Inflammation response type bionic hydrogel as well as preparation method and application thereof

Abstract

The invention provides inflammation response type bionic hydrogel as well as a preparation method and application thereof, and belongs to the field of biomedicine. The inflammation response type bionic hydrogel is prepared by taking a cross-linked polymer, hyaluronic acid, platelet lysate and nanoparticles as raw materials in an aqueous solution. The inflammation response type bionic hydrogel can be used as a stent for remodeling a pathological environment, converting an infarct part into a regeneration promoting state and promoting vascular regeneration so as to regulate and control survival and differentiation of neural stem cells and promote endogenous regeneration and repair, and has a good application prospect in treatment of ischemic stroke.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an inflammation-responsive bionic hydrogel and a preparation method and application thereof. Background Art

[0002] Ischemic stroke (IS) is a leading cause of death and disability worldwide. IS occurs when blood oxygen supply to local brain tissue is interrupted, resulting in cerebral ischemic damage. Currently, reperfusion therapy, such as intravenous thrombolysis and thrombectomy, remains the best management method for the hyperacute phase of IS, but only 10% of patients can benefit from it due to its narrow treatment time window of 6 hours. For most patients, IS progresses to a chronic stage, bringing about a series of cellular and molecular consequences, including mitochondrial dysfunction, reactive oxygen species (ROS) accumulation, metabolic disorders, inflammatory responses, and ultimately brain cell death.

[0003] In normal brain tissue, endogenous antioxidant enzymes are able to easily eliminate ROS. However, at the site of cerebral ischemic injury, these enzymes have difficulty in clearing excessive ROS, resulting in persistent oxidative stress, mitochondrial dysfunction, and neuronal apoptosis. In addition, matrix metalloproteinases (MMPs) are activated due to the immune response after ischemic injury. This activation degrades the extracellular matrix (ECM) and destroys the tight junctions between endothelial cells, leading to blood-brain barrier disruption and inflammatory response. In addition, re-establishing blood supply is an important protective mechanism to promote neural regeneration and functional recovery during the pathophysiological process of stroke. Although the adult brain may repair itself through neurogenesis after stroke, this self-repair ability is limited by the harsh neuropathological microenvironment. The immune response after ischemic injury clears necrotic debris, leaving behind a stroke cavity without ECM and cells, which has an adverse effect on neural repair and regeneration. Summary of the invention

[0004] The purpose of the present invention is to provide an inflammation-responsive bionic hydrogel and a preparation method and use thereof.

[0005] The invention provides a bionic hydrogel, which is prepared in an aqueous solution using cross-linked polymer, hyaluronic acid, platelet lysate and nanoparticles as raw materials.

[0006] Furthermore, the mass ratio of the cross-linked polymer, hyaluronic acid, platelet lysate and nanoparticles is 1:1-3:1-3:0.01-0.05.

[0007] Furthermore, the mass ratio of the cross-linked polymer, hyaluronic acid, platelet lysate and nanoparticles is 1:2:1:0.03.

[0008] Furthermore, the cross-linked polymer is prepared according to the following method: 3-aminophenylboronic acid and epoxy compound-polyethylene glycol-epoxy compound are reacted in an organic solvent, and the cross-linked polymer is obtained by purification;

[0009] The platelet lysate is prepared according to the following method: whole blood is centrifuged for the first time to obtain plasma, and the plasma is subjected to freeze-thaw cycles and a second centrifugation to obtain platelet lysate;

[0010] The nanoparticles are prepared by using a thin film hydration method to combine a nonionic surfactant and polyunsaturated fatty acids.

[0011] Furthermore, in the method for preparing a cross-linked polymer, the epoxy compound-polyethylene glycol-epoxy compound is epoxy compound-polyethylene glycol 500-epoxy compound; the organic solvent is tetrahydrofuran; the reaction temperature is 40° C.-80° C.; the reaction time is 36-72 hours;

[0012] The thin film hydration method comprises the following steps: mixing a nonionic surfactant and a polyunsaturated fatty acid in an organic solvent, concentrating, adding water to react, and drying to obtain nanoparticles; the nonionic surfactant is monostearate triglyceride; the polyunsaturated fatty acid is docosahexaenoic acid; the organic solvent is chloroform; and the reaction time is 0.5-2 hours. Furthermore, in the method for preparing a cross-linked polymer, the reaction temperature is 60°C; and the reaction time is 48 hours;

[0013] In the thin film hydration method, the reaction time is 1 hour.

[0014] The present invention also provides a method for preparing the bionic hydrogel, which comprises the following steps: mixing hyaluronic acid, platelet lysate and nanoparticles in an aqueous solution, and finally adding a cross-linked polymer and mixing evenly to obtain the bionic hydrogel.

[0015] The present invention also provides a use of the bionic hydrogel in preparing a hydrogel scaffold for treating ischemic stroke.

[0016] Further, the hydrogel scaffold promotes nerve repair.

[0017] Furthermore, the hydrogel scaffold is a hydrogel scaffold that promotes the formation of new blood vessels.

[0018] The present invention has achieved the following beneficial effects:

[0019] The inflammatory response bionic hydrogel provided by the present invention is used as a scaffold to reshape the pathological environment, convert the infarct site to a regeneration-promoting state, regulate the survival and differentiation of neural stem cells, and promote the endogenous regeneration and repair process; the infarct site is endowed with the ability to restore blood supply; the monostearate glyceryl nanoparticles loaded with docosahexaenoic acid can be enzymatically cleaved by matrix metalloproteinases overexpressed in the cerebral infarction site, thereby improving the pathological microenvironment of the infarction site. The inflammatory response bionic hydrogel scaffold provided by the present invention has biodegradability and long-term retention at the infarction site, can enhance neurogenesis, has a strong neuroprotective effect, significantly promotes angiogenesis, improves motor function and nerve defects, can effectively repair nerves, and treat ischemic stroke.

[0020] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0021] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .Schematic diagram of hydrogel preparation and inflammation-responsive release.

[0023] Figure 2 .EP-PEG 500 -EP, B and B-PEG 500 -B's NMR mass spectrum (left) and IR spectrum (right).

[0024] Figure 3 .Sol-gel transition image, SEM image and EDS element mapping of pls gel.

[0025] Figure 4 Frequency sweep test (left) and strain sweep test (right) of hydrogel.

[0026] Figure 5 .Self-healing scanning test (left) and viscosity test (right) of hydrogel.

[0027] Figure 6 .TEM image of TD (left) and TEM image of TD after incubation with MMP-9 (right).

[0028] Figure 7 Size distribution intensity (left) and PDI (right) of TD and TD after incubation with MMP-9.

[0029] Figure 8 .Drug release profiles of hydrogels under different environments.

[0030] Fig. 9 .Scavenging efficiency of hydrogel on DPPH and ABTS free radicals.

[0031] Fig.10 .The effect of different addition amounts of TGMS@DHA NPs in hydrogel on HT22 cell viability (left figure) and the effect of different concentrations of pls in hydrogel on HUVECs cell viability.

[0032] Fig.11 .HT22 cell viability (left) and HUVECs cell viability (right) after OGD and 48 hours after hydrogel treatment; the names of the four groups of hydrogels are: gel = HA hydrogel; gel@TD = HA hydrogel encapsulating TD nanoparticles; plsgel = HA and pls composite hydrogel; pls gel@TD = HA and pls composite hydrogel encapsulating TD nanoparticles. * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001.

[0033] Fig.12 .ELISA quantitative analysis of TNF-α and IL-1β secretion by microglia after OGD and 48 h of hydrogel treatment.

[0034] Fig.13 .Semi-quantitative analysis results of 4-HNE and Caspase-3 immunofluorescence staining in the infarct area of ​​mice.

[0035] Fig.14 .Semi-quantitative analysis results of CD31 / VEGF immunofluorescence staining in the infarct area of ​​mice.

[0036] Fig.15 . Representative immunofluorescence staining of Nestin / Ki67 and DCX / Ki67 in mice. LV refers to lateral ventricle, and SVZ refers to subventricular zone.

[0037] Fig.16 .Semi-quantitative analysis results of mouse Nestin / Ki67 and DCX / Ki67 immunofluorescence staining. DETAILED DESCRIPTION

[0038] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0039] Hyaluronic acid (HA) and triglyceride monostearate (TGMS) were purchased from MacLean (China); Epoxy-polyethylene glycol 500-epoxy (EP-PEG 500-EP) and docosahexaenoic acid (DHA) were purchased from Aladdin; 3-aminophenylboronic acid (BA) was purchased from Adamas (China).

[0040] All experimental protocols involving animals complied with the institutional animal care standards of Sichuan University.

[0041] Example 1. Preparation of composite biomimetic hydrogel scaffolds for inflammatory response

[0042] 1. B-PEG 500 -B Synthesis of polymer

[0043] 2 g of 3-aminophenylboronic acid was dissolved in 20 mL of tetrahydrofuran at 25 °C, and then 4 mL of epoxide-polyethylene glycol 500-epoxide (EP-PEG 500 -EP), stirred at 60°C for 48 hours. After the reaction, the product was purified by rotary evaporation and recrystallization from anhydrous ether to obtain B-PEG 500 -B polymer.

[0044] 2. Preparation of Platelet Lysate

[0045] Whole blood from rabbits was centrifuged at 1500 rpm for 15 minutes to obtain platelet-rich plasma (PRP). PRP was subjected to three freeze-thaw cycles (-80°C / 30°C, 10 minutes each) to destroy the platelet membrane, and then centrifuged at 2000g for 10 minutes to remove cell debris to obtain platelet lysate (abbreviated as PLS). Finally, it was freeze-dried for later use.

[0046] 3. Preparation of Composite Nanoparticles

[0047] Nanoparticles were prepared by thin film hydration method: 10 mg of monostearate triglyceride (TGMS) and 4 μL of docosahexaenoic acid (DHA) were dissolved in 3 mL of chloroform, the solution was treated by rotary evaporation at 35 ° C for 10 minutes, and then 10 mL of deionized water was added to react in a 200W ultrasonic water bath at 35 ° C for 1 hour. After filtering through a 0.45 μM syringe filter (Millipore, USA), composite nanoparticles of monostearate triglyceride and docosahexaenoic acid (abbreviated as TD nanoparticles) were obtained and freeze-dried for use.

[0048] 4. Preparation of Composite Biomimetic Hydrogel

[0049] Phosphate buffered saline (PBS) was used as the solvent to prepare HA solutions containing 25 mg / mL HA and 100 mg / mL B-PEG. 500 -B-PEG 500-B solution, pls solution containing 100 mg / mL pls, 300 μg of freeze-dried TD nanoparticles. 100 μL of pls solution and 800 μL of HA solution were mixed and stirred, 300 μg of freeze-dried TD nanoparticles were added, and then 100 μL of B-PEG was added. 500 -B solution was rapidly stirred to form a HA and pls composite hydrogel encapsulating TD nanoparticles (referred to as pls gel@TD).

[0050] Example 2: Preparation of composite biomimetic hydrogel scaffolds for inflammatory response

[0051] The method of Example 1 was referred to, except that in step 4, 300 μg of TD nanoparticles was changed to 100 μg, and an inflammatory responsive composite biomimetic hydrogel scaffold was prepared.

[0052] Example 3. Preparation of composite biomimetic hydrogel scaffolds for inflammatory response

[0053] The method of Example 1 was referred to, except that in step 4, 300 μg of TD nanoparticles was changed to 200 μg, and an inflammatory responsive composite biomimetic hydrogel scaffold was prepared.

[0054] Example 4. Preparation of composite biomimetic hydrogel scaffolds for inflammatory response

[0055] The method of Example 1 was referred to, except that in step 4, 300 μg of TD nanoparticles was changed to 400 μg, and an inflammatory responsive composite biomimetic hydrogel scaffold was prepared.

[0056] Example 5. Preparation of composite biomimetic hydrogel scaffolds for inflammatory response

[0057] Referring to the method of Example 1, the only difference is that in step 4, the concentration of the pls solution is modified to 50 mg / mL to prepare an inflammatory responsive composite bionic hydrogel scaffold.

[0058] Example 6. Preparation of composite biomimetic hydrogel scaffolds for inflammatory response

[0059] The method of Example 1 was referred to, except that in step 4, the concentration of the pls solution was modified to 150 mg / mL to prepare an inflammatory responsive composite bionic hydrogel scaffold.

[0060] Example 7. Preparation of composite biomimetic hydrogel scaffolds for inflammatory response

[0061] Referring to the method of Example 1, the only difference is that in step 4, the concentration of the pls solution is modified to 200 mg / mL to prepare an inflammatory responsive composite bionic hydrogel scaffold.

[0062] Example 8. Preparation of composite biomimetic hydrogel scaffolds for inflammatory response

[0063] The method of Example 1 was referred to, except that in step 4, the concentration of the pls solution was modified to 250 mg / mL to prepare an inflammatory responsive composite bionic hydrogel scaffold.

[0064] The following is the preparation method of the control sample.

[0065] Comparative Example 1: Preparation of HA hydrogel

[0066] Phosphate buffered saline (PBS) was used as the solvent to prepare 25 mg / mL hyaluronic acid (HA) solution and 100 mg / mL B-PEG solution. 500 -B solution. Add 100 μL of B-PEG to 900 μL of HA solution. 500 -B solution, and rapidly stirred to form HA hydrogel (abbreviated as gel).

[0067] Comparative Example 2: Preparation of HA hydrogel encapsulating TD nanoparticles

[0068] Phosphate buffered saline (PBS) was used as the solvent to prepare 25 mg / mL hyaluronic acid (HA) solution, 100 mg / mL B-PEG solution and 100 mg / mL PEG solution. 500 -B solution. Add 300 μg of freeze-dried TD nanoparticles to 900 μL of HA solution, and then add 100 μL of B-PEG 500 -B solution, which was rapidly stirred to form HA hydrogel encapsulating TD nanoparticles (abbreviated as gel@TD).

[0069] Comparative Example 3: Preparation of HA and PLS composite hydrogel

[0070] Phosphate buffered saline (PBS) was used as the solvent to prepare 25 mg / mL hyaluronic acid (HA) solution and 100 mg / mL B-PEG solution. 500 -B solution. Mix 100 μL of pls solution and 800 μL of HA solution and stir, then add 100 μL of B-PEG 500 -B solution, and rapidly stirred to form a HA and pls composite hydrogel (pls gel for short).

[0071] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0072] Experimental Example 1: Characterization of the structure and antioxidant capacity of the composite bionic hydrogel scaffold with inflammatory response

[0073] 1. Experimental methods

[0074] The inflammatory response composite bionic hydrogel scaffold used in this experiment was prepared in Example 1.

[0075] (1) Through nuclear magnetic resonance ( 1 H NMR) and infrared spectroscopy (FTIR) confirmed that B-PEG 500 -Synthesis of B.

[0076] (2) The morphology and size of TD nanoparticles were measured by transmission electron microscopy (TEM) and dynamic light scattering (DLS).

[0077] (3) The fracture structure and element distribution of the freeze-dried hydrogel were observed by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) mapping. The modulus of the hydrogel was measured by a rheometer (MCR302, Anton Paar GmbH, AT). The oscillation frequency of the frequency scan was 1 to 100 rad / s, and the fixed strain was 1%. The strain scan had a strain range of 0.1% to 1000%, and the fixed oscillation frequency was 1 rad / s.

[0078] (4) Detecting drug release from hydrogels by UV-visible light. 1 mL of hydrogel was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa. The dialysis bag was immersed in 5 mL of PBS and PBS containing 10 mM H2O2 and 100 ng / mL MMP-9. The mixture was incubated on a shaker. 1 mL of dialysate was taken and the released drug was detected using UV-visible light (UV-vis) at specific time points.

[0079] (5) The in vitro antioxidant capacity of the hydrogel was verified by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-nitrobis-(3-ethylbenzothiazole-6-sulfonate) (ABTS) free radical scavenging experiments. DPPH and ABTS were dissolved in ethanol to prepare DPPH working solution (2.0 mL) and ABTS working solution (2.0 mL), respectively. 100 μL of pls gel@TD was incubated with DPPH working solution and ABTS working solution, respectively. Then, the absorbance of DPPH and ABTS was detected using UV-vis at the set time point.

[0080] 2. Experimental results

[0081] Preparation and responsive chemical formula of composite biomimetic hydrogels Figure 1 shown.

[0082] B-PEG 500 The benzene ring of -B (7.4ppm) confirmed that 3-aminophenylboronic acid was present in EP-PEG 500 -EP grafting. FTIR results confirmed that B-PEG 500-B's BO stretching vibration peak (1347cm-1) further verifies the crosslinking agent B-PEG 500 -B Synthesis ( Figure 2 ).like Figure 3 As shown, HA-pls and B-PEG were mixed 500 -B solution was immediately formed into pls gel, showing a uniform porous structure with a pore size of approximately 100 μm in the SEM image. EDS element mapping detected C, N, O and B elements in the hydrogel, further verifying the successful formation of pls gel.

[0083] Subsequently, frequency sweep tests revealed the stability and frequency-dependent effects of gel and pls gel@TD within the test frequency range. An elastic modulus of about 270 Pa was obtained by strain sweep, which is consistent with the modulus of the cerebral cortex. In addition, 381% strain is the critical point from the gel state to the quasi-liquid state, which means that the hydrogel has undergone a transition from the gel state to the quasi-liquid state ( Figure 4 ).

[0084] Self-healing experiments performed alternately at 1% and 700% strains showed that the storage modulus (G′) of the hydrogel suddenly dropped at 700% strain, and the loss modulus (G″) exceeded G′, and then G′ and G″ immediately recovered at 1% strain, indicating that the hydrogel has self-healing properties. The viscosity of the hydrogel continued to decrease with increasing shear rate, proving the shear thinning property ( Figure 5 ).

[0085] TD in PBS showed a uniform spherical structure. After incubation with MMP-9, the structure of TD was destroyed due to the enzyme responsiveness of TGMS ( Figure 6 ).

[0086] Dynamic light scattering (DLS) results showed that the average hydrodynamic diameter of TD nanoparticles was 115.7 nm. In addition, the aggregation index (PDI) of the nanoparticles was 0.179, indicating a good size distribution. After incubation with MMP-9, the size distribution intensity changed, and the PDI was 0.84, indicating a change in size distribution after incubation with MMP-9 ( Figure 7 ).

[0087] Since DHA has no significant UV absorption peak, curcumin was selected to simulate DHA in the drug release test, and their molecular weights are similar. The results showed that 85.4% of the drug was released in PBS containing 10mM H2O2 and 100ng / mL MMP-9, which was significantly higher than 47.1% in PBS, indicating that DHA is rapidly released in the inflammatory microenvironment ( Figure 8 ).

[0088] The in vitro free radical scavenging ability of pls gel@TD was verified by ultraviolet-visible spectroscopy (UV-vis). The UV-vis spectrum showed that the absorption peaks of DPPH and ABTS free radicals dropped sharply after incubation with pls gel@TD. The free radical scavenging activity was calculated. The DPPH and ABTS scavenging activities increased with the extension of incubation time, reaching 61.7% and 88.5% respectively at 25 minutes. Fig. 9 ).

[0089] The above experiments prove that the present invention successfully prepares pls gel@TD, and pls gel@TD has self-healing properties, can quickly release DHA in the inflammatory microenvironment, and has excellent in vitro free radical scavenging ability.

[0090] Experimental Example 2: Screening of the ratio of composite bionic hydrogel scaffolds for inflammatory response

[0091] 1. Experimental methods

[0092] Hippocampal neuron (HT22) cells, microglia (BV2) cells, and human umbilical vein endothelial (HUVECs) cell lines were purchased from Procell Life Science & Technology Co., Ltd. Cells were routinely cultured in complete medium (high glucose DMEM, 10% FBS, and 1% penicillin / streptomycin). In order to establish an OGD model, the entire culture medium was replaced with glucose-free DMEM and exposed to a container (37°C, 5% CO2, 94% nitrogen 2, and 1% O2) for 2 hours. The culture medium was then replaced with complete culture medium, and the cells were placed in a conventional environment. Control cells were cultured using conventional procedures. CCK-8 was used to assess 48 hours of cell compatibility and metabolic activity. Inflammatory cytokines secreted by BV2 cells were detected using ELISA kits.

[0093] 2. Experimental results

[0094] The effects of different TD NPs addition amounts on the viability of HT22 cells were determined by CCK-8. Fig.10 It can be seen that the composite bionic hydrogel scaffolds prepared in Examples 1-4 of the present invention can effectively improve the viability of hippocampal neuronal cells, and with the increase in the amount of TD NPs added, the viability improvement effect shows a trend of first increasing and then decreasing. Among them, the composite bionic hydrogel scaffold obtained in Example 1 has the best improvement effect, achieving unexpected technical effects.

[0095] The effects of different amounts of PLS ​​added to the hydrogel scaffolds on the viability of HUVECs were determined by CCK-8. Fig.10It can be seen that the composite bionic hydrogel scaffolds prepared in Example 1 and Examples 5-8 can improve the viability of HUVECs cells, and with the increase in the amount of pls added, the viability improvement effect shows a trend of first increasing and then decreasing. Among them, the composite bionic hydrogel scaffold obtained in Example 1 has the best improvement effect, achieving unexpected technical effects.

[0096] In summary, the composite bionic hydrogel scaffold prepared under the conditions of Example 1 has the best effect on improving the viability of hippocampal neuron cells and HUVECs cells, and achieved unexpected technical effects.

[0097] Experimental Example 3: In vitro performance test of composite bionic hydrogel scaffold

[0098] 1. Experimental methods

[0099] The composite bionic hydrogel scaffold used in this experiment was prepared by Example 1 and Control Examples 1-3.

[0100] 2. Experimental results

[0101] (1) In vitro cell compatibility test

[0102] The HT22 cell line was selected to evaluate the cytocompatibility of the hydrogel treatment.

[0103] The OGD model was established to simulate ischemic injury in vitro. After OGD, the viability of HT22 cells decreased to 59.6%. Notably, pls gel@TD increased the cell viability to 67.0% after 48 h of treatment, indicating that the hydrogel has good cell compatibility and improves the cell viability after OGD ( Fig.11 In addition, the hydrogel has similar pro-proliferation properties in HUVECs. The CCK-8 results showed that the cell viability damaged after OGD was 59.5%. After 48 hours of hydrogel treatment, the cell viability reached 64.3%, 63.7%, 66.6% and 71.6%, respectively, indicating that pls gel@TD can significantly improve the cell viability after OGD ( Fig.11 ).

[0104] (2) In vitro anti-inflammatory performance test

[0105] Cerebral ischemic injury is accompanied by the activation of inflammatory pathways and the release of proinflammatory cytokines such as IL-1β and TNF-α, which can trigger severe brain neuroinflammatory responses. Quantitative analysis showed that the levels of IL-1β and TNF-α increased after the OGD model, while the plsgel@TD hydrogel significantly reduced the levels of inflammatory cytokines, indicating its potential anti-inflammatory properties ( Fig.12 ).

[0106] In summary, the results show that the composite bionic hydrogel scaffold of the present invention has good cell compatibility, can significantly reduce the level of pro-inflammatory cytokines, and has excellent anti-inflammatory effects.

[0107] Experimental Example 4: In vivo performance test of composite bionic hydrogel scaffold

[0108] 1. Experimental methods

[0109] All animal experiments were in accordance with the institutional animal care standards of Sichuan University. Mice were divided into control group, stroke group, gel group, gel@TD group, pls gel group, and pls gel@TD (prepared in Example 1) group (n=7). The photochemical cerebral thrombosis (PT) animal model is a known experimental model for simulating ischemic stroke. In this experiment, focal cortical ischemia was induced using a cortical photothrombosis stroke model. First, mice were anesthetized with isoflurane and injected intraperitoneally with Rose Bengal (1%, 10 μL / g body weight). After 15 minutes, the mice were fixed in a stereotaxic apparatus, and a 3 mm bone window was made 2 mm lateral to the anterior fontanelle using a cranial drill. Subsequently, the motor cortex area was illuminated through the bone window by a cold light illuminator (100 mW) for 8 minutes. After illumination, the incision was sutured. A 25 μL syringe loaded with hydrogel was injected into the infarct cavity at a rate of 1 μL / min. During the recovery period, the health status of the mice was monitored daily. Immunofluorescence staining was performed to evaluate the expression of related proteins (Nestin / ki67, DCX / ki67, 4-HNE, Caspase-3, and CD31 / VEGF) in the infarct site.

[0110] 2. Experimental results

[0111] (1) Antioxidant, anti-inflammatory, and angiogenesis in vivo

[0112] The specific operation of the PT stroke model was as described in our previous report. Lipid peroxidation occurs when free oxygen radicals oxidize polyunsaturated fatty acids of phospholipids, producing toxic phospholipid byproducts such as 4-hydroxynonenal (4-HNE). 4-HNE staining was used to assess oxidative stress, and Caspase-3 was used as an early indicator of apoptosis. The least 4-HNE and Caspase-3 fluorescence was observed in the control group, while the significant increase in these two markers in the stroke group indicated enhanced oxidative stress and apoptosis in the infarct area. The hydrogel-treated group showed a significant reduction in 4-HNE and Caspase-3 fluorescence, while the pls gel@TD group showed the most significant reduction in these oxidative stress and apoptosis markers, indicating a strong neuroprotective effect ( Fig.13). Double staining of CD31 and VEGF as markers of angiogenesis showed a significant increase in these markers in the pls gel and pls gel@TD groups, indicating significant angiogenesis in vivo, thus providing a favorable regenerative environment for nerve repair ( Fig.14 ). This favorable angiogenesis may be due to the physical support of the hydrogel for endothelial cell growth and the release of potential pro-angiogenic factors.

[0113] (2) Neurogenesis in vivo

[0114] Representative immunofluorescence (IF) staining of Nestin (NSCs marker) and Ki67 (proliferation marker) showed that the number of double-positive cells in the subventricular zone (SVZ) of the plsgel@TD group increased significantly, indicating the active proliferation of NSCs ( Fig.15 ). Similarly, the number of DCX (neural progenitor cell marker) and Ki67 double-positive cells in the pls gel@TD group increased significantly, indicating enhanced neural progenitor cell and neurogenesis ( Fig.16 ). These results suggest that the pls-gel@TD group had a significant effect on the proliferation of NSCs and progenitor cells, which may benefit from the optimized microenvironment and increased blood supply to the infarct-related area.

[0115] The above results indicate that pls gel@TD can significantly improve the microenvironment at the infarct site, promote angiogenesis, enhance neurogenesis, have a strong neuroprotective effect, and can effectively treat ischemic stroke.

[0116] In summary, the present invention provides an inflammation-responsive bionic hydrogel and its preparation method and use. The inflammation-responsive bionic hydrogel is prepared in an aqueous solution using cross-linked polymers, hyaluronic acid, platelet lysate, and nanoparticles as raw materials. The inflammation-responsive bionic hydrogel can be used as a scaffold to reshape the pathological environment, convert the infarct site to a regeneration-promoting state, regulate the survival and differentiation of neural stem cells, promote endogenous regeneration and repair, rescue mitochondrial dysfunction, reduce neuronal apoptosis, and promote neovascularization, and has good application prospects in the treatment of ischemic stroke.

Claims

1. A bionic hydrogel, characterized in that: The bionic hydrogel is prepared in an aqueous solution using cross-linked polymer, hyaluronic acid, platelet lysate and nanoparticles as raw materials.

2. The biomimetic hydrogel according to claim 1, characterized in that: The mass ratio of the cross-linked polymer, hyaluronic acid, platelet lysate and nanoparticles is 1:1-3:1-3:0.01-0.

05.

3. The biomimetic hydrogel according to claim 2, characterized in that: The mass ratio of the cross-linked polymer, hyaluronic acid, platelet lysate and nanoparticles is 1:2:1:0.

03.

4. The biomimetic hydrogel according to claim 1, characterized in that: The cross-linked polymer is prepared according to the following method: 3-aminophenylboronic acid and epoxy compound-polyethylene glycol-epoxy compound are reacted in an organic solvent, and the cross-linked polymer is obtained by purification; The platelet lysate is prepared according to the following method: whole blood is centrifuged for the first time to obtain plasma, and the plasma is subjected to freeze-thaw cycles and a second centrifugation to obtain platelet lysate; The nanoparticles are prepared by using a thin film hydration method to combine a nonionic surfactant with polyunsaturated fatty acids.

5. The biomimetic hydrogel according to claim 4, characterized in that: In the method for preparing a cross-linked polymer, the epoxy compound-polyethylene glycol-epoxy compound is epoxy compound-polyethylene glycol 500-epoxy compound; the organic solvent is tetrahydrofuran; the reaction temperature is 40° C.-80° C.; the reaction time is 36-72 hours; The thin film hydration method comprises the following steps: mixing a nonionic surfactant and a polyunsaturated fatty acid in an organic solvent, concentrating, adding water to react, and drying to obtain nanoparticles; the nonionic surfactant is monostearate triglyceride; the polyunsaturated fatty acid is docosahexaenoic acid; the organic solvent is chloroform; and the reaction time is 0.5-2 hours.

6. The biomimetic hydrogel according to claim 5, characterized in that: In the method for preparing a cross-linked polymer, the reaction temperature is 60° C. and the reaction time is 48 h. In the thin film hydration method, the reaction time is 1 hour.

7. A method for preparing the biomimetic hydrogel according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: mixing hyaluronic acid and platelet lysate in an aqueous solution, then adding nanoparticles, and finally adding a cross-linked polymer, and mixing them evenly to obtain the product.

8. Use of the biomimetic hydrogel according to any one of claims 1 to 6 in preparing a hydrogel scaffold for treating ischemic stroke.

9. The use according to claim 8, characterized in that The hydrogel scaffold promotes the hydrogel scaffold of nerve repair.

10. The use according to claim 8, characterized in that The hydrogel scaffold is a hydrogel scaffold that promotes the formation of new blood vessels.

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