Low-temperature injectable hydrogel as well as preparation method and application thereof

Abstract

The invention discloses low-temperature injectable hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. Hydrogel is prepared from high-molecular-weight hyaluronic acid grafted with vitamin E and poly (N-isopropylacrylamide), the hydrogel is in an injectable liquid state at 4 DEG C, and a drug capsaicin (CPZ) wrapped by micelle is loaded and injected into the brain from the brain in a stereotactic mode. The pharmaceutical composition can temporarily maintain local low temperature of brain tissue and release capsaicin, reduce local metabolic activity of ischemic brain tissue, temporarily inhibit biogenesis, reduce NADH, FADH2 and other reducing equivalents and ADP accumulation, reduce the possibility of electron leakage in an electron transport chain, and improve the bioavailability of ischemic brain tissue. Vitamin E is added to reduce superoxide anions (O2 <.->) generated by reduction reaction of oxygen and leaked electrons in the reperfusion process, and vitamin E can promote ROS removal, synergistically alleviate oxidative stress and subsequent inflammation, and comprehensively improve cerebral ischemia reperfusion injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a method for preparing and applying a low-temperature injectable hydrogel for reducing reperfusion injury in ischemic stroke. Background Technology

[0002] Ischemic stroke (IS) is mainly caused by cerebral artery blockage due to thrombosis, accounting for approximately 65.3% (62.4-67.7%) of stroke cases. IS causes 11.6% of deaths worldwide, making it the second leading cause of death and disability globally. Currently, thrombolysis and neuroprotection are two important interventions for IS. However, the time window for thrombolytic therapy is narrow. Even if blood supply is fully restored, reperfusion can still trigger a series of pathological reactions, including a surge in the production of oxygen free radicals, inducing oxidative stress-induced tissue damage, and activation of inflammatory pathways. This means that most stroke patients do not benefit from thrombolysis and may also be accompanied by related complications. More than 60% of survivors will still have physical disabilities.

[0003] In ischemia-reperfusion injury (ISI), all cell types within the ischemic area, including neurons, microglia, peripheral cells, and endothelial cells, are affected. Among these cells, neurons have the highest energy demand and the most abundant and vulnerable mitochondria; their death is the most significant factor contributing to IS-related mortality and disability. During ischemia, to provide energy to neurons, the brain compensatorily accumulates large amounts of metabolites, including lactate, NADH, FADH2, and ADP. During reperfusion, the sudden influx of oxygen exacerbates the mitochondrial reactive oxygen species (mtROS) burst caused by electron leakage in the mitochondrial electron transport chain during oxygen recovery. mtROS directly damages the mitochondrial membrane, triggering cytochrome C-induced intrinsic neuronal apoptosis and inflammatory cascade-mediated extrinsic apoptosis, representing a crucial pathway for neuronal injury.

[0004] TRPV1 is a member of the TRPV (Vanilloid) subfamily within the TRP channel family. It is a non-selective cation channel permeable to sodium, calcium, and other substances. TRPV1 channels are typically located on the cell membrane as tetramers; they are also expressed on the mitochondrial membrane. In the nervous system, TRPV1 mediates the body's response to endogenous and exogenous chemical stimuli and physical stimuli such as temperature by regulating cellular calcium signaling or cell membrane depolarization, leading to the transmission of nociceptive signals to the central nervous system and the generation of pain. Early studies have shown that TRPV1 activation increases calcium signaling in muscle cells and the expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), promoting fatty acid oxidation and mitochondrial energy metabolism, and promoting mitochondrial biosynthesis. However, under ischemic conditions, impaired oxidative phosphorylation and increased metabolic activity lead to the accumulation of metabolites such as lactate, NADH, FADH2, and ADP. Impaired function of the oxidative respiratory electron transport chain results in electron leakage, and the influx of oxygen during blood flow restoration leads to a large amount of superoxide anions (O2·g·m3). ﹣ The generation of ).

[0005] TRPV1, a non-selective cation channel, can be activated by various noxious stimuli (such as thermal stimuli, chemical ligands including vanillin compounds, and protons) and plays a key regulatory role in various pathological pain processes. Although TRPV1 antagonists have been shown to have the potential to become novel analgesics, none of these drugs have yet been successfully approved for clinical use. A significant factor hindering their clinical translation is that many TRPV1 antagonists acting on the vanillin-binding domain can cause significant changes in core body temperature (CBT), some inducing hyperthermia, while others anomalously induce hypothermia similar to that caused by TRPV1 agonists (such as capsaicin and resin toxins). Currently, the specific mechanism by which TRPV1 ligands regulate CBT is not fully understood, but several hypotheses exist. One view suggests that TRPV1 expressed in vascular endothelium or smooth muscle can participate in CBT regulation by modulating peripheral vascular tone (contraction or diastole), affecting the body's heat dissipation process. However, a recent study showed that specifically eliminating TRPV1 in peripheral sensory neurons (while preserving TRPV1 in vascular smooth muscle cells) completely blocked the CBT changes induced by TRPV1 ligands. This result indicates that the regulatory effect of TRPV1 ligands on CBT mainly depends on TRPV1 channels expressed in peripheral sensory neurons.

[0006] Currently, delivery strategies such as physical targeting to promote intracerebral delivery, electrostatic adsorption-mediated transcytosis, biomimetic targeting, and receptor / ligand-mediated targeting of intracerebral drugs are gradually being used in the treatment of stroke. Eliminating existing reactive oxygen species (ROS) and dysfunctional mitochondria to reduce the continued production of ROS has also become an important neuroprotective strategy. However, the pathophysiological mechanisms after stroke are complex and involve numerous targets. Current research methods are still limited by problems such as low BBB passage rate, low cell selectivity, high cytotoxicity, non-specific binding of endogenous ligands, and difficulties in large-scale production. Hydrogels, as an excellent drug carrier, have attracted widespread attention due to their good biocompatibility and controllable release characteristics. Injectable hydrogels can be implanted into lesions through minimally invasive methods to achieve local sustained drug release, increase target drug concentration, and reduce systemic side effects. Low-temperature injectable hydrogels possess unique "sol-gel" transformation characteristics, maintaining fluidity before injection and gelling in situ after injection, perfectly fitting the soft, closed, and precise drug delivery environment of brain tissue.

[0007] For example, patent CN119524209A discloses a silk fibroin-hyaluronic acid thermosensitive hydrogel scaffold for traumatic brain injury repair. This scaffold, loaded with bone marrow mesenchymal stem cells and paeoniflorin, is implanted into the injured site after surgery to promote nerve regeneration. While this technology overcomes some blood-brain barrier limitations, its design relies on surgically implanted pre-formed scaffolds, lacking the ability for in-situ intervention via minimally invasive injection in the acute phase. Furthermore, its effect focuses on tissue repair and regeneration, rather than addressing the root cause of oxidative stress in the early stages of ischemia-reperfusion injury. Another example is patent CN119345119A, which discloses a multifunctional injectable hydrogel. This hydrogel uses gelatin and thiolated hyaluronic acid to construct a dual network, possessing reactive oxygen species (ROS) scavenging capabilities and loading exosomes for the treatment of cerebral hemorrhage. This technology emphasizes the removal of existing ROS and anti-inflammation, but its mechanism of action is "post-incident clearance," failing to intervene at the upstream source of ROS outbreaks in neuronal mitochondria during reperfusion—namely, the electron leakage process in the electron transport chain. In addition, the exosomes loaded on it, as bioactive substances, present challenges such as complex preparation, difficulty in standardization, and relatively weak controllability of drug delivery.

[0008] Therefore, finding a comprehensive treatment method that can directly reduce neuronal damage, rationally control neuroinflammation, and reduce brain tissue damage, thereby improving the prognosis of ischemic brain injury and enhancing the quality of life for patients with cerebral infarction, is a pressing clinical challenge. Summary of the Invention

[0009] This invention addresses the technical problem of the lack of a comprehensive hydrogel drug delivery system in the existing technology that can be minimally invasively intervened in the acute phase and can simultaneously combat ischemia-reperfusion injury from both the "source inhibition" and "process clearance" levels. It proposes a low-temperature injectable hydrogel, its preparation method and application.

[0010] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0011] On one hand, the present invention provides a method for preparing a low-temperature injectable hydrogel, comprising the following steps:

[0012] (1) Dissolve vitamin E succinate in an organic solvent, add an activator to obtain solution I;

[0013] (2) ADH was dissolved in distilled water, and solution I was added dropwise during stirring to carry out the reaction. After extraction and rotary evaporation, ADH-VE was obtained.

[0014] (3) Dissolve HA in distilled water, add an activator and react to obtain solution II;

[0015] (4) After dissolving the ADH-VE prepared in step (2) in an organic solvent, add it to solution II for reaction. After the reaction is completed, HA-VE is obtained by dialysis, centrifugation and drying.

[0016] (5) After dissolving PNIPAM in deionized water, add HA-VE prepared in step (4), stir and react to obtain a low-temperature injectable hydrogel;

[0017] The low temperature condition is 0-4℃.

[0018] The organic solvent mentioned in step (1) is DMF, the activator is HATU, the concentration of vitamin E succinate in solution I is 0.02-0.05 g / mL, and the mass ratio of vitamin E succinate to activator is 10:13;

[0019] In step (2), the mass ratio of ADH to vitamin E succinate is 13:20, and the initial concentration of ADH is 0.12-0.15 g / mL; the reaction is carried out under an argon protective atmosphere.

[0020] The molecular weight of HA in step (3) is 90-100 kDa; the concentration of HA in solution II is 0.01-0.02 g / mL, and the mass ratio of HA to activator is 8:5; the activator is EDC.

[0021] The organic solvent in step (4) is DMF, the initial concentration of ADH-VE is 0.1-0.2 g / mL, and the mass ratio of ADH-VE to HA is 5:3; the reaction is carried out under an argon protective atmosphere, and the drying is freeze drying;

[0022] In step (5), the mass ratio of PNIPAM to HA-VE is 2:1, the initial concentration of PNIPAM is 0.015-0.025 g / mL, and the stirring reaction time is 12-14 h.

[0023] This invention provides a low-temperature injectable hydrogel prepared using the above-described preparation method.

[0024] On the other hand, the present invention provides a method for preparing a low-temperature injectable drug-loaded hydrogel, comprising the following steps:

[0025] (1) Dissolve vitamin E succinate in an organic solvent, add an activator to obtain solution I;

[0026] (2) ADH was dissolved in distilled water, and solution I was added dropwise during stirring to carry out the reaction. After extraction and rotary evaporation, ADH-VE was obtained.

[0027] (3) Dispose of HA separately 3600 and HA 90-100k Dissolved in distilled water, and reacted with an activator, HA is obtained. 3600 Solution and HA 90-100k Solution;

[0028] (4) After dissolving the ADH-VE prepared in step (2) in an organic solvent, add HA respectively. 3600 Solution and HA 90-100k The reaction is carried out in solution, and after completion, HA is obtained by dialysis, centrifugation, and drying. 3600 -VE and HA 90-100k -VE;

[0029] (5) HA 3600 -VE and CPZ are both dissolved in acetone. After dissolution, stirring, sonication, and rotary evaporation, a thin film is formed. PNIPAM is dissolved in deionized water and then re-dissolved to form a film to obtain drug-loaded micelles. HA is then added. 90-100 k -VE, after stirring and reaction, drug-loaded hydrogel is obtained.

[0030] The organic solvent mentioned in step (1) is DMF, the activator is HATU, the concentration of vitamin E succinate in solution I is 0.02-0.05 g / mL, and the mass ratio of vitamin E succinate to activator is 10:13;

[0031] In step (2), the mass ratio of ADH to vitamin E succinate is 13:20, and the initial concentration of ADH is 0.12-0.15 g / mL; the reaction is carried out under an argon protective atmosphere.

[0032] The HA mentioned in step (3) 3600 The mass ratio of HA to activator is 8:5. 3600 In HA 3600 The concentration in the solution is 0.01-0.02 g / mL; the HA 90-100k The mass ratio of HA to activator is 8:5. 90-100k In HA 90-100k The concentration in the solution is 0.01-0.02 g / mL; the activator is EDC;

[0033] The organic solvent in step (4) is DMF, the initial concentration of ADH-VE is 0.1-0.2 g / mL, and the mass ratio of ADH-VE to HA is 5:3; the reaction is carried out under an argon protective atmosphere, and the drying is freeze drying;

[0034] The HA mentioned in step (5) 3600 The mass ratio of VE to CPZ is 100:11; the initial concentration of PNIPAM is 0.015-0.025 g / mL; and the PNIPAM and HA... 90-100 k The mass ratio of -VE is 2:1; the loading of CPZ is 4-5 wt%; and the stirring reaction time is 12-14 h.

[0035] This invention provides a low-temperature injectable drug-loaded hydrogel prepared using the above-described preparation method;

[0036] The low-temperature injectable drug-loaded hydrogel can release capsaicin (CPZ), which can synergistically inhibit the metabolic activity of local cells, thereby reducing the accumulation of reducing equivalents such as lactate, NADH, and FADH2, as well as ADP in the ischemic area. This reduces the possibility of electron leakage in the electron transport chain and decreases the superoxide anion (O2·) produced by the reduction reaction between the large influx of oxygen and the leaked electrons during cerebral ischemia-reperfusion. - );

[0037] The cryogenic injectable hydrogel or cryogenic injectable drug-loaded hydrogel helps to reduce the explosive generation of reactive oxygen species (ROS) during blood reperfusion and promotes the clearance of ROS.

[0038] The aforementioned low-temperature injectable hydrogel or low-temperature injectable drug-loaded hydrogel is a comprehensive treatment method that can directly reduce neuronal damage, rationally control neuroinflammation, reduce brain tissue damage, and improve the prognosis of ischemic brain injury.

[0039] The present invention also provides the application of the aforementioned low-temperature injectable hydrogel or low-temperature injectable drug-loaded hydrogel in the preparation of drugs for treating nerve injury repair, reducing brain tissue damage and ischemic stroke.

[0040] The present invention also provides the use of the aforementioned low-temperature injectable hydrogel or low-temperature injectable drug-loaded hydrogel in the preparation of drugs for alleviating or treating ischemia-reperfusion injury.

[0041] The low-temperature injectable hydrogel or low-temperature injectable drug-loaded hydrogel is administered via local injection at a temperature of 4°C.

[0042] The beneficial effects of this invention are:

[0043] 1. This invention constructs a thermosensitive hydrogel based on high molecular weight hyaluronic acid grafted with vitamin E and poly(N-isopropylacrylamide). This system maintains an injectable sol state at low temperatures (e.g., 4°C) and can be precisely delivered to ischemic lesions via minimally invasive methods such as stereotactic brain localization. After injection, it rapidly transforms into a gel at body temperature, achieving in-situ drug retention and long-term sustained release. The low-temperature injectable hydrogel loaded with CPZ NPs prepared in this invention can transiently lower the temperature of ischemic brain tissue, reduce metabolic activity in ischemic brain tissue, reduce the accumulation of harmful metabolites during ischemia, reduce ROS production, promote ROS elimination, and salvage damaged neurons. It also has repair functions against inflammation, vascular injury, and damaged blood-brain barrier, ultimately achieving functional recovery of brain tissue after ischemic stroke. Directly targeting the core mechanism of reperfusion injury in the acute phase of ischemic stroke, it achieves early intervention through minimally invasive injection, aiming to directly protect vulnerable neurons, reduce oxidative stress and inflammation, create a favorable microenvironment for subsequent recovery, and thus more comprehensively improve neurological prognosis.

[0044] 2. This invention is the first to synergistically combine local hypothermia physical therapy with capsaicin pharmacochemotherapy and vitamin E antioxidant therapy. Local hypothermia itself can reduce the metabolic rate of brain tissue and temporarily inhibit biosynthesis, thereby reducing the abnormal accumulation of reducing equivalents and energy substrates such as NADH, FADH2, and ADP during ischemia, thus reducing the driving force of electron leakage during reperfusion from the source. The loaded capsaicin is encapsulated in micelles, and after continuous release, it can antagonize the neuronal mitochondrial TRPV1, further precisely regulating the mitochondrial metabolic state, inhibiting electron leakage, and reducing superoxide anion (O2·)⁻. ﹣ The generation of [a specific substance] achieves "source inhibition" of ROS; the vitamin E grafted into the hydrogel framework can continuously clear the generated ROS. Thus, a multi-level synergistic intervention of "metabolic regulation-source inhibition-process clearance" is achieved for ischemia-reperfusion injury. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 Performance testing of the low-temperature injectable hydrogel prepared in Example 1 of this invention; wherein, A is the 1H NMR spectrum of the typical peak of HA-VE; B is the particle size distribution diagram of empty micelles and drug-loaded micelles and the TEM image of the drug-loaded micelles taken by transmission electron microscopy; C is the gelation performance and SEM image of the synthesized low-temperature injectable drug-loaded hydrogel; D is the rheological performance test of the drug-loaded hydrogel.

[0047] Figure 2 The following tests were conducted to assess the cooling performance and in vivo degradation of the low-temperature injectable hydrogel prepared in Example 1 of this invention; wherein, A is the test of the hydrogel's ability to reduce local temperature; and B is the test of the hydrogel's degradation in the brain.

[0048] Figure 3 The images show the cerebral infarction volume and short-term behavioral improvement in experimental rats after drug administration; where A is a TTC staining image; B is a statistical graph of cerebral infarction volume; and C is a statistical graph of neurological deficit scores.

[0049] Figure 4 The figures show the changes in neurological function and behavior of experimental rats after drug administration over time. A is a heatmap of the open field test trajectory; B is a statistical chart of the number of times the central region was traversed; C is a statistical chart of the total distance and time traveled in the open field test, as well as the distance and time traveled in the inner edge regions; D is a trajectory diagram of the Y-maze test; E is a statistical chart of the spontaneous alternation rate; F is a trajectory diagram of the Morris water maze; G and H are statistical charts of the distance and time traveled during the latency period of the orientation navigation test; Figure I is a statistical chart of the percentage of distance traveled in the target quadrant; and J is a statistical chart of the percentage of time spent in the target quadrant.

[0050] Figure 5 The study included monitoring of intracranial blood flow in rats from each drug-treated group.

[0051] Figure 6 H&E staining and Nissl staining were performed on the brain tissue of experimental mice in each drug-treated group.

[0052] Figure 7The study aimed to detect relevant metabolites in the brains of experimental rats in each drug-treated group. Specifically, A showed the detection of lactate content, NADH / NAD+ ratio, and ATP / ADP ratio in the ischemic brain tissue 2 h after ischemia-reperfusion treatment with low-temperature hydrogel; B showed the detection of lactate content, NADH / NAD+ ratio, and ATP / ADP ratio in the ischemic brain tissue 24 h after ischemia-reperfusion treatment with low-temperature hydrogel.

[0053] Figure 8 The morphology of mitochondria in the brain tissue of rats in each drug-treated group.

[0054] Figure 9 The images show the detection of oxidative stress and inflammation in the brains of experimental rats in each drug-treated group. A represents the detection of MDA content; B represents the detection of CAT activity; C represents the immunohistochemical images of inflammatory factors IL-6, TNF-α, and IL-10 in the ischemic brain tissue; and D represents the statistical graph of the immunohistochemical images of inflammatory factors IL-6, TNF-α, and IL-10.

[0055] Figure 10 The images show the detection of apoptosis in the brains of rats in each drug-treated group. A shows the immunohistochemical images of endogenous and extrinsic apoptotic proteins Caspase 9 and Caspase 8 in the ischemic brain tissue; B shows the statistical graph of the immunohistochemical results of Caspase 9 and Caspase 8; and C shows the inverted fluorescence image of apoptotic cells in the ischemic brain tissue. Detailed Implementation

[0056] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0057] The main experimental reagents, instruments, animals, and consumables used in this invention are as follows:

[0058] Hyaluronic acid (HA, 3600 Da and 90-100 kDa) was purchased from Shandong Freda Biotechnology Co., Ltd., poly(N-isopropylacrylamide) was purchased from Aladin Chemical Reagent Inc. of Shanghai, and vitamin E succinate was purchased from Bid Pharmaceutical Co., Ltd., all of which were ready for use without further purification. All other reagents used in the synthesis were purchased from Sigma Chemical Co. (St. Louis, MO) or Aladin Chemical Reagent Inc. of Shanghai. Male SD rats and C57BL / 6 were purchased from Henan Skbex Biotechnology Co., Ltd.

[0059] MCAO sutures for 230-260 g rats and 20-25 g mice were purchased from Beijing Xinong Technology Co., Ltd.; TTC was purchased from Sigma-Aldrich, USA; other reagents and drugs not specified were all commonly used analytical grade products in the laboratory and will not be described further.

[0060] Cerebral blood flow monitoring was performed using a Rayward laser speckle instrument.

[0061] Example 1

[0062] A method for preparing a low-temperature injectable hydrogel, wherein the synthetic route of HA-VE is as follows:

[0063]

[0064] The specific steps are as follows:

[0065] (1) Weigh 200 mg of vitamin E succinate (VE) into a beaker, add 5 mL of DMF and stir until it dissolves and becomes clear. Add 260 mg of activator HATU at room temperature and react for 1 h to activate the hydroxyl groups on VE to obtain solution I.

[0066] (2) Weigh 130 mg of ADH into a flask, add 1 mL of distilled water and sonicate to dissolve it completely. Then, slowly add solution I dropwise while stirring. React under an argon atmosphere for 6 h. After the reaction is complete, extract with ethyl acetate and rotary evaporate at 70 °C to obtain ADH-VE.

[0067] (3) Weigh 160 mg of HA into a flask, add 8 mL of distilled water and stir until the solution is clear. Then add 100 mg of activator EDC at 0℃ and react at room temperature for 30 min to activate the hydroxyl groups on HA.

[0068] (4) Dissolve 270 mg of ADH-VE prepared in step (2) in 2.7 mL of DMF and slowly add the HA solution from step (3). React for 4 h under argon protection. After the reaction is complete, dialyze with distilled water for 24 h, collect the liquid in the dialysis bag, centrifuge, and freeze-dry the supernatant to obtain HA-VE.

[0069] Example 2

[0070] A method for preparing a low-temperature injectable hydrogel, the specific steps of which are as follows:

[0071] (1) Weigh 200 mg of vitamin E succinate (VE) into a beaker, add 10 mL of DMF and stir until it dissolves and becomes clear. Add 260 mg of activator HATU at room temperature and react for 1 h to activate the hydroxyl groups on VE to obtain solution I.

[0072] (2) Weigh 130 mg of ADH into a flask, add 1.08 mL of distilled water and sonicate to dissolve it completely. Then, slowly add solution I dropwise while stirring. React under an argon atmosphere for 6 h. After the reaction is complete, extract with ethyl acetate and rotary evaporate at 70 °C to obtain ADH-VE.

[0073] (3) Weigh HA 90 -100 k 160 mg was placed in a flask, 16 mL of distilled water was added and stirred until the solution was clear. Then, 100 mg of activator EDC was added at 0 °C and the mixture was reacted at room temperature for 30 min to activate the hydroxyl groups on HA.

[0074] (4) Dissolve 270 mg of ADH-VE prepared in step (2) in 2.7 mL of DMF, and slowly add the HA from step (3). 90-100 k The solution was reacted under argon protection for 4 hours. After the reaction, the solution was dialyzed with distilled water for 24 hours. The liquid in the dialysis bag was collected, centrifuged, and the supernatant was freeze-dried to obtain HA. 90-100 k -VE;

[0075] (5) Dissolve 0.1 g PNIPAM in 5 mL of deionized water at 4℃, stir until clear, and then add 0.05 g HA at room temperature. 90-100 k -VE was mixed at a concentration of 1 wt% and stirred at room temperature for 12 h to obtain an injectable hydrogel. The next day, the mixture was placed at 4 °C for 2 h to obtain an injectable hydrogel LT Gel in a low-temperature state.

[0076] Example 3

[0077] A method for preparing a low-temperature injectable drug-loaded hydrogel, the specific steps of which are as follows:

[0078] (1) Weigh 200 mg of vitamin E succinate (VE) into a beaker, add 10 mL of DMF and stir until it dissolves and becomes clear. Add 260 mg of activator HATU at room temperature and react for 1 h to activate the hydroxyl groups on VE to obtain solution I.

[0079] (2) Weigh 130 mg of ADH into a flask, add 1.08 mL of distilled water and sonicate to dissolve it completely. Then, slowly add solution I dropwise while stirring. React under an argon atmosphere for 6 h. After the reaction is complete, extract with ethyl acetate and rotary evaporate at 70 °C to obtain ADH-VE.

[0080] (3) Weigh out 160 mg of HA respectively 3600 and 160 mg of HA 90-100 k In separate flasks, 8 mL and 16 mL of distilled water were added and stirred until the solutions became clear. Then, 100 mg of activator EDC was added to each flask at 0°C, and the reactions were carried out at room temperature for 30 min to activate the hydroxyl groups on HA, yielding HA. 3600 Solution and HA 90-100 k Solution;

[0081] (4) Dissolve 270 mg of ADH-VE prepared in step (2) in 2.7 mL of DMF, and slowly add the HA obtained in step (3). 3600 Solution and HA 90-100 k The solution was reacted under argon protection for 4 hours. After the reaction, the solution was dialyzed with distilled water for 24 hours. The liquid in the dialysis bag was collected, centrifuged, and the supernatant was freeze-dried to obtain HA. 3600 -VE and HA 90-100 k -VE;

[0082] (5) Take 0.1 g of HA 3600 -VE and 0.011 g of CPZ were dissolved together in 5 mL of acetone. After dissolution, stirring, sonication, and rotary evaporation, a thin film was formed. 0.1 g of PNIPAM was dissolved in 5 mL of deionized water at 4 °C, stirred until clear, and then reconstituted to form a thin film, yielding CPZ NPs micelles. Subsequently, 0.05 g of HA was added at room temperature. 90-100 k -VE was mixed at a concentration of 1 wt% and stirred at room temperature for 14 h to obtain a drug-loaded hydrogel. The next day, the mixture was placed at 4 °C for 2 h to obtain a drug-loaded hydrogel CPZ NPs@LT Gel at a low temperature.

[0083] The hydrogel prepared in this embodiment was characterized and its gel-forming properties were tested. The results are as follows: Figure 1 As shown, Figure 1A shows the 1H NMR spectrum of HA-ADH-VE (HA-VE). It can be seen that HA-VE has the characteristic peaks of HA at 1.9 and 3.0-4.0 ppm, which are the signal peaks of the methyl proton of the N-acetyl group and the methylene and hydroxyl groups, respectively; there is also a double peak at 0.85 ppm, which belongs to the four terminal methyl groups in the vitamin E structure, proving that VE was successfully grafted onto the hyaluronic acid chain in this embodiment. Figure 1 B shows the particle size distribution of empty micelles (NPs) and drug-loaded micelles (CPZ NPs), as well as the TEM image of the drug-loaded micelles. It can be seen that HA... 3600 -VE was stirred to form nanomicelle particles with a particle size of about 300 nm. After loading the drug, the particle size became larger than that of the empty micelles. The above results prove that this embodiment successfully grafted VE onto the hyaluronic acid chain. Figure 1 C shows the gelation properties and SEM image of the synthesized low-temperature injectable drug-loaded hydrogel. It can be seen that the gel is in a liquid state at 4℃ and will change from a solution state to a gel state as the temperature rises to about 37℃. The SEM image of the hydrogel shows that the hydrogel has a loose and porous structure, which is conducive to encapsulating nanomicelle drugs.

[0084] Example 4

[0085] A method for preparing a low-temperature injectable drug-loaded hydrogel, the specific steps of which are as follows:

[0086] (1) Weigh 200 mg of vitamin E succinate (VE) into a beaker, add 10 mL of DMF and stir until it dissolves and becomes clear. The concentration of VE in the VE solution is 0.02 g / mL. Add 260 mg of activator HATU at room temperature and react for 1 h to activate the hydroxyl groups on VE to obtain solution I.

[0087] (2) Weigh 130 mg of ADH into a flask, add 0.87 mL of distilled water and sonicate to dissolve it completely. The concentration of ADH in the ADH solution is 0.15 g / mL. Then, slowly add solution I dropwise while stirring. React under an argon atmosphere for 6 h. After the reaction is complete, extract with ethyl acetate and rotary evaporate at 70 °C to obtain ADH-VE.

[0088] (3) Weigh out 160 mg of HA respectively 3600 and 160 mg of HA 90-100 k Add 12 mL of distilled water to each flask and stir until the solution is clear. HA 3600 In HA 3600 The concentration in the solution is 0.013 g / mL, HA 90-100 k In HA 90-100 kThe concentration in the solution was 0.013 g / mL. Then, 100 mg of each activator EDC was added at 0℃, and the reaction was carried out at room temperature for 30 min to activate the hydroxyl groups on HA, thus obtaining HA. 3600 Solution and HA 90-100 k Solution;

[0089] (4) Take 270 mg of ADH-VE prepared in step (2) in two separate solutions and dissolve them in 1.35 mL of DMF. The concentration of ADH-VE in the ADH-VE solution is 0.2 g / mL. Slowly add the HA from step (3) to each solution. 3600 Solution and HA 90-100 k The reaction was carried out in solution under argon protection for 4 hours. After the reaction was completed, the solution was dialyzed with distilled water for 24 hours. The liquid in the dialysis bag was collected, centrifuged, and the supernatant was freeze-dried to obtain HA. 3600 -VE and HA 90-100k -VE;

[0090] (5) Take 0.1 g of HA 3600 -VE and 0.011 g of CPZ were dissolved together in 5 mL of acetone. After dissolution, stirring, sonication, and rotary evaporation, a thin film was formed. 0.1 g of PNIPAM was dissolved in 6.67 mL of deionized water at 4℃, with an initial PNIPAM concentration of 0.015 g / mL. After stirring until clear, the film was reconstituted to obtain CPZ NPs micelles. Subsequently, 0.05 g of HA was added at room temperature. 90-100k -VE was mixed at a concentration of 1 wt% and stirred at room temperature for 12 h to obtain a drug-loaded hydrogel. The next day, the mixture was placed at 4 °C for 2 h to obtain a drug-loaded hydrogel CPZ NPs@LT Gel at a low temperature.

[0091] Example 5

[0092] A method for preparing a low-temperature injectable drug-loaded hydrogel, the specific steps of which are as follows:

[0093] (1) Weigh 200 mg of vitamin E succinate (VE) into a beaker, add 4 mL of DMF and stir until it dissolves and becomes clear. The concentration of VE in the VE solution is 0.05 g / mL. Add 260 mg of activator HATU at room temperature and react for 1 h to activate the hydroxyl groups on VE to obtain solution I.

[0094] (2) Weigh 130 mg of ADH into a flask, add 1.08 mL of distilled water and sonicate to dissolve it completely. The concentration of ADH in the ADH solution is 0.12 g / mL. Then, slowly add solution I dropwise while stirring. React under an argon atmosphere for 6 h. After the reaction is complete, extract with ethyl acetate and rotary evaporate at 70 °C to obtain ADH-VE.

[0095] (3) Weigh out 160 mg of HA respectively 3600 and 160 mg of HA 90-100 k Add 8 mL of distilled water to each flask and stir until the solution is clear. HA 3600 In HA 3600 The concentration in the solution is 0.02 g / mL, HA 90-100 k In HA 90-100 k The concentration in the solution was 0.02 g / mL. Then, 100 mg of activator EDC was added to each at 0℃, and the reaction was carried out at room temperature for 30 min to activate the hydroxyl groups on HA, thus obtaining HA. 3600 Solution and HA 90-100 k Solution;

[0096] (4) Take 270 mg of ADH-VE prepared in step (2) in two separate solutions and dissolve them in 1.8 mL of DMF. The concentration of ADH-VE in the ADH-VE solution is 0.15 g / mL. Slowly add the HA from step (3) to each solution. 3600 Solution and HA 90-100 k The solution was reacted under argon protection for 4 hours. After the reaction, the solution was dialyzed with distilled water for 24 hours. The liquid in the dialysis bag was collected, centrifuged, and the supernatant was freeze-dried to obtain HA. 3600 -VE and HA 90-100k -VE;

[0097] (5) Take 0.1 g of HA 3600 -VE and 0.011 g of CPZ were dissolved together in 5 mL of acetone. After dissolution, stirring, sonication, and rotary evaporation, a thin film was formed. 0.1 g of PNIPAM was dissolved in 4 mL of deionized water at 4 °C, with an initial PNIPAM concentration of 0.025 g / mL. After stirring until clear, the film formed by resolution yielded CPZ NPs micelles. Subsequently, 0.05 g of HA was added at room temperature. 90-100k -VE was mixed at a concentration of 1 wt% and stirred at room temperature for 14 h to obtain a drug-loaded hydrogel. The next day, the mixture was placed at 4 °C for 2 h to obtain a drug-loaded hydrogel CPZ NPs@LT Gel at a low temperature.

[0098] Example of implementation effect 1

[0099] The performance of the low-temperature injectable hydrogel prepared in Example 1 was tested, and the results are as follows: Figure 1 As shown, Figure 1D represents the rheological properties of the hydrogel prepared at 37℃. The results show that within a certain strain range, the storage modulus (G') of the hydrogel is greater than the loss modulus (G"). Frequency scanning indicates that the hydrogel maintains a gel state within the linear viscoelastic region, and the shear-thinning characteristics demonstrate that the hydrogel is injectable. These results indicate that the hydrogel prepared in this invention is in a liquid state at low temperatures, and transitions from a solution state to a gel state as the temperature gradually increases to 37℃.

[0100] Figure 2 The results of local cooling and in vivo degradation tests were performed on the low-temperature injectable hydrogel prepared in Example 1. Figure 2 A is the performance test of the hydrogel in reducing local temperature, proving that the hydrogel prepared by the present invention can reduce the temperature of the surrounding brain tissue after being injected into the brain; the surrounding brain tissue injected with the hypothermic injectable hydrogel increased from 24.78°C to 30.39°C within 1.5 hours, with an average temperature recovery of 3.74°C per hour; the temperature of the damaged brain tissue that underwent hypothermia treatment was expected to remain at a local hypothermia of about 3.5 hours before recovering to the normal temperature of 37°C. Figure 2 B represents the degradation test of the hydrogel in the brain. The hydrogel injected into the brain showed almost no fluorescence after 21 days, proving that the hydrogel prepared in this invention can be completely degraded in brain tissue.

[0101] Application Example 1: Reducing the volume of cerebral infarction in tMCAO rats

[0102] Right middle cerebral artery occlusion-reperfusion surgery was performed in SD rats and c57 mice using the Longa modified suture occlusion method, as detailed below:

[0103] Animals were fasted for 12 hours prior to surgery, but water intake was not restricted. SD rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg) according to body weight; the anesthesia should not be too deep. After anesthesia, the rats were fixed in a supine position. A longitudinal incision of about 1 cm was made in the middle of the rat's neck, along the right side of the lower part of the mandible. Muscles, fascia, nerves, and blood vessels were bluntly dissected to expose the right common carotid artery (CCA). The external carotid artery (ECA) and internal carotid artery (ICA) were separated at the distal end of the common carotid artery. A 4-0 suture was inserted and tied tightly at the proximal end of the external carotid artery (ECA). An arterial clamp was used to clamp the distal end of the external carotid artery, and the external carotid artery was cut by hot ironing between the suture and the arterial clamp. Subsequently, the arterial clamp was used to clamp the proximal end of the common carotid artery and the internal carotid artery. At the same time, a small incision was made in the external carotid artery, and a 28 μm diameter 4-0 nylon suture plug was inserted through the external carotid artery incision to block blood flow. To prevent bleeding from the vascular incision site, the suture was ligated below the incision site. The ECA stump was then aligned with the ICA to facilitate the suture's entry into the internal carotid artery. Insertion was stopped when resistance was felt at a depth of approximately 18.5 ± 0.5 mm. Excess suture length was cut, and the hemostatic clips holding the common carotid and internal carotid arteries were removed. The separated tissues were restored to their original state, sutured, and a heating pad was placed for warmth. Postoperatively, the rats were warmed by a warm lamp or a heating pad. After 1.5 hours of occlusion, the rat brain was reperfused by removing the suture. Finally, 1 hour after middle cerebral artery occlusion, 10 μL of 0.9% NaCl, Gel, LT Gel, and CPZ NPs@LT Gel were injected into the right cerebral cortex of each group of rats at a rate of 2 μL / min. The sham-operated group received preoperative anesthesia, and the CCA, ECA, and ICA were separated. Only a thin suture was tied to the ECA without inserting a suture, serving as a no-operation control.

[0104] This experiment was divided into 5 groups: Sham group, Model group, Gel group, LT Gel group, and CPZ NPs@LT Gel group. The drugs used in the Gel group, LT Gel group, and CPZ NPs@LT Gel group were Gel, LT Gel, and CPZ NPs@LT Gel, respectively, with a dosage volume of 10 μL for each group. The drugs were administered once 30 minutes before reperfusion. The drug used in the Model group was physiological saline.

[0105] 1. Measurement of cerebral infarction

[0106] The ischemic time after MCAO surgery was 1.5 h. According to the grouping, rats in each group were injected with the drug stereotactically into the brain 1 h after ischemia (i.e. 0.5 h before reperfusion). 24 h later, rat brain tissue was taken, frozen to a mousse state, and evenly cut into 5 equal parts. The tissue was stained with 2% triphenyltetrazole ammonium chloride (TTC) solution, and the infarcted area was quantified using ImageJ to quantitatively evaluate the infarcted brain tissue (n=3).

[0107] 2. The effect of hydrogel on short-term behavioral recovery in tMCAO rats was evaluated using the mNSS scoring method. The specific criteria for the mNSS scoring are shown in Table 1.

[0108] Table 1. Specific criteria for mNSS scoring

[0109]

[0110] Experimental results are as follows Figure 3 As shown, Figure 3 As shown in A and B, the mean infarct volume in the Model group was 18.64%, while the mean infarct volume in the low-temperature drug-free injectable hydrogel LT Gel group and the low-temperature drug-loaded injectable hydrogel CPZ NPs@LT Gel group decreased to 6.3% and 2.8%, respectively (n=3). The infarct volume was significantly different from that in the Model group. Figure 3 C indicates that the neurological deficit score after low-temperature hydrogel treatment was significantly lower than that of the model group, indicating that the CPZ NPs@LT Gel prepared in this invention has a significant effect in reducing ischemia-reperfusion injury (n=7).

[0111] Application Example 2: Improving Learning, Memory, and Motor Recovery in Rats After Treatment

[0112] Following the tMCAO model construction and stereotactic drug delivery method described in Application Example 1, the learning, memory, and motor functions of rats were assessed at 21, 22, and 28 days using the open field test, Y-maze test, and Morris water maze test (n=7).

[0113] The results are as follows Figure 4 As shown, by Figure 4 As shown in B, after treatment, the cerebral ischemia rats exhibited significantly increased motor activity in the open field test, with increased movement distance and speed. Figure 4 C showed that after treatment, the curiosity of the cerebral ischemia rats to their environment significantly increased, indicating that their learning and spontaneous motor functions recovered well. The spontaneous alternation rate was statistically analyzed in the Y-maze test. Figure 4 E indicates that in the Y-maze experiment, the spontaneous alternation rate of treated rats significantly increased, suggesting improved spatial working memory and spontaneous activity abilities. Figure 4As can be seen from F, in the Morris water maze test, the motion trajectory diagram shows that the tMCAO rats moved along the maze walls and rarely explored to find the target platform. Figure 4 G and H showed that, compared with normal mice, tMCAO rats had a significantly longer escape latency, indicating dyskinesia and delayed plateau detection. Figure 4 I and J showed that motor and navigation abilities improved to varying degrees in all groups after treatment. In particular, SD rats exhibited longer movement distances and dwell times in the target quadrant after CPZ NPs@LT Gel treatment, indicating improved learning and memory functions.

[0114] Application Example 3: Improving the Restoration of Cerebral Blood Flow After Ischemia-Reperfusion

[0115] Following the tMCAO model construction and stereotactic drug delivery method described in Application Example 1, a mouse tMCAO model was constructed (total ischemia duration 1 h, 2 μL drug administered 0.5 h before reperfusion). Cerebral blood flow recovery was monitored at 0.5 h, 1 h, 6 h, and 24 h after the onset of ischemia. Results are as follows... Figure 5 As shown, cerebral blood flow (CBF) was recorded at baseline and at 0.5 h, 1 h, 6 h, and 24 h after stroke. Compared with the sham-operated group, the ischemic injury group showed a significant reduction in CBF within 0-1 h. After 6 h, the hypothermic mice showed a significant increase in vascular density within the stroke cavity. This indicates that local hypothermia treatment with hydrogel promotes the normalization and recovery of cerebral blood flow (n=3).

[0116] Application Example 4: Reducing Neuronal Damage in the Hippocampus

[0117] Following the tMCAO model construction and stereotactic brain drug delivery method described in Application Example 1, ischemic brain tissue was collected 24 hours after reperfusion. Paraffin sections of the brain tissue were prepared, and hematoxylin-eosin (H&E) and Nissl staining were performed to observe the neuronal damage in each group. The results are as follows: Figure 6 As shown, compared with the model group, in the ischemic penumbra region of rats in the CPZ NPs@LT Gel group, H&E staining showed a significant reduction in necrotic cells, and Nissl staining showed a significant reduction in atrophied neurons.

[0118] Application Example 5: Reducing Metabolic Damage

[0119] 1. Detection of metabolites related to brain metabolic activity

[0120] Following the tMCAO model construction and stereotactic brain drug delivery method described in Application Example 1, ischemic brain tissue (n=3) was obtained 2 h and 24 h after reperfusion. Brain tissue homogenates were prepared, and lactate content and NADH / NAD ratio were measured using a kit. +The ratio and ATP / ADP ratio were measured, and the results are as follows: Figure 7 As shown in Figure A, it can be seen that the hydrogel injected at low temperatures can reduce the production of lactic acid during the acute phase of ischemia (2 h) and reduce the accumulation of harmful substances during ischemia; it can resist the increase in the NADH / NAD+ and ATP / ADP ratios during ischemia, but does not impair energy supply function. Figure 7 As shown in Figure B, at 24 h, the NADH / NAD+ and ATP / ADP ratios tended to be similar to those in the sham-operated group.

[0121] 2. Observation of mitochondrial morphology in brain tissue

[0122] Following the tMCAO model construction and stereotactic brain drug delivery method described in Application Example 1, ischemic brain tissue (n=3) was obtained 2 hours after reperfusion. Thin sections were prepared, fixed in transmission electron microscopy fixative for 48 hours, and then sent for examination. The results are as follows: Figure 8 As shown, cryohydrogel treatment reduced damage to mitochondrial morphology, and... Figure 7 The results corroborate each other, showing that cryohydrogel therapy salvaged the damaged mitochondrial morphology and function, laying the foundation for reducing neuronal damage.

[0123] Application Example 6: Reducing Oxidative Stress and Inflammatory Cascades in the Brain

[0124] 1. Experimental detection of reduced oxidative stress in the brain

[0125] Following the tMCAO model construction and stereotactic brain drug delivery method described in Application Example 1, ischemic brain tissue (n=3) was collected 24 h after reperfusion. Brain tissue homogenates were prepared, and MDA content was detected using an MDA assay kit, while CAT activity was detected using a CAT assay kit. The effect of low-temperature hydrogel therapy on oxidative stress in tMCAO model rats was evaluated. Results are as follows: Figure 9 As shown in A and B, cryohydrogel treatment reduced the level of oxidative stress in the brain tissue of rats undergoing ischemia-reperfusion.

[0126] 2. Reduce the level of inflammatory cascades in the brain.

[0127] Following the tMCAO model construction and stereotactic brain drug delivery method described in Application Example 1, ischemic brain tissue (n=3) was collected 24 h after reperfusion. Paraffin sections of the brain tissue were prepared, and immunohistochemical staining was performed to observe the expression of inflammatory factors in the brain tissue. The results are as follows: Figure 9 As shown in C and D, cryohydrogel treatment reduced the level of the inflammatory cascade in the brain tissue of rats undergoing ischemia-reperfusion.

[0128] Application Example 7: Reducing Brain Cell Apoptosis

[0129] Following the tMCAO model construction and stereotactic brain drug delivery method described in Application Example 1, ischemic brain tissue (n=3) was obtained 24 h after reperfusion. Paraffin sections of the brain tissue were prepared, and immunohistochemistry of extrinsic apoptosis marker proteins Caspase 8 and Caspase 9, as well as TdT-mediated dUTP nickel-terminal labeling (TUNEL) staining, were performed to observe the expression of apoptosis-related proteins and the apoptosis status of brain cells in each group. Results are as follows: Figure 10 As shown, Figure 10 A shows that the expression of apoptosis-related proteins is reduced in brain tissue treated with cryohydrogel, by Figure 10 As shown in B, TUNEL staining also revealed a significant reduction in apoptotic cells.

[0130] Based on the above test results, it can be seen that the low-temperature injectable hydrogel or low-temperature injectable drug-loaded hydrogel prepared by the present invention can be used to treat ischemic stroke, indicating that it has good clinical significance and application value for the treatment of stroke.

[0131] 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 low-temperature injectable hydrogel, characterized in that, Includes the following steps: (1) Dissolve vitamin E succinate in an organic solvent, add an activator to obtain solution I; (2) ADH was dissolved in distilled water, and solution I was added dropwise during stirring to carry out the reaction. After extraction and rotary evaporation, ADH-VE was obtained. (3) Dissolve HA in distilled water, add an activator and react to obtain solution II; (4) After dissolving the ADH-VE prepared in step (2) in an organic solvent, add it to solution II for reaction. After the reaction is completed, HA-VE is obtained by dialysis, centrifugation and drying. (5) After dissolving PNIPAM in deionized water, add HA-VE prepared in step (4), stir and react to obtain a low-temperature injectable hydrogel.

2. The method for preparing the low-temperature injectable hydrogel according to claim 1, characterized in that: The organic solvent mentioned in step (1) is DMF, the activator is HATU, the concentration of vitamin E succinate in solution I is 0.02-0.05 g / mL, and the mass ratio of vitamin E succinate to activator is 10:13; In step (2), the mass ratio of ADH to vitamin E succinate is 13:20, and the initial concentration of ADH is 0.12-0.15 g / mL; the reaction is carried out under an argon protective atmosphere.

3. The method for preparing the low-temperature injectable hydrogel according to claim 2, characterized in that: The molecular weight of HA in step (3) is 90-100 kDa; the concentration of HA in solution II is 0.01-0.02 g / mL, and the mass ratio of HA to activator is 8:5; the activator is EDC. The organic solvent in step (4) is DMF, the initial concentration of ADH-VE is 0.1-0.2 g / mL, and the mass ratio of ADH-VE to HA is 5:3; the reaction is carried out under an argon protective atmosphere, and the drying is freeze drying; In step (5), the mass ratio of PNIPAM to HA-VE is 2:1, the initial concentration of PNIPAM is 0.015-0.025 g / mL, and the stirring reaction time is 12-14 h.

4. The low-temperature injectable hydrogel prepared by the preparation method according to claim 3.

5. A method for preparing a low-temperature injectable drug-loaded hydrogel, characterized in that, Includes the following steps: (1) Dissolve vitamin E succinate in an organic solvent, add an activator to obtain solution I; (2) ADH was dissolved in distilled water, and solution I was added dropwise during stirring to carry out the reaction. After extraction and rotary evaporation, ADH-VE was obtained. (3) HA 3600 and HA 90-100k Dissolved in distilled water, and reacted with an activator, HA is obtained. 3600 Solution and HA 90-100k Solution; (4) After dissolving the ADH-VE prepared in step (2) in an organic solvent, add HA respectively. 3600 Solution and HA 90-100k The reaction is carried out in solution, and after completion, HA is obtained by dialysis, centrifugation, and drying. 3600 -VE and HA 90-100k -VE; (5) HA 3600 -VE and CPZ are both dissolved in acetone. After dissolution, stirring, sonication, and rotary evaporation, a thin film is formed. PNIPAM is dissolved in deionized water and then re-dissolved to form a film to obtain drug-loaded micelles. HA is then added. 90-100k -VE, after stirring and reaction, drug-loaded hydrogel is obtained.

6. The method for preparing the low-temperature injectable drug-loaded hydrogel according to claim 5, characterized in that: In step (1), the mass ratio of vitamin E succinate to activator is 10:13, and the concentration of vitamin E succinate in solution I is 0.02-0.05 g / mL; the organic solvent is DMF, and the activator is HATU; In step (2), the mass ratio of ADH to vitamin E succinate is 13:20, and the initial concentration of ADH is 0.12-0.15 g / mL; the reaction is carried out under an argon protective atmosphere.

7. The method for preparing a low-temperature injectable drug-loaded hydrogel according to claim 6, characterized in that: The HA mentioned in step (3) 3600 The mass ratio of HA to activator is 8:

5. 3600 In HA 3600 The concentration in the solution is 0.01-0.02 g / mL; the HA 90-100k The mass ratio of HA to activator is 8:

5. 90-100k In HA 90-100k The concentration in the solution is 0.01-0.02 g / mL; the activator is EDC; The organic solvent mentioned in step (4) is DMF, the initial concentration of ADH-VE is 0.1-0.2 g / mL, and ADH-VE and HA 3600 The mass ratio is 5:3, ADH-VE and HA 90-100k The mass ratio is 5:3; the reaction is carried out under an argon protective atmosphere, and the drying is freeze-drying; The HA mentioned in step (5) 3600 The mass ratio of VE to CPZ is 100:11; the initial concentration of PNIPAM is 0.015-0.025 g / mL; and the PNIPAM and HA... 90-100 k The mass ratio of -VE is 2:1; the loading of CPZ is 4-5 wt%; and the stirring reaction time is 12-14 h.

8. The low-temperature injectable drug-loaded hydrogel prepared by the preparation method of claim 7.

9. The use of the low-temperature injectable hydrogel of claim 4 or the low-temperature injectable drug-loaded hydrogel of claim 8 in the preparation of medicaments for treating nerve injury repair, reducing brain tissue damage and ischemic stroke.

10. The use of the cryogenic injectable hydrogel of claim 4 or the cryogenic injectable drug-loaded hydrogel of claim 8 in the preparation of a medicament for alleviating or treating ischemia-reperfusion injury.

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