a promising exogenous supplement. However, due to the presence of the blood-brain barrier, intravenous administration to the brain remains a huge challenge and may lead to off-target effects outside the brain or even cause systemic adverse reactions. In situ hydrogels offer a possibility to deliver drugs without these side effects. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a multifunctional injectable hydrogel, its preparation method, and its application. This invention constructs an in-situ hydrogel platform with good biocompatibility for the co-delivery of DSPE-se-se-PEG@AC NPs and NGF, reshaping the pathological microenvironment of the cerebral infarction region, thereby alleviating cerebral ischemia-reperfusion injury.

[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for preparing a multifunctional injectable hydrogel is provided, comprising the following steps:

[0007] (1) Dissolve carboxymethyl chitosan in an aqueous solution containing sodium hydroxide and stir at 20-30℃ for 3-5 hours. Then add allyl glycidyl ether and stir at 70-90℃ for 2-4 days. After the reaction is terminated, neutralize with hydrochloric acid and finally dialyze and freeze dry to obtain allyl glycidyl ether modified carboxymethyl chitosan (CMC-AGE).

[0008] (2) Dextran and sodium periodate were dissolved in deionized water and stirred at 20-30℃ for 5-7 h. The reaction was then terminated with ethylene glycol. After dialyzing and freeze-drying, oxidized dextran was obtained. The oxidized dextran was then added to ethanol and stirred. L-cysteine ​​and 2-pyridine-borane were added successively and stirred for 24 h. Finally, the mixture was washed three times with distilled water and ethanol respectively and dried at 30-50℃ to obtain L-cysteine-modified oxidized dextran (OD-SH).

[0009] (3) Phospholipid-diselenyl bond-polyethylene glycol (DSPE-se-se-PEG) and atorvastatin calcium (AC) were stirred and dissolved in a mixture of chloroform and methanol. The mixture was dried in a rotary evaporator at 30-50℃ for 10-20 min, then dried in an oven at 30-50℃ for 10-30 min. Deionized water was added, and the mixture was ultrasonically bathed at 50-70℃ for 1 h and then freeze-dried to obtain DSPE-se-se-PEG@AC NPs nanoparticles.

[0010] (4) Dissolve the allyl glycidyl ether modified carboxymethyl chitosan obtained in step (1) and the L-cysteine ​​modified oxidized dextran obtained in step (2) in PBS buffer, mix them to form a hydrogel, and then add the DSPE-se-se-PEG@AC NPs nanoparticles and / or β-nerve growth factor obtained in step (3) to form a multifunctional injectable hydrogel.

[0011] Furthermore, in step (1), the mass-to-volume ratio of carboxymethyl chitosan, allyl glycidyl ether, and sodium hydroxide aqueous solution is 4-6g:5-7g:250mL.

[0012] Furthermore, in step (1), the mass-volume ratio of carboxymethyl chitosan, allyl glycidyl ether, and sodium hydroxide aqueous solution is 5g:6g:250mL.

[0013] Furthermore, in step (1), the sodium hydroxide solution has a mass concentration of 4-6 wt%.

[0014] Furthermore, in step (2), the mass ratio of dextran to sodium periodate is 5:2-3.

[0015] Furthermore, in step (2), the mass ratio of dextran to sodium periodate is 5:2.5.

[0016] Furthermore, in step (2), the mass-volume ratio of oxidized dextran, L-cysteine, 2-pyridine-borane and ethanol is 1g:7-8g:2-3g:100mL.

[0017] Furthermore, in step (2), the mass-volume ratio of oxidized dextran, L-cysteine, 2-pyridine-borane and ethanol is 1g:7.57g:2.67g:100mL.

[0018] Furthermore, in step (3), the mass-volume ratio of phospholipid-diselement bond-polyethylene glycol, atorvastatin calcium, chloroform and methanol is 5-7 mg: 1-1.4 mg: 4 mL: 1 mL.

[0019] Furthermore, in step (3), the mass-volume ratio of phospholipid-diselenyl bond-polyethylene glycol, atorvastatin calcium, chloroform and methanol is 6 mg:1.2 mg:4 mL:1 mL.

[0020] Furthermore, in step (4), the allyl glycidyl ether modified carboxymethyl chitosan and the L-cysteine ​​modified oxidized dextran were dissolved in PBS buffer at concentrations of 6.67 wt% and 10 wt%, respectively, with a volume ratio of 0.5:0.35.

[0021] Furthermore, in step (4), the mass ratio of DSPE-se-se-PEG@AC NPs nanoparticles to β-nerve growth factor is 0.1-0.3 mg: 200 ng.

[0022] Furthermore, in step (4), the mass ratio of DSPE-se-se-PEG@AC NPs nanoparticles to β-nerve growth factor is 0.2 mg: 200 ng.

[0023] The multifunctional injectable hydrogel prepared by the above method is a multifunctional hydrogel.

[0024] The above-mentioned multifunctional injectable hydrogels are used in biomaterials that can remodel the repair microenvironment of cerebral infarction areas.

[0025] A biomaterial capable of reshaping the repair microenvironment of the infarct region, comprising the aforementioned multifunctional injectable hydrogel.

[0026] The present invention has the following beneficial effects:

[0027] 1. This invention constructs an in-situ hydrogel platform for the co-delivery of anti-inflammatory / antioxidant and neurotrophic factors, which holds promise as a new and promising treatment method for post-stroke neurological function recovery. This invention selects carboxymethyl chitosan (CMC) and dextran, which have good biocompatibility and biodegradability, as the hydrogel matrix. L-cysteine-modified oxidized dextran (OD-SH) and allyl glycidyl ether-modified carboxymethyl chitosan (CMC-AGE) are crosslinked via a thiol-enol click reaction to form the hydrogel. The absence of a photoinitiator avoids the potential biotoxicity of photoinitiators to the brain. Next, ROS-responsive phospholipid-diselement-polyethylene glycol drug-loaded nanoparticles (DSPE-se-se-PEG@AC NPs) loaded with the anti-inflammatory / antioxidant atorvastatin calcium are prepared to achieve rapid release of AC under high ROS conditions. The amphiphilic triblock nanoshell ensures high encapsulation efficiency of the hydrophobic AC drug, while the diselement bonds ensure ROS-responsive release of AC. To further enhance the angiogenesis and nerve repair functions of the hydrogel, NGF was introduced during its preparation. Carboxymethyl chitosan, a positively charged polymer, served as the hydrogel matrix. Due to its binding affinity, it was an ideal material for transporting negatively charged NGF. This direct loading method ensured maximum retention of NGF, ultimately achieving sustained release rather than burst release.

[0028] 2. A multifunctional hydrogel containing DSPE-se-se-PEG@AC NPs and NGF alleviates cerebral ischemia-reperfusion injury after oGD by scavenging free radicals, rescuing mitochondrial membrane potential and function, inhibiting neuronal apoptosis, and alleviating neuroinflammation. In an ischemic stroke model, the hydrogel modulus matches that of soft tissue and can be injected in situ within the stroke cavity. Furthermore, the multifunctional hydrogel effectively modulates the inflammatory response, rescues apoptotic neurons, promotes angiogenesis, and significantly reduces cerebral infarction. More importantly, in long-term observation, this hydrogel significantly improved neurological deficits following cerebral ischemia-reperfusion injury in rats. Both in vitro and in vivo experimental results confirm that the in-situ hydrogel delivery platform combined with DSPE-se-se-PEG@AC NPs and NGF can alleviate cerebral ischemia-reperfusion injury through multiple pathways, providing an effective strategy for the clinical treatment of ischemic stroke.

[0029] 3. This invention constructs a biocompatible in-situ hydrogel delivery platform for the co-delivery of DSPE-se-se-PEG@AC NPs and NGF, reshaping the pathological microenvironment of the brain and thus alleviating ischemic brain injury. Cellular experiments demonstrate that the multifunctional hydrogel reverses the adverse consequences induced by OGD, such as increased ROS levels, decreased mitochondrial membrane potential, insufficient ATP supply, neuronal apoptosis, and upregulation of inflammatory mediators. In a rat model of ischemic stroke, the multifunctional hydrogel reduces the infarct area by regulating microglia, rescuing apoptotic neurons, and promoting angiogenesis. Furthermore, mNSS, rotarod, cylinder, and MWM experiments demonstrate that, in long-term observation, the hydrogel significantly improves neurological deficits following ischemic brain injury in rats. Attached Figure Description

[0030] Figure 1 TEM images of dssp nps and dssp@ac nps in 1mM H2O2;

[0031] Figure 2 UV-vis images for dssp nps and dssp@ac nps;

[0032] Figure 3 Particle size distribution diagrams for dssp nps and dssp@ac nps;

[0033] Figure 4 The ζ potentials of dssp nps and dssp@ac nps;

[0034] Figure 5 This is a schematic diagram of the sol-gel transition.

[0035] Figure 6 Images show the injectability and self-healing properties of the hydrogel;

[0036] Figure 7 SEM images of the lyophilized hydrogel;

[0037] Figure 8 This is the in vitro AC release curve;

[0038] Figure 9 This is the in vitro NGF release curve;

[0039] Figure 10 The in vitro degradation curve is shown.

[0040] Figure 11 This is a schematic diagram of the frequency scanning results of the hydrogel;

[0041] Figure 12 This is a schematic diagram of the amplitude scanning results of the hydrogel;

[0042] Figure 13This is the result of the hydrogel's self-healing mechanism;

[0043] Figure 14 These are representative images of cell compatibility and metabolic activity.

[0044] Figure 15 Results of CCK-8 assay in HT22 cells treated with different hydrogels;

[0045] Figure 16 A quantitative fluorescence intensity map of DCFH-DA in HT22 cells;

[0046] Figure 17 The red-green fluorescence ratio of JC-1;

[0047] Figure 18 The relative ATP content in HT22 cells under different treatments;

[0048] Figure 19 The BCL-2 / BAX ratio in HT22 cells;

[0049] Figure 20 ELISA quantitative determination results of IL-6 in BV2 cells under different treatments;

[0050] Figure 21 ELISA quantitative determination results of IL-1β in BV2 cells under different treatments;

[0051] Figure 22 ELISA quantification results of IL-10 in BV2 cells under different treatments;

[0052] Figure 23 A timeline diagram for animal experiments and representative images of hematoxylin and eosin (H&E) staining;

[0053] Figure 24 Representative immunofluorescence staining results for iNOS / Iba1 and Arg-1 / Iba1;

[0054] Figure 25 The results of quantitative analysis of inos-positive microglia;

[0055] Figure 26 The results of quantitative analysis of arg-1 positive microglia;

[0056] Figure 27 Representative immunofluorescence staining results of Glut-1 in the periinfarct cortex at 4 weeks and 6 weeks after hydrogel treatment;

[0057] Figure 28 The results of quantitative analysis of Tunel-positive neurons;

[0058] Figure 29 The results of quantitative analysis of the Glut-1 area;

[0059] Figure 30 The results included the mNSS scores of different groups of rats, the dwell time of rats until they fell from the rotundus device, and the percentage of rats touching their left paws.

[0060] Figure 31 A representative swimming trajectory for spatial learning ability;

[0061] Figure 32 To determine the escape latency of rats searching for hidden platforms on training days;

[0062] Figure 33 A schematic diagram showing the swimming time of the rat in the target quadrant and the number of times it crossed the platform in the target quadrant;

[0063] Figure 34 Enrichment analysis of the KEGG pathway;

[0064] Figure 35 KEGG pathway classification analysis;

[0065] Figure 36 A volcano diagram for regulating genes;

[0066] Figure 37 The table shows the first 20 altered genes and their corresponding Log2FC (log2-fold change) tables.

[0067] Figure 38 A heatmap showing changes in gene expression in pathways of inflammation, apoptosis, and neurological function recovery. Detailed Implementation

[0068] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0069] Carboxymethyl chitosan (CMC), dextran, allyl glycidyl ether (AGE), L-cysteine, and sodium periodate were purchased from Adamas (China). Atorvastatin calcium (AC) was purchased from Macklin (China). β-nerve growth factor (NGF) was purchased from Peprotech (USA). DSPE-se-se-PEG was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (China). ROS assay kit, JC-1 assay kit, and ATP assay kit were purchased from Tianhua Biotechnology (China). DMEM medium, fetal bovine serum (FBS), and penicillin-streptomycin dual antibiotics were purchased from Gibco (USA). Cell counting kit (CellCounting Kit-8, CCK-8) was purchased from Dojindo Molecular Technologies (Japan).

[0070] Example 1

[0071] A multifunctional injectable hydrogel, the preparation method of which includes the following steps:

[0072] (1) Dissolve 5g of carboxymethyl chitosan in 250mL of 5wt% sodium hydroxide solution and stir at 25℃ for 4h. Then add 6g of allyl glycidyl ether and stir at 80℃ for 3d. After the reaction is terminated, neutralize with hydrochloric acid. Finally, after dialysis and freeze-drying, carboxymethyl chitosan modified with allyl glycidyl ether (CMC-AGE) is obtained.

[0073] (2) Dissolve 5g of dextran and 2.5g of sodium periodate in deionized water and stir at 25°C for 6h. Then terminate the reaction with ethylene glycol (1mL). After dialyzing and freeze-drying, oxidized dextran is obtained. Then add 1g of oxidized dextran to 100mL of ethanol and stir. Then add 7.57g of L-cysteine ​​and 2.67g of 2-pyridine-borane and stir for 24h. Finally, wash three times with distilled water and ethanol and dry at 40°C to obtain L-cysteine-modified oxidized dextran (OD-SH).

[0074] (3) 6 mg of phospholipid-diselenyl bond-polyethylene glycol (DSPE-se-se-PEG) and 1.2 mg of atorvastatin calcium (AC) were stirred and dissolved in a mixture of 4 mL chloroform and 1 mL methanol. The mixture was dried in a rotary evaporator at 40 °C for 15 min, then dried in an oven at 40 °C for 20 min. Deionized water was added, and the mixture was ultrasonically bathed at 60 °C for 1 h and then freeze-dried to obtain DSPE-se-se-PEG@AC NPs nanoparticles.

[0075] (4) The carboxymethyl chitosan CMC-AGE (6.67wt%, 0.5mL) modified with allyl glycidyl ether obtained in step (1) and the oxidized dextran OD-SH (10wt%, 0.35mL) modified with L-cysteine ​​obtained in step (2) were dissolved in PBS buffer and mixed to form a hydrogel. Then, 0.2mg of DSPE-se-se-PEG@AC NPs nanoparticles obtained in step (3) and / or 200ng of β-nerve growth factor were added to form a multifunctional injectable hydrogel.

[0076] Experimental Example 1: Material Characterization

[0077] DSPE-se-se-PEG@AC NPs nanoparticles or β-nerve growth factor (NGF) were added to the hydrogels formed by CMC-AGE and OD-SH. The resulting hydrogels were denoted as Gel@AC or Gel@NGF, respectively. The hydrogels with both DSPE-se-se-PEG@AC NPs nanoparticles and β-nerve growth factor (NGF) were denoted as Gel@AC&NGF, while the blank hydrogel was denoted as Gel.

[0078] (1) The morphology and size of blank NPs and AC-loaded NPs in deionized water and 1 mM H2O2 were observed using TEM, respectively. Figure 1 As shown, its UV-vis image, particle size distribution, and zeta potential were simultaneously acquired, as shown in the figures below. Figure 2-4 As shown. Among them, Figure 2 In the middle, from top to bottom, they are ac, dssp@ac NPs, and dssp NPs; Figure 3 In the middle, the highest peaks from top to bottom are dsspNPs, dssp@ac NPs, dssp NPs-H2O2 and dssp@ac NPs-H2O2; Figure 4 In the middle, from left to right, they are dssp NPs, dssp NPs-H2O2, dssp@ac NPs, and dssp@ac NPs-H2O2.

[0079] Depend on Figure 1 It can be seen that the diameter of AC-loaded NPs is slightly larger than that of blank NPs. Both blank NPs and DSPE-se-se-PEG@ACNPs can disintegrate in 1mM H2O2, which indicates that nanoparticles are sensitive to ROS due to diselenide bonds.

[0080] Depend on Figure 2-4The UV-vis results show that AC was introduced into the DSPE-se-se-PEG NPs with a loading efficiency of 28.5%. The intensity distribution results indicate that the AC-loaded NPs (220.0 nm) have a larger diameter than the blank NPs (122.0 nm), which may be attributed to the hydrophobic drug-loading space within the DSPE segment. Both the blank NPs and the AC-loaded NPs exhibit dispersed particle size distributions due to structural disintegration in 1 mM H₂O₂, consistent with the TEM images. Furthermore, the zeta potential of the blank NPs is -8.9 mV, while that of the AC-loaded NPs is -13.4 mV, which changes to -15.0 mV in 1 mM H₂O₂.

[0081] (2) Use the inverted vial method to present photographs of the hydrogel sol-gel transition, such as... Figure 5 As shown; images of the injectability and self-healing properties of the hydrogel and SEM images of the lyophilized hydrogel are shown in the figures below. Figure 6-7 As shown.

[0082] Depend on Figure 5-7 It can be seen that by reconnecting the two hydrogels, the self-healing of the hydrogels was observed, and the hydrogels healed well. By observing the internal fracture structure of the freeze-dried hydrogels by SEM, the structural information of the four groups of hydrogels is similar, and the pore size is close to 150μm.

[0083] (3) To determine the release efficiency of AC and NGF from the hydrogel under physiological and pathological ROS conditions, in vitro drug release experiments were conducted. 1 mL of Gel@AC&NGF (1 mL) was placed in a dialysis bag and immersed in 5 mL of PBS 7.4 and 5 mL of PBS 7.4 and 1 mM H2O2 solution, respectively (n = 3). At 0, 2, 4, 6, 8, 20, 24, 48, 72, and 168 h, 2 mL of dialysis solution was taken and the amount of released AC was detected using UV-vis. 0.2 mL of dialysis solution was taken and the amount of released NGF was detected using an ELISA kit, with 0.2 mL of fresh PBS used as a substitute. The results are as follows: Figure 8-9 As shown. Among them, Figure 8-9 In the diagram, the lines above all represent PBS + 1mM H2O2.

[0084] Depend on Figure 8-9It was observed that in PBS 7.4, the proportions of AC released by Gel@AC & NGF at 24h and 48h were approximately 34.9% and 44.4%, respectively, while in 1mM H2O2, the proportions of AC released at 24h and 48h exceeded 56.4% and 75.9%, respectively. The variation in AC release efficiency between PBS 7.4 and 1mM H2O2 environments is attributed to the ROS-responsive diselenyl bonds, ensuring rapid AC release to reconstruct the early inflammatory microenvironment after stroke. NGF exhibited sustained release, accumulating approximately 55% release within 7 days. This release kinetics may be attributed to the slow diffusion and degradation of the porous hydrogel, which ensures the continued angiogenesis and neurorepair functions of NGF in nerve repair.

[0085] (4) In vitro degradation experiment: The lyophilized hydrogel was weighed as W0 and placed in artificial cerebrospinal fluid at 37℃. The hydrogel was collected and lyophilized at predetermined time points, and the weighted average was W0. t Degradation rate (%) = (W0 - W) t ) / W0*100%; the result is as follows Figure 10 As shown in the figure. Rheological tests were performed on the hydrogel, and the results of frequency scan, amplitude scan, and self-healing experiments are as follows. Figure 11-13 As shown.

[0086] Depend on Figure 10 It can be seen that the degradation rate of the hydrogel was approximately 28.8% and 43.4% on the 7th and 13th days, respectively.

[0087] Depend on Figure 11-13 The frequency scan results show that within the frequency range of 0.1-10 rad / s, the storage modulus (G') is consistently greater than the loss modulus (G”), indicating the gel-forming state of the hydrogel at the test frequency. The hydrogel's modulus is 0.2-0.5 kPa, which matches the modulus of the rat cerebral cortex (0.03-1.75 kPa), thus preventing compression of the brain parenchyma. Amplitude scan results show that G' and G” are constant under small strains, but when the strain exceeds 100%, the G' and G” curves intersect, indicating that 100% strain is the critical point between the gel and solution states. Cyclic application of 1% and 300% strains to the hydrogel shows that the hydrogel undergoes a transition between gel and sol states under different strain conditions; switching the strain from 300% to 1% causes the solution to self-heal into a hydrogel, exhibiting excellent self-healing properties.

[0088] In summary, the multifunctional injectable hydrogel of the present invention has good drug release efficiency and modulus matching that of rat cerebral cortex, enabling in situ intracavitary injection in stroke patients.

[0089] Experiment 2: Mitochondrial protection, anti-apoptosis, and anti-inflammatory effects.

[0090] An OGD model was established to simulate ischemic injury: mouse hippocampal neurons (HT22), mouse microglia (BV2), and human umbilical vein endothelial cells (HUVECs) were purchased from Procell Life Sciences, Inc. Cells were routinely cultured in complete medium (DMEM high glucose, 10% fetal bovine serum, 1% penicillin / streptomycin). To establish the OGD model, the complete medium was replaced with d-glucose-free DMEM, and the cells were exposed for 2 h at 37°C, 5% CO2, 94% N2, and 1% O2; then the medium was replaced with complete medium, and the cells were placed in the normal environment. Control group cells were cultured as usual.

[0091] After co-culturing with hydrogel for 48 h, the cell compatibility and metabolic activity of HT22 cells were evaluated using CCK-8 and Live / Dead staining methods. Figure 14 As shown. Among them, Figure 14 In the image, Figure A shows representative images of HT22 cells stained with FDA / PI (live / dead) under different hydrogel treatments, scale bar = 50 μm; Figure B shows representative images of HT22 cells stained with DCFH-DA to detect ROS levels under different treatments, scale bar = 100 μm; Figure C shows HT22 cells stained with JC-1 observed under a confocal microscope, scale bar = 20 μm.

[0092] Simultaneously, CCK-8 levels in HT22 cells treated with different hydrogels were measured. The fluorescence density of DCFH-DA in HT22 cells, the red-green fluorescence ratio of JC-1 (n=3), the relative ATP content in HT22 cells under different treatments (n=3), and the BCL-2 / BAX ratio in HT22 cells (n=3) were quantitatively analyzed using a microplate reader. The results are as follows: Figure 15-19 As shown. And the ELISA quantitative determination of IL-6, IL-1β, and IL-10 in BV2 cells under different treatments (n=3), as shown below. Figure 20-22 As shown. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0093] Depend on Figure 14-22 The results of CCK-8 assays on HT22 cells treated with different hydrogels showed that the number of HT22 cells increased after co-culturing with Gel@AC, Gel@NGF, and Gel@AC&NGF for 48 hours, indicating that the multifunctional hydrogel has good cell compatibility and proliferation properties. Figure 14(Figure A). Compared with the control group, the viability of HT22 cells decreased to 57.4% after OGD. Notably, the control hydrogel did not affect cell viability. After co-culturing with Gel@AC, Gel@NGF, and Gel@AC&NGF for 48 h, cell viability reached 65.2%, 72.9%, and 74.8%, respectively, indicating that the hydrogel has good cell compatibility and can improve the metabolic activity of HT22 cells after OGD. Figure 15 Representative images of DCFH-DA staining show ( Figure 14 (Figure B) The green fluorescence intensity increased after OGD treatment, while the fluorescence intensity decreased after Gel@AC and Gel@AC&NGF treatment, indicating that the hydrogel can eliminate excess ROS induced by OGD in HT22 cells; this superoxide scavenging result may be due to AC increasing the activity of antioxidant enzymes. Quantitative analysis showed ( Figure 16 Treatment of HT22 cells and HUVECs with Gel@AC and Gel@AC&NGF significantly reduced DCFH-DA density, demonstrating the hydrogel's ability to scavenge intracellular ROS. The JC-1 probe is widely used to detect MMPs. At high MMP levels, JC-1 aggregates in the mitochondrial matrix, forming red aggregates; at low MMP levels, JC-1 monomers (green) appear. The red-to-green ratio indicates the proportion of mitochondrial depolarization and the degree of apoptosis. In the control group, HT22 cells showed strong red fluorescence and weak green fluorescence. After OGD, red fluorescence weakened while green fluorescence increased, indicating a decrease in MMPs and early apoptosis after OGD. Treatment of HT22 cells with Gel@AC, Gel@NGF, and Gel@AC&NGF for 48 hours increased MMPs, indicating that MMPs and apoptosis were rescued after treatment with the multifunctional hydrogel. Figure 14 Figure C in the middle Figure 17 Notably, co-culturing with Gel@AC&NGF increased ATP levels, suggesting that the recovery of mitochondrial function may depend on a combination of anti-inflammatory, antioxidant, and neurotrophic effects. Figure 18 Compared with the OGD group, the Bcl-2 / Bax ratio was significantly increased in the Gel@AC&NGF group, indicating that Gel@AC&NGF has strong anti-apoptotic properties. Figure 19 Pro-inflammatory mediators are upregulated in microglia after OGD. Figure 20-22 The results showed that the levels of pro-inflammatory factors IL-1β and IL-6 in BV2 cells increased after OGD treatment, while Gel@AC, Gel@NGF and Gel@AC&NGF downregulated the levels of IL-1β and IL-6, indicating that the hydrogel has a strong anti-inflammatory ability. In addition, the anti-inflammatory factor IL-10 in BV2 cells was upregulated after Gel@AC&NGF treatment, further demonstrating the inflammatory regulatory properties of Gel@AC&NGF.

[0094] Experimental Example 3: Hydrogel can alleviate the inflammatory response after cerebral ischemia-reperfusion injury in rats.

[0095] A photothrombotic (PT) stroke model was established. Five days after the PT model was established, different hydrogels or saline were injected into a stereotaxic apparatus. Rats were randomly divided into 6 groups: sham-operated group, PT stroke saline group, PT stroke gel group, PT stroke Gel@AC group, PT stroke Gel@NGF group, and PT stroke Gel@AC&NGF group; these groups are abbreviated as Sham, PT, Gel, Gel@AC, Gel@NGF, and Gel@AC&NGF, respectively. Timeline diagrams of the animal experiments and representative images of hematoxylin and eosin (H&E) staining are shown below. Figure 23 As shown. Representative immunofluorescence staining results for iNOS / Iba1 and Arg-1 / Iba1 are as follows. Figure 24 As shown. Simultaneously, quantitative analysis of inos-positive microglia and arg-1-positive microglia was performed, as shown below. Figure 25-26 As shown.

[0096] in, Figure 23 In the diagram, Figure A shows the timeline of the animal experiment, and Figure B shows a representative image of hematoxylin and eosin (H&E) staining. Figure 24 The top image shows representative immunofluorescence staining results for iNOS / Iba1, and the bottom image shows representative immunofluorescence staining results for Arg-1 / Iba1. The scale bar is 50 μm. **** indicates p < 0.0001.

[0097] Depend on Figure 23 It was found that the PT group showed significant cerebral infarction compared to the sham surgery group. Both Gel@AC and Gel@NGF treatments reduced the size of cerebral infarctions to some extent, with the most significant reduction observed in the Gel@AC&NGF group. This may be related to the combined effects of anti-inflammatory, antioxidant, angiogenic, and neuroprotective properties. Furthermore, representative images of other organs showed no pathological damage, indicating good biocompatibility of the hydrogel in vivo.

[0098] Depend on Figure 24-26 It was found that, compared with other treatment groups, the Gel@AC&N GF group showed more M2 microglia and fewer M1 microglia, indicating that Gel@AC&N GF modulates microglia polarization after stroke to suppress inflammation.

[0099] Experiment Example 4: Hydrogels reduce neuronal apoptosis and promote angiogenesis after cerebral ischemia in rats.

[0100] Tunel / Neun immunofluorescence staining observation 4 thChanges in apoptotic neurons within the week, and representative immunofluorescence staining results of Tunel / Neun at 4 weeks and 6 weeks after hydrogel treatment in the periinfarct cortex, as shown in... Figure 27 As shown; then, apoptotic cells were labeled with TUNEL, neurons with NEUN, cell nuclei with DAPI, and microvessels with Glut-1. Quantitative analysis results of TUNEL-positive neurons and Glut-1 area are shown below. Figures 28-29 As shown. Among them, Figure 27 The top image shows representative immunofluorescence staining results of Tunel / Neun 4 weeks after hydrogel treatment, and the bottom image shows representative immunofluorescence staining results of Glut-1 in the peri-infarct cortex 6 weeks after hydrogel treatment; **** indicates p<0.0001.

[0101] Depend on Figure 27-29 It was found that the PT group showed more Tunel-positive neurons compared to the sham-operated group. Treatment with gel and Gel@NGF slightly reduced the number of Tunel-positive neurons; however, treatment with Gel@AC and Gel@AC&NGF significantly reduced the number of Tunel-positive neurons, attributed to the anti-inflammatory and antioxidant capabilities of AC. Compared to the PT group, the Gel@NGF and Gel@AC&NGF groups showed a significant increase in microvessels, while other groups did not show significant microvascular function, indicating that the NGF-loaded hydrogel has a significant angiogenic effect. These results demonstrate that Gel@AC&NGF has satisfactory anti-apoptotic and pro-angiogenic effects in vivo.

[0102] Experimental Example 5: Neurological Function Recovery

[0103] The neurological recovery of stroke-affected rats was further assessed using a double-blind method, employing the mNSS, rotating bar test, cylinder test, and Morris water maze (MWM). Neurobehavioral function was dynamically monitored from preoperatively to 8 weeks post-injection. mNSS scores (n=3) were recorded every 2 weeks for different groups of rats; the time rats spent in the rotating bar test until falling from the device (n=3); and the percentage of rats touching the cylinder test with their left paw (n=3). The results are as follows: Figure 30 As shown.

[0104] in, Figure 30 In the figure, A represents the mNSS score, B represents the time the rat remained in the rat until it fell from the rotundus device, and C represents the percentage of time the rat touched the device with its left paw.

[0105] Depend on Figure 30 It can be seen that the rats in the Gel@AC&NGF group had the lowest mNSS score, the longest rotation time in the rotator, and the best recovery of nerve function. The percentage of left paw contact was increased after Gel@AC&NGF treatment.

[0106] The spatial learning and memory abilities of rats after ischemia were evaluated using the MWM method, and representative traces in spatial learning tests were identified, such as... Figure 31 As shown; the escape latency of rats searching for hidden platforms on training days, such as Figure 32 As shown; subsequently, the underwater platform was removed, and the rats' memory abilities were assessed, including swimming time in the target quadrant and the number of times they crossed the platform in the target quadrant, as shown. Figure 33 As shown. Among them, Figure 33 In the diagram, the left graph represents the swimming time spent in the target quadrant, and the right graph represents the number of times the target quadrant was crossed. ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0107] Depend on Figures 31-33 It was found that, compared with the PT group, the average escape latency of the Gel@AC&NGF group was significantly shorter, indicating a substantial improvement in learning ability. Among all treatment groups, the Gel@AC&NGF group had the longest swimming time in the target quadrant and the highest frequency of crossing platform positions, indicating a significant recovery in memory ability.

[0108] In summary, all neurobehavioral tests indicate that Gel@AC & NGF significantly improve neurological function caused by ischemic injury, and that treatment with Gel@AC or Gel@NGF also aids in recovery to some extent.

[0109] Experiment 6: Transcriptome Sequencing

[0110] like Figure 34 As shown, enrichment pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes database. Among these pathways, inflammation-related pathways included natural killer cell-mediated cytotoxicity, B cell receptor signaling, nf-κB signaling, and chemokine signaling. Apoptosis-related pathways included PI3K-Akt signaling, apoptosis, and phagosomes. Endothelial cell-related pathways included gap junctions, adhesion junctions, Rap1 signaling, VEGF signaling, and ecm receptor interactions. Neurofunctional pathways included neuroactive ligand-receptor interactions, neurotrophic factor signaling pathways, dopaminergic synapses, and the apelin pathway. Figure 35 As shown, in the KEGG classification, pathways rich in differentially expressed genes (deg) are divided into cellular processes, environmental information processing, genetic information processing, metabolism, and body systems, indicating that Gel@AC&NGF plays multiple roles in the treatment of ischemic injury.

[0111] The volcano diagram and the top 20 regulated genes are as follows: Figures 36-37 As shown. And heatmaps of gene expression changes in the inflammation, apoptosis, and neurological function recovery pathways (n=3), as shown. Figure 38 As shown. Figure 38In the middle, from left to right, are inflammation, apoptosis, and recovery of nerve function.

[0112] Depend on Figures 36-37 As shown in the volcano plot, compared with the PT group, 807 genes in the Gel@AC&NGF group were statistically significant, including 326 downregulated genes and 481 upregulated genes. These genes are related to cell migration, proliferation, differentiation, apoptosis, lipid metabolism, and protein metabolism. The heatmap results of regulatory genes from the Gel@AC&NGF and PT groups indicate that the mechanism of action of multifunctional hydrogels in vivo mainly includes anti-inflammatory, anti-apoptotic, and neurological function recovery.

[0113] KEGG analysis confirmed that Gel@AC&NGF significantly upregulated anti-inflammatory genes, including CXCL13, IL10ra, and IL18. Compared with the PT group, Gel@AC&NGF significantly upregulated the expression of Bcl-2a1 and Avp genes in the PI3K / Akt anti-apoptotic pathway, and downregulated the expression of bok gene, which is associated with mitochondrial apoptosis.

[0114] In cerebral ischemia-reperfusion injury, the apelin pathway plays a neuroprotective role against apoptosis by protecting neuronal survival. Upregulation of genes related to the apelin pathway, such as Aplenr and Adcy7, may be involved in neural repair and functional recovery. Gel@AC&NGF upregulates Ngfr, Bdnf, and Tp73 in the neurotrophic factor signaling pathway. In this pathway, released NGF is recognized by cell receptors such as Trk and p75NTR, further activating the downstream PI3K / akt cell cycle pathway, promoting cell proliferation and neural development. Gel@AC&NGF significantly upregulates the expression of Adora2a, Aplenr, Drd2, and Tac1 genes in neuroactive ligand-receptor interactions, confirming their involvement in learning and memory functions.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a multifunctional injectable hydrogel, characterized in that, Includes the following steps: (1) Dissolve carboxymethyl chitosan in an aqueous solution containing sodium hydroxide and stir at 20-30℃ for 3-5 hours. Then add allyl glycidyl ether and stir at 70-90℃ for 2-4 days. After the reaction is terminated, neutralize with hydrochloric acid and finally dialyze and freeze dry to obtain carboxymethyl chitosan modified with allyl glycidyl ether. (2) Dissolve dextran and sodium periodate in deionized water and stir at 20-30℃ for 5-7h. Then terminate the reaction with ethylene glycol. After dialyzing and freeze drying, oxidized dextran is obtained. Then add oxidized dextran to ethanol and stir. L-cysteine ​​and 2-pyridine-borane are added successively and stirred for 24h. Finally, wash three times with distilled water and ethanol respectively and dry at 30-50℃ to obtain L-cysteine ​​modified oxidized dextran. (3) Phospholipid-diselenyl bond-polyethylene glycol and atorvastatin calcium were stirred and dissolved in a mixture of chloroform and methanol. The mixture was concentrated in a rotary evaporator at 30-50℃ for 10-20 min, then dried in an oven at 30-50℃ for 10-30 min. Deionized water was added, and the mixture was ultrasonically bathed at 50-70℃ for 1 h and then freeze-dried to obtain DSPE-se-se-PEG@ACNPs nanoparticles. (4) The carboxymethyl chitosan modified with allyl glycidyl ether obtained in step (1) and the oxidized dextran modified with L-cysteine ​​obtained in step (2) are dissolved in PBS buffer, mixed to form a hydrogel, and then DSPE-se-se-PEG@AC NPs nanoparticles and β-nerve growth factor obtained in step (3) are added to form a multifunctional injectable hydrogel.

2. The method for preparing the multifunctional injectable hydrogel as described in claim 1, characterized in that, In step (1), the mass-volume ratio of carboxymethyl chitosan, allyl glycidyl ether and sodium hydroxide aqueous solution is 4-6g:5-7g:250mL.

3. The method for preparing the multifunctional injectable hydrogel as described in claim 1, characterized in that, In step (2), the mass ratio of dextran to sodium periodate is 5:2-3.

4. The method for preparing the multifunctional injectable hydrogel as described in claim 1, characterized in that, In step (2), the mass-volume ratio of oxidized dextran, L-cysteine, 2-pyridine-borane and ethanol is 1g:7-8g:2-3g:100mL.

5. The method for preparing the multifunctional injectable hydrogel as described in claim 1, characterized in that, In step (3), the mass-volume ratio of phospholipid-diselenyl bond-polyethylene glycol, atorvastatin calcium, chloroform and methanol is 5-7 mg: 1-1.4 mg: 4 mL: 1 mL.

6. The method for preparing the multifunctional injectable hydrogel as described in claim 1, characterized in that, In step (4), the allyl glycidyl ether modified carboxymethyl chitosan and the L-cysteine ​​modified oxidized dextran were dissolved in PBS buffer at concentrations of 6.67 wt% and 10 wt%, respectively, with a volume ratio of 0.5:0.

35.

7. The method for preparing the multifunctional injectable hydrogel as described in claim 1, characterized in that, In step (4), the mass ratio of DSPE-se-se-PEG@AC NPs nanoparticles to β-nerve growth factor is 0.1-0.3 mg: 200 ng.

8. The multifunctional hydrogel prepared by the method of any one of claims 1-7.

9. The application of the multifunctional injectable hydrogel of claim 8 in biomaterials for remodeling the repair microenvironment of cerebral infarction areas.

10. A biomaterial capable of reshaping the repair microenvironment of a cerebral infarction region, characterized in that, Including the multifunctional injectable hydrogel as described in claim 8.

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