Inflammation-responsive biomimetic hydrogel and preparation method and use thereof

Abstract

The application provides an inflammation response type biomimetic hydrogel as well as a preparation method and application thereof, and belongs to the biomedical field. The inflammation response type biomimetic hydrogel is prepared from crosslinked polymers, hyaluronic acid, platelet lysate and nanoparticles in an aqueous solution. The inflammation response type biomimetic hydrogel can be used as a scaffold to remodel a pathological environment, convert an infarction site into a state promoting regeneration, promote vascular regeneration, regulate the survival and differentiation of neural stem cells, and promote endogenous regeneration repair, and has a good application prospect in the treatment of ischemic stroke.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular 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 the blood oxygen supply to localized brain tissue is interrupted, resulting in cerebral ischemic damage. Currently, reperfusion therapy, such as intravenous thrombolysis and thrombectomy, remains the best management approach for the hyperacute phase of IS, but only 10% of patients benefit from it due to its narrow 6-hour treatment window. In most patients, IS progresses to a chronic stage, with a range of cellular and molecular consequences, including mitochondrial dysfunction, accumulation of reactive oxygen species (ROS), metabolic disorders, inflammatory responses, and ultimately brain cell death.

[0003] In normal brain tissue, endogenous antioxidant enzymes can easily eliminate ROS. However, at the site of cerebral ischemic injury, these enzymes have difficulty clearing excessive ROS, leading to 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 breakdown and inflammatory response. In addition, re-establishing blood supply is an important protective mechanism that promotes neural regeneration and functional recovery in 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 biomimetic hydrogel and a preparation method and use thereof.

[0005] The invention provides a biomimetic hydrogel, which is prepared in an aqueous solution using a 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 a first time to obtain plasma, the plasma is subjected to a freeze-thaw cycle and a second centrifugation to obtain platelet lysate;

[0010] The nanoparticles are prepared by combining nonionic surfactant and polyunsaturated fatty acid through a thin film hydration method.

[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.; and 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 the 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 above-mentioned biomimetic 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 them uniformly to obtain the biomimetic hydrogel.

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

[0016] Furthermore, 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 inflammation-responsive biomimetic hydrogel provided by the present invention acts as a scaffold to reshape the pathological environment, transforming the infarct site into a pro-regenerative state, regulating the survival and differentiation of neural stem cells, and promoting endogenous regenerative repair processes. It also endows the infarct site with the ability to restore blood supply. DHA-loaded glyceryl monostearate nanoparticles can be enzymatically cleaved by matrix metalloproteinases overexpressed in the infarct site, thereby improving the pathological microenvironment of the infarct site. The inflammation-responsive biomimetic hydrogel scaffold provided by the present invention is biodegradable and has long-term retention at the infarct site, can enhance neurogenesis, has a strong neuroprotective effect, significantly promotes angiogenesis, improves motor function and neurological deficits, and can effectively repair nerves and treat ischemic stroke.

[0020] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0021] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within 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 panel) and PDI (right panel) of TD and TD after incubation with MMP-9.

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

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

[0031] Figure 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] Figure 11 HT22 cell viability (left) and HUVEC cell viability (right) after OGD and 48 hours after hydrogel treatment. The four hydrogel groups are designated as follows: 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, and **** indicates p < 0.0001.

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

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

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

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

[0037] Figure 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 and are obtained by purchasing commercially available products.

[0039] Hyaluronic acid (HA) and triglyceride monostearate (TGMS) were purchased from MacLean (China); Epoxide-polyethylene glycol 500-epoxide (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 Synthesis of -B polymer

[0043] 2 g of 3-aminophenylboronic acid was dissolved in 20 mL of tetrahydrofuran at 25 °C, and then 4 mL of epoxy compound-polyethylene glycol 500-epoxy compound (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] Rabbit whole blood was centrifuged at 1500 rpm for 15 minutes to obtain platelet-rich plasma (PRP). The PRP was subjected to three freeze-thaw cycles (-80°C / 30°C, 10 minutes each) to disrupt the platelet membranes. The platelet lysate (PLS) was then centrifuged at 2000 g for 10 minutes to remove cell debris. Finally, the platelet lysate was freeze-dried for later use.

[0046] 3. Preparation of Composite Nanoparticles

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

[0048] 4. Preparation of Composite Bionic Hydrogel

[0049] Phosphate buffered saline (PBS) was used as the solvent to prepare HA solutions containing 25 mg / mL HA, 100 mg / mL B-PEG, and 100 mg / mL PEG. 500 -B 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, and 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 responsive to inflammation

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

[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, to prepare an inflammation-responsive composite biomimetic hydrogel scaffold.

[0054] Example 4. Preparation of composite biomimetic hydrogel scaffolds responsive to inflammation

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

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

[0057] The method of Example 1 was referred to, except that in step 4, the concentration of the PLS solution was modified to 50 mg / mL to prepare an inflammation-responsive composite biomimetic 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 biomimetic hydrogel scaffold.

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

[0061] The method of Example 1 was referred to, except that in step 4, the concentration of the PLS solution was modified to 200 mg / mL to prepare an inflammation-responsive composite biomimetic 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 inflammation-responsive composite biomimetic 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, and rapidly stirred to form HA hydrogel encapsulating TD nanoparticles (abbreviated as gel@TD).

[0069] Control 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 (referred to as pls gel).

[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 inflammatory response composite biomimetic hydrogel scaffold

[0073] 1. Experimental methods

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

[0075] (1) Through nuclear magnetic resonance ( 1 H NMR) and FTIR spectroscopy confirmed the presence of 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 elemental distribution of the freeze-dried hydrogels were observed by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) mapping. The modulus of the hydrogels was measured using a rheometer (MCR302, Anton Paar GmbH, AT). The frequency sweep was performed with an oscillation frequency of 1 to 100 rad / s and a fixed strain of 1%. The strain sweep was performed with a strain range of 0.1% to 1000% and a fixed oscillation frequency of 1 rad / s.

[0078] (4) Drug release from the hydrogel was detected by UV-visible spectroscopy. 1 mL of hydrogel was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa. The 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 collected and the released drug was detected using UV-visible spectroscopy 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. The absorbance of DPPH and ABTS was then detected using UV-vis at the set time points.

[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 (1347 cm-1) further verified the cross-linking agent B-PEG 500 -B synthesis ( Figure 2 ).like Figure 3 As shown, HA-pls and B-PEG were mixed 500 -B solution immediately formed pls gel, showing a uniform porous structure with a pore size of about 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 dependence of gel and pls gel@TD within the test frequency range. An elastic modulus of approximately 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, with a PDI of 0.84, indicating that the size distribution changed 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, as they have similar molecular weights. 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 decreased 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. Figure 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 Biomimetic 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% nitrogen2 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. Figure 10 It can be seen that the composite biomimetic 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 biomimetic 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 scaffold on the viability of HUVECs were determined by CCK-8. Figure 10It can be seen that the composite biomimetic hydrogel scaffold prepared in Example 1, Examples 5-8 can improve the viability of HUVECs cells, and with the increase of the amount of pls, the viability improvement effect appears to increase first and then decrease, among which the composite biomimetic hydrogel scaffold prepared in Example 1 has the best improvement effect, and unexpected technical effects are achieved.

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

[0097] Experimental Example 3, in vitro performance test of composite biomimetic hydrogel scaffold

[0098] 1. Experimental method

[0099] The composite biomimetic hydrogel scaffold used in this experiment is prepared in Example 1, Comparative Examples 1-3.

[0100] 2. Experimental results

[0101] (1) In vitro cell compatibility test

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

[0103] OGD model was established to simulate ischemic injury in vitro. After OGD, the HT22 cell viability decreased to 59.6%, and it is worth noting that pls gel@TD improved the cell viability to 67.0% after 48 hours of treatment, indicating that the hydrogel has good cell compatibility and improves the cell viability after OGD( Figure 11 ). In addition, the hydrogel has similar proliferation-promoting properties in HUVECs, and CCK-8 results show that the cell viability of damaged cells after OGD is 59.5%, and after 48 hours of hydrogel treatment, the cell viability reaches 64.3%, 63.7%, 66.6% and 71.6% respectively, indicating that pls gel@TD can significantly improve the cell viability after OGD( Figure 11 ).

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

[0105] Brain ischemic injury is accompanied by activation of inflammatory pathways and release of pro-inflammatory cytokines such as IL-1β and TNF-α, which can trigger severe brain inflammation. Quantitative analysis shows that the levels of IL-1β and TNF-α increase after OGD model, while the pls gel@TD hydrogel significantly reduces the levels of inflammatory cytokines, indicating its potential anti-inflammatory properties( Figure 12 ).

[0106] In summary, the results show that the composite biomimetic 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 biomimetic hydrogel scaffold

[0108] 1. Experimental methods

[0109] All animal experiments complied 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 well-known experimental model for simulating ischemic stroke. In this experiment, focal cortical ischemia was induced by the cortical photothrombosis stroke model. First, the 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 with 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 area.

[0110] 2. Experimental results

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

[0112] The specific operation of the PT stroke model was as described in our previous report. Lipid peroxidation occurs when free oxygen radicals oxidize the 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 a 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 decrease in 4-HNE and Caspase-3 fluorescence, while the pls gel@TD group showed the most significant decrease in these oxidative stress and apoptosis markers, indicating a strong neuroprotective effect ( Figure 13Double 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 ( Figure 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 was significantly increased, indicating the active proliferation of NSCs ( Figure 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 ( Figure 16 These results indicate that the pls-gel@TD group had a significant effect on the proliferation of NSCs and progenitor cells, which may be due to 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 into 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 biomimetic hydrogel, characterized in that: The biomimetic hydrogel is prepared in an aqueous solution using cross-linked polymers, hyaluronic acid, platelet lysate, and nanoparticles as raw materials; 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 nanoparticles are prepared by using a thin film hydration method to combine a nonionic surfactant and a polyunsaturated fatty acid; The nonionic surfactant is triglyceride monostearate; The polyunsaturated fatty acid is docosahexaenoic acid.

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 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.

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.; and 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 for reaction, and drying to obtain nanoparticles; 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 hours; 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 uniformly 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 is a hydrogel scaffold that promotes 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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