A pH-responsive injectable drug-loaded hydrogel, its preparation method and application

Abstract

This invention specifically discloses a pH-responsive injectable drug-loaded hydrogel, its preparation method, and its applications, relating to the field of biomedical technology. The hydrogel provided by this invention efficiently releases ICA at a pH of 6.0–7.0 and exhibits excellent injectability, adhesion, and good blood compatibility.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a pH-responsive injectable drug-loaded hydrogel, its preparation method, and its application. Background Technology

[0002] Spinal cord injury, abbreviated as SCI, is a severe neurological disorder with an extremely high rate of disability. The pathological process of SCI includes primary and secondary injury. Primary injury is an irreversible physical injury; while secondary injury involves a series of complex, reversible pathophysiological changes, including inflammatory responses, oxidative stress, ischemia, and apoptosis. Among these, the cytokine storm caused by the inflammatory response and the overexpression of reactive oxygen species due to oxidative stress play crucial roles in secondary injury. Early intervention in inflammatory and oxidative stress responses to reduce the neurotoxic harm caused by secondary injury is essential for promoting the recovery of neurological and motor function in SCI patients.

[0003] Icariin, abbreviated as ICA, is the main active ingredient of the traditional Chinese medicine Epimedium. It possesses various pharmacological activities, including anti-inflammatory, antioxidant, anti-apoptotic, and nerve regeneration-promoting activities. Existing studies have shown that ICA can promote the recovery of motor function by inhibiting the mitochondrial-mediated apoptosis pathway in a mouse SCI model, reducing the release of pro-inflammatory factors and oxidative stress levels. However, ICA has poor water solubility and low bioavailability, affecting its efficacy in clinical applications.

[0004] Cyclodextrins are a class of water-soluble oligosaccharides whose hydrophobic cavities can encapsulate ICA through host-guest interactions, thereby improving drug solubility. By using β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and methylated-β-cyclodextrin to form inclusion complexes with ICA, the water solubility of ICA can be increased by 1.2-6.3 times. In comparison, γ-cyclodextrin (abbreviated as γ-CD) has higher water solubility and a larger cavity volume, resulting in a more significant solubilizing effect on ICA. Chinese patent application CN116370653A discloses an inclusion complex of hydroxypropyl-γ-CD and ICA and its preparation method. This inclusion complex is prepared by complexation of hydroxypropyl-γ-CD and ICA with trace amounts of water-soluble polymers, resulting in an 80-fold increase in the water solubility of ICA. While this technology effectively improves the problem of low ICA solubility, the provided inclusion complex is only suitable for oral administration, and the ICA release rate is too fast, lacking sustained-release capability, making it difficult to achieve its long-term therapeutic effect at the site of SCI injury.

[0005] CN106309380A discloses a pH-responsive controlled-release microsphere and its preparation method. The controlled-release microsphere exhibits good pH responsiveness, showing no significant degradation in the low-pH environment of the stomach, resulting in a low drug release. In the higher-pH environment of the colon, it adheres to the colonic surface, continuously releasing the drug, thus promoting drug release, prolonging colonic retention time, increasing drug absorption, and improving local bioavailability for oral administration of drugs to treat colonic diseases. In vitro drug release studies show that the cumulative release rate of icariin-targeted sustained-release microspheres in simulated gastric juice is only about 10% after 2 hours, while the cumulative release rate in simulated colonic juice can reach 65%. The icariin pH-responsive biocontrolled-release microspheres have the drug loaded into the core, resulting in a high drug loading capacity. The outer layer of the drug is coated with chitosan, which can fix the drug, thereby reducing drug loss during in vivo transport. However, its early release rate at the target site is low, and it cannot act rapidly.

[0006] Compared to oral dosage forms, injectable dosage forms release drugs directly at the lesion site, avoiding the first-pass effect of the liver. Furthermore, their sustained-release properties can maintain drug concentration over a long period, achieving the goal of reducing toxicity and increasing efficacy. For example, the icariin injection disclosed in CN113440512A simply mixes icariin with an organic solvent, without altering the water solubility of the active ingredient, and cannot achieve sustained and stable release.

[0007] Therefore, how to achieve a high dose loading of ICA and ensure its continuous and stable release at the SCI site is a key issue in improving the efficacy of ICA in treating SCI. Summary of the Invention

[0008] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a pH-responsive injectable drug-loaded hydrogel, comprising a carrier loaded with icariin; said carrier is formed by a hydrazone-coupled reaction of carboxymethyl poria polysaccharide grafted with adipic acid dihydrazide and aldehyde-modified γ-cyclodextrin. The hydrogel provided by this invention efficiently releases ICA at a pH of 6.0–7.0 and exhibits excellent injectability, adhesion, and good blood compatibility.

[0009] (II) Technical Solution To achieve the above objectives, the present invention provides a pH-responsive injectable drug-loaded hydrogel (Gel@ICA) comprising a carrier loaded with icariin (ICA); The carrier is formed by a hydrazone coupling reaction of carboxymethyl poria cocos polysaccharide grafted with adipic acid dihydrazide (CMP-ADH) and aldehyde-modified γ-cyclodextrin (γCD-CHO).

[0010] In one embodiment, the concentration of icariin is 0.05~3.4 mg / mL.

[0011] In one embodiment, the concentration of icariin is 0.39~3.4 mg / mL.

[0012] In one embodiment, the concentration of icariin is 1.17 mg / mL.

[0013] In one embodiment, the structural formula of the CMP-ADH is: ; Where X is 0.16~0.31 and M is 0.88.

[0014] In one embodiment, the CMP-ADH has a weight-average molecular weight of 206 kDa to 224 kDa; the degree of carboxymethyl substitution M is 0.88 based on the number of moles of carboxymethyl groups per mole repeating unit; and the degree of adipic acid dihydrazide substitution X is 0.16 to 0.31 based on the number of moles of adipic acid dihydrazide groups per mole repeating unit.

[0015] In one embodiment, the structural formula of the γCD-CHO is: Where Y ranges from 2.02 to 3.07.

[0016] In one embodiment, the degree of aldehyde substitution Y of the γCD-CHO is 2.02 to 3.07, based on the number of aldehyde groups on each γ-cyclodextrin molecule.

[0017] In another aspect, the present invention provides a method for preparing the above-mentioned Gel@ICA, comprising: S1: The CMP-ADH was prepared by reacting carboxymethyl poria cocos polysaccharide (CMP) with adipic dihydrazide (ADH), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); S2: The γCD-CHO was prepared by reacting γ-cyclodextrin (γCD) with Desmond-Martin reagent (DMP); S3: The γCD-CHO was mixed with ICA to prepare the aldehyde-modified γ-cyclodextrin inclusion complex of icariin (γCD-CHO@ICA). S4: The γCD-CHO@ICA is mixed and reacted with CMP-ADH to obtain the Gel@ICA.

[0018] In one embodiment, the preparation method specifically includes: S1: Weigh CMP and dissolve it in an aqueous solution of 2-(N-morpholino)ethanesulfonic acid (MES). Then, add ADH, EDC, and NHS sequentially and dissolve them. React at room temperature in the dark. Subsequently, place the reaction solution in a dialysis bag with a molecular weight cutoff of 8kDa~14kDa and dialyze it with deionized water. Collect the dialysate in the dialysis bag, dry it, and obtain CMP-ADH. S2: Prepare a γCD solution using anhydrous dimethyl sulfoxide (DMSO) and a DMP solution using DMSO. Add the DMP solution dropwise to the γCD solution to react and obtain a reaction solution. Add 5 times the volume of cold acetone to the reaction solution for precipitation. Centrifuge to obtain a crude product. Dissolve the crude product in deionized water and precipitate it again using 5 times the volume of cold acetone. Repeat this process three times. Dry the crude product collected in the last step to obtain γCD-CHO. S3: Dissolve γCD-CHO in deionized water to obtain an aqueous solution of γCD-CHO, and dissolve ICA in anhydrous ethanol to obtain an ethanol solution of ICA; slowly add the ethanol solution of ICA to the aqueous solution of γCD-CHO while stirring. During this process, γCD-CHO will encapsulate ICA. Filter and collect the filtrate, and dry it to obtain γCD-CHO@ICA. S4: Mix the aqueous solution of γCD-CHO@ICA and the aqueous solution of CMP-ADH, and incubate to obtain the Gel@ICA.

[0019] In one embodiment, the molar ratio of the CMP repeating unit, ADH, EDC, and NHS is 1:5:0.21~0.4:0.21~0.4.

[0020] In one embodiment, the concentration of the MES aqueous solution is 10 mM to 50 mM.

[0021] In one embodiment, the reaction time of S1 is 12h to 48h.

[0022] In one embodiment, the molar ratio of γCD to DMP is 1:5~10.

[0023] In one embodiment, the reaction time of S2 is 2h to 4h.

[0024] In one embodiment, the molar ratio of γCD-CHO to ICA is 1:1.

[0025] In one embodiment, the incubation temperature is 20°C to 37°C, and the incubation time is 0.017h to 12h.

[0026] In one embodiment, the molar ratio of hydrazine groups in CMP-ADH to aldehyde groups in γCD-CHO@ICA is 1:1. When the aldehyde to hydrazine ratio is 1:1, the resulting hydrazone-bonded hydrogel network is more stable.

[0027] The pH-responsive injectable drug-loaded hydrogel provided by this invention uses CMP-ADH and γCD-CHO as carrier materials, and loads ICA onto the carriers. The pH-responsive injectable drug-loaded hydrogel is prepared through a hydrazone coupling reaction. The carrier is formed by the coupling reaction of CMP-ADH and γCD-CHO with a high degree of aldehyde substitution. Since hydrazone bonds have acid-responsive properties, they hydrolyze more rapidly at pH ≤ 6.5. Therefore, the pH-responsive injectable drug-loaded hydrogel can efficiently release ICA in a weakly acidic environment of pH 6.0~7.0. Due to inflammatory responses and abnormal cell metabolism, the pH value at the lesion site in SCI decreases to 6.0~7.0, exhibiting a slightly acidic environment, which matches the pH-responsive characteristics of the pH-responsive injectable drug-loaded hydrogel. Therefore, the pH-responsive injectable drug-loaded hydrogel can continuously and stably release ICA at the lesion site of SCI, achieving the purpose of continuous treatment. The ICA loaded on the carrier is the active ingredient for treating SCI. The CMP-ADH has a relative molecular mass of 206 kDa to 224 kDa. Based on the number of moles of carboxymethyl groups per mole of repeating unit, the degree of carboxymethyl substitution M is 0.88, and based on the number of moles of adipic acid dihydrazide groups per mole of repeating unit, the degree of ADH substitution X is 0.16 to 0.31. The γCD-CHO has a degree of aldehyde substitution Y of 2.02 to 3.07 based on the number of aldehyde groups on each γCD molecule. This carrier, through the inclusion effect of the macrocyclic host molecule γCD, efficiently loads the hydrophobic drug ICA while maintaining its good stability, thus preparing a pH-responsive injectable drug-loaded hydrogel.

[0028] In another aspect, the present invention also provides a pH-responsive injectable drug-loaded hydrogel, which is obtained by the above preparation method.

[0029] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) The above-mentioned pH-responsive injectable drug-loaded hydrogel is used in a therapeutically effective amount; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0030] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0031] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0032] In one embodiment, the pharmaceutical product is a vial or box.

[0033] In another aspect, the present invention also provides the use of the above-mentioned pH-responsive injectable drug-loaded hydrogels, pharmaceutical compositions, pharmaceutical formulations and / or pharmaceutical products in the preparation of drugs for the prevention and / or treatment of spinal cord injury.

[0034] (III) Beneficial Effects This invention provides a pH-responsive injectable drug-loaded hydrogel, its preparation method, and its applications. Compared with existing technologies, it has the following advantages: 1. The pH-responsive injectable drug-loaded hydrogel provided by this invention releases 53.1% ICA on day 1, 10.8% on day 2, 11.1% on day 3, 8.7% on day 5, and 7.2% on day 7, for a total release of 90.9% ICA in the first 7 days. Therefore, Gel@ICA can rapidly release ICA to treat SCI in the early stages of injection and also continuously and stably release ICA after injection, prolonging the duration of drug efficacy and improving the bioavailability of ICA. Thus, the pH-responsive injectable drug-loaded hydrogel of this invention can achieve precise release of ICA at the SCI lesion site, resulting in highly efficient treatment of SCI.

[0035] 2. The inclusion of ICA with γCD overcomes the problem that ICA is poorly soluble in water and difficult to prepare into a solution. This is the key point for pH-responsive injectable drug-loaded hydrogels to deliver ICA for the treatment of SCI.

[0036] 3. The pH-responsive injectable drug-loaded hydrogel exhibits rapid network recovery after damage, demonstrating significant shear-thinning properties, and can be successfully injected through a 22G needle. This indicates that the pH-responsive injectable drug-loaded hydrogel possesses excellent injectability. Therefore, this invention provides a novel injection treatment method for SCI treatment requiring ICA.

[0037] 4. The pH-responsive injectable drug-loaded hydrogel provided by this invention can be directly injected into the SCI site via a convenient minimally invasive drug delivery method, thereby reducing surgical risks, alleviating patient suffering, and reducing drug distribution in non-damaged areas. Furthermore, the hydrogel provided by this invention is pH-responsive, enabling controlled drug release in the slightly acidic environment of the SCI site. This allows the ICA to maintain an effective drug concentration at the lesion site for a longer period, reducing the frequency of administration and improving treatment convenience and compliance.

[0038] 5. The pH-responsive injectable drug-loaded hydrogel provided by this invention also has excellent adhesion, antioxidant properties and good blood compatibility, and has broad application prospects in the field of SCI repair. Attached Figure Description

[0039] 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.

[0040] Figure 1 The 1H NMR spectrum of CMP-ADH in this invention; Figure 2 This is the 1H NMR spectrum of the γCD-CHO carboxylate in this invention; Figure 3 This is the standard curve diagram of ICA in this invention; Figure 4 This is a diagram illustrating the preparation process of the pH-responsive injectable drug-loaded hydrogel in this invention. Figure 5 Fourier transform infrared spectra of CMP-ADH, γCD-CHO and pH-responsive injectable drug-loaded hydrogels in this invention; Figure 6 These are rheological time scans of pH-responsive injectable drug-loaded hydrogels with different solid contents in embodiments of the present invention; Figure 7 This is a rheological frequency scan of the pH-responsive injectable drug-loaded hydrogel in Example 1 of the present invention; Figure 8 The following are rheological amplitude scans and high / low strain cyclic scans of the pH-responsive injectable drug-loaded hydrogel in Embodiment 1 of the present invention: where a is the rheological amplitude scan of the pH-responsive injectable drug-loaded hydrogel; b is the high / low strain cyclic scan of the pH-responsive injectable drug-loaded hydrogel. Figure 9 This is a viscosity scan of the pH-responsive injectable drug-loaded hydrogel in Example 1 of the present invention at different shear rates; Figure 10 This is an injection demonstration diagram of the pH-responsive injectable drug-loaded hydrogel in Example 1 of the present invention; Figure 11 This is an adhesion test diagram of the pH-responsive injectable drug-loaded hydrogel in this invention; Figure 12 This is a mass degradation curve of the pH-responsive injectable drug-loaded hydrogel in Example 1 of the present invention; Figure 13 This is a release curve of ICA in the pH-responsive injectable drug-loaded hydrogel of Example 1 of the present invention; Figure 14 The results of the blood compatibility assessment of the pH-responsive injectable drug-loaded hydrogel in this invention; Figure 15The results of hemolysis rate determination for the pH-responsive injectable drug-loaded hydrogel in this invention; Figure 16 The total antioxidant capacity test results of the pH-responsive injectable drug-loaded hydrogel in Example 1 of this invention; Figure 17 This is the Trolox standard curve for the total antioxidant capacity test of the pH-responsive injectable drug-loaded hydrogel in Example 1 of the present invention; Figure 18 This is a graph showing the reactive oxygen species scavenging capacity of the pH-responsive injectable drug-loaded hydrogel in mouse spinal cord tissue. Figure 19 This is a BMS score graph showing how the pH-responsive injectable drug-loaded hydrogel promotes the recovery of motor function in SCI mice according to the present invention. Figure 20 This is an immunofluorescence result of the pH-responsive injectable drug-loaded hydrogel promoting nerve regeneration in the SCI region of mice in this invention. Figure 21 This figure shows the biosafety assessment results of the pH-responsive injectable drug-loaded hydrogel in mice. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, 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.

[0042] Terms and Definitions As used herein, the term "pharmaceutical composition" refers to a composition comprising Gel@ICA formulated with one or more pharmaceutically acceptable carriers.

[0043] As used in this paper, the term "carboxymethyl pachyman (CMP)" refers to a water-soluble polysaccharide derivative obtained by carboxymethylation of water-insoluble β-pachyman from Poria cocos. This can be referenced in the literature Yan M, Cheng H, Jingjing C, et al. The extraction, structure characterization and hydrogel construction of a water-insoluble β-glucan from Poria cocos[J]. Carbohydrate Research, 2023, 534: 108960 and Miao C, Xun X, Dodd L J. Inverse Vulcanization with SiO2-Embedded Elemental Sulfur for Superhydrophobic, Anticorrosion, and Antibacterial Coatings[J]. ACS Applied Polymer Materials, 2025, 7(9): 9180-9193.

[0044] The formulation of a pharmaceutical composition can be adjusted according to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals.

[0045] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0046] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0047] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0048] The active substance in the product disclosed in this invention accounts for 0.001-99.9 wt% of the total weight of the composition, with the remainder being pharmaceutically acceptable carriers and other additives.

[0049] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0050] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0051] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0052] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0053] As used in this article, the term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0054] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0056] Example 1: Preparation of pH-responsive injectable drug-loaded hydrogel #1: 1. Preparation of CMP-ADH: CMP containing 0.87 mmol of glucose repeating units and a carboxymethyl substitution degree M = 0.88 was dissolved in 40 mL of 10 mM MES aqueous solution. Then, 4.35 mmol of ADH, 0.18 mmol of EDC, and 0.18 mmol of NHS were added sequentially and the solution was reacted at room temperature in the dark for 24 h. The reaction solution was then placed in a dialysis bag with a molecular weight cutoff (MWCO) of 8 kDa and dialyzed with deionized water for 3 days. The dialysate was collected, dried, and CMP-ADH was obtained, with an ADH substitution degree X = 0.16 and a weight-average molecular weight of 206 kDa.

[0057] 2. Preparation of CMP-ADH solution: Weigh 30 mg of CMP-ADH and dissolve it in 0.97 mL of 10 mM PBS buffer (pH 7.4) to obtain a CMP-ADH solution with a mass concentration of 3 wt%.

[0058] 3. Preparation of γCD-CHO: Weigh 0.77 mmol of γCD and add it to 20 mL of anhydrous DMSO to prepare a γCD solution. Simultaneously, weigh 7.7 mmol of DMP and add it to 10 mL of DMSO to prepare a DMP solution. Add the DMP solution dropwise to the γCD solution. React at room temperature for 4 h. Then, add 5 times the volume of cold acetone to the reaction solution for precipitation, and centrifuge to obtain the crude product. Dissolve the crude product in deionized water and precipitate it again using 5 times the volume of cold acetone. Repeat the precipitation process three times. Dry the last collected precipitate to obtain γCD-CHO with an aldehyde substitution degree Y = 3.07.

[0059] 4. Preparation of γCD-CHO@ICA: 0.07 mmol of γCD-CHO was dissolved in 50 mL of deionized water to obtain a γCD-CHO solution, and 0.07 mmol of ICA was dissolved in 10 mL of anhydrous ethanol to obtain an ICA ethanol solution. The ICA ethanol solution was added dropwise to the γCD-CHO solution, stirred for 24 h, filtered, and the filtrate was collected. After drying, the drug inclusion complex γCD-CHO@ICA was obtained.

[0060] 5. Preparation of γCD-CHO@ICA solution: Weigh 30 mg of γCD-CHO@ICA into a container, add 0.97 mL of 10 mM PBS buffer (pH=7.4) to dissolve it, and obtain a γCD-CHO@ICA solution with a concentration of 3 wt%.

[0061] 6. Preparation of pH-responsive injectable drug-loaded hydrogel: Take 100 μL of CMP-ADH solution and 32 μL of γCD-CHO@ICA solution, mix them well, and incubate at 37℃ for 0.017 h. A pH-responsive injectable drug-loaded hydrogel with a solid content of 3 wt% and an ICA concentration of 1.17 mg / mL is formed through hydrazone coupling reaction.

[0062] Example 2: Preparation of pH-responsive injectable drug-loaded hydrogel #2: 1. Preparation of CMP-ADH: CMP containing 0.87 mmol of glucose repeating units and a carboxymethyl substitution degree M = 0.88 was dissolved in 40 mL of 30 mM MES aqueous solution. Then, 4.35 mmol of ADH, 0.18 mmol of EDC, and 0.18 mmol of NHS were added sequentially and the solution was reacted at room temperature in the dark for 12 h. The reaction solution was then placed in a dialysis bag with a molecular weight cutoff (MWCO) of 10 kDa and dialyzed with deionized water for 3 days. After drying, CMP-ADH was obtained, with an ADH substitution degree X = 0.16 and a weight-average molecular weight of 206 kDa.

[0063] 2. Preparation of CMP-ADH solution: Weigh 40 mg of CMP-ADH and dissolve it in 0.96 mL of 10 mM PBS buffer (pH=7.4) to obtain a 4 wt% CMP-ADH solution.

[0064] 3. Preparation of γCD-CHO: Weigh 0.77 mmol of γCD and add it to 20 mL of anhydrous DMSO to prepare a γCD solution. Simultaneously, weigh 7.7 mmol of DMP and add it to 10 mL of DMSO to prepare a DMP solution. Add the DMP solution dropwise to the γCD solution. React at room temperature for 4 h. Then, add 5 times the volume of cold acetone to the reaction solution for precipitation, and centrifuge to obtain the crude product. Dissolve the crude product in deionized water and precipitate it again using 5 times the volume of cold acetone. Repeat the precipitation process three times. Dry the last collected precipitate to obtain γCD-CHO with an aldehyde substitution degree Y = 3.07.

[0065] 4. Preparation of γCD-CHO@ICA: 0.07 mmol of γCD-CHO was dissolved in 50 mL of deionized water to obtain a γCD-CHO solution, and 0.07 mmol of ICA was dissolved in 10 mL of anhydrous ethanol to obtain an ICA ethanol solution. The ICA ethanol solution was added dropwise to the γCD-CHO solution, stirred for 24 h, filtered, and the filtrate was collected. After drying, the drug inclusion complex γCD-CHO@ICA was obtained.

[0066] 5. Preparation of γCD-CHO@ICA solution: Weigh 40 mg of γCD-CHO@ICA into a container, add 0.96 mL of 10 mM PBS buffer (pH=7.4) to dissolve it, and obtain a γCD-CHO@ICA solution with a concentration of 4 wt%.

[0067] 6. Preparation of pH-responsive injectable drug-loaded hydrogel: Take 100 μL of CMP-ADH solution and 32 μL of γCD-CHO@ICA solution, mix them well, and incubate at 27℃ for 0.5 h. A pH-responsive injectable drug-loaded hydrogel with a solid content of 4 wt% and an ICA concentration of 1.56 mg / mL is formed through hydrazone coupling reaction.

[0068] Example 3: Preparation of pH-responsive injectable drug-loaded hydrogel #3: 1. Preparation of CMP-ADH: CMP containing 0.87 mmol of glucose repeating units and a carboxymethyl substitution degree M = 0.88 was dissolved in 40 mL of 50 mM MES aqueous solution. Then, 4.35 mmol of ADH, 0.18 mmol of EDC, and 0.18 mmol of NHS were added sequentially and the solution was reacted at room temperature in the dark for 48 h. The reaction solution was then placed in a dialysis bag with a molecular weight cutoff (MWCO) of 14 kDa and dialyzed with deionized water for 3 days. The dialysate was collected, dried, and CMP-ADH was obtained, with an ADH substitution degree X = 0.16 and a weight-average molecular weight of 206 kDa.

[0069] 2. Preparation of CMP-ADH solution: Weigh 20 mg of CMP-ADH and dissolve it in 0.98 mL of 10 mM PBS buffer (pH=7.4) to obtain a 2 wt% CMP-ADH solution.

[0070] 3. Preparation of γCD-CHO: Weigh 0.77 mmol of γCD and add it to 20 mL of anhydrous DMSO to prepare a γCD solution. Simultaneously, weigh 7.7 mmol of DMP and add it to 10 mL of DMSO to prepare a DMP solution. Add the DMP solution dropwise to the γCD solution. React at room temperature for 4 h. Then, add 5 times the volume of cold acetone to the reaction solution for precipitation, and centrifuge to obtain the crude product. Dissolve the crude product in deionized water and precipitate it again using 5 times the volume of cold acetone. Repeat the precipitation process three times. Dry the last collected precipitate to obtain γCD-CHO with an aldehyde substitution degree Y = 3.07.

[0071] 4. Preparation of γCD-CHO@ICA: 0.07 mmol of γCD-CHO was dissolved in 50 mL of deionized water to obtain a γCD-CHO solution, and 0.07 mmol of ICA was dissolved in 10 mL of anhydrous ethanol to obtain an ICA ethanol solution. The ICA ethanol solution was added dropwise to the γCD-CHO solution, stirred for 24 h, filtered, and the filtrate was collected. After drying, the drug inclusion complex γCD-CHO@ICA was obtained.

[0072] 5. Preparation of γCD-CHO@ICA and γCD-CHO solution: Weigh 20 mg of γCD-CHO@ICA into a container, add 0.98 mL of 10 mM PBS buffer (pH=7.4) to dissolve it, and obtain a γCD-CHO@ICA solution with a concentration of 2 wt%.

[0073] 6. Preparation of pH-responsive injectable drug-loaded hydrogel: Take 100 μL of CMP-ADH solution, 16 μL of γCD-CHO@ICA solution and 16 μL of γCD-CHO solution from step 4, mix them well, and incubate at 25℃ for 1 h. A pH-responsive injectable drug-loaded hydrogel with a solid content of 2 wt% and an ICA concentration of 0.39 mg / mL is formed through hydrazone coupling reaction.

[0074] Example 4: Preparation of pH-responsive injectable drug-loaded hydrogel #4 1. Preparation of CMP-ADH: CMP containing 0.87 mmol of glucose repeating units and a carboxymethyl substitution degree M = 0.88 was dissolved in 40 mL of 10 mM MES aqueous solution. Then, 4.35 mmol of ADH, 0.35 mmol of EDC, and 0.35 mmol of NHS were added sequentially and the solution was reacted at room temperature in the dark for 24 h. The reaction solution was then placed in a dialysis bag with a molecular weight cutoff (MWCO) of 14 kDa and dialyzed with deionized water for 3 days. The dialysate was collected, dried, and CMP-ADH was obtained, with an ADH substitution degree X = 0.31 and a weight-average molecular weight of 224 kDa.

[0075] 2. Preparation of CMP-ADH solution: Weigh 30 mg of CMP-ADH and dissolve it in 0.97 mL of 10 mM PBS buffer (pH=7.4) to obtain a 3 wt% CMP-ADH solution.

[0076] 3. Preparation of γCD-CHO: Weigh 0.77 mmol of γCD and add it to 20 mL of anhydrous DMSO to prepare a γCD solution. Simultaneously, weigh 3.85 mmol of DMP and add it to 10 mL of DMSO to prepare a DMP solution. Add the DMP solution dropwise to the γCD solution. React at room temperature for 2 h. Then, add 5 times the volume of cold acetone to the reaction solution for precipitation, and centrifuge to obtain the crude product. Dissolve the crude product in deionized water and precipitate it again using 5 times the volume of cold acetone. Repeat the precipitation process three times. Dry the precipitate collected in the last step to obtain γCD-CHO with an aldehyde substitution degree Y = 2.02.

[0077] 4. Preparation of γCD-CHO@ICA: 0.07 mmol of γCD-CHO was dissolved in 50 mL of deionized water to obtain a γCD-CHO solution, and 0.07 mmol of ICA was dissolved in 10 mL of anhydrous ethanol to obtain an ICA ethanol solution. The ICA ethanol solution was added dropwise to the γCD-CHO solution, stirred for 24 h, filtered, and the filtrate was collected. After drying, the drug inclusion complex γCD-CHO@ICA was obtained.

[0078] 5. Preparation of γCD-CHO@ICA solution: Weigh 30 mg of γCD-CHO@ICA into a container, add 0.97 mL of 10 mM PBS buffer (pH=7.4) to dissolve it, and obtain a γCD-CHO@ICA solution with a concentration of 3 wt%.

[0079] 6. Preparation of pH-responsive injectable drug-loaded hydrogel: Take 100 μL of CMP-ADH solution and 94 μL of γCD-CHO@ICA solution, mix them well, and incubate at 20℃ for 12 h. A pH-responsive injectable drug-loaded hydrogel with a solid content of 3 wt% and an ICA concentration of 3.4 mg / mL is formed through hydrazone coupling reaction.

[0080] Comparative Example 1: Preparation of pH-responsive injectable drug-loaded hydrogel #5 1. Preparation of CMP-ADH: 0.87 mmol of CMP with a degree of carboxymethyl substitution M=0.88 was dissolved in 40 mL of 10 mM MES aqueous solution. Then, 4.35 mmol of ADH, 0.18 mmol of EDC, and 0.18 mmol of NHS were added sequentially and the solution was dissolved. The reaction was carried out at room temperature in the dark for 24 h. Subsequently, the reaction solution was placed in a dialysis bag with a molecular weight cutoff MWCO=10 kDa and dialyzed with deionized water for 3 days. After drying, CMP-ADH was obtained with a degree of substitution X=0.16 and a weight-average molecular weight of 206 kDa.

[0081] 2. Preparation of CMP-ADH / ICA mixed solution: Weigh 90 mg of CMP-ADH and 1.5 mg of icariin, add 2.91 mL of 10 mM PBS buffer (pH=7.4) to dissolve, and prepare a 3 wt% CMP-ADH / ICA solution.

[0082] 3. Preparation of glutaraldehyde / ICA mixed solution: Weigh 90 mg of glutaraldehyde and 1.5 mg of icariin, add 2.91 mL of 10 mM PBS buffer (pH 7.4) to dissolve, and prepare a glutaraldehyde / ICA mixed solution with a concentration of 3 wt%.

[0083] 4. Preparation of pH-responsive injectable drug-loaded hydrogel: Take 100 μL of CMP-ADH / ICA mixed solution and 12 μL of glutaraldehyde / ICA mixed solution, mix well, and incubate at 37℃ for 0.017 h. A pH-responsive injectable drug-loaded hydrogel with a solid content of 3 wt% and an ICA concentration of 0.05 mg / mL is formed through hydrazone coupling reaction.

[0084] Example 6 Characterization of pH-responsive injectable drug-loaded hydrogels: 1. Determination of the degree of substitution of CMP-ADH and γCD-CHO: The CMP-ADH prepared in Example 1 was dissolved in D2O to prepare a 20 mg / mL CMP-ADH solution. 10 mg / mL of L-phenylalanine was added as an internal control to obtain the CMP-ADH test solution. The γCD-CHO prepared in Example 1 was completely converted to γCD carboxylate (γCD-COOH) using an excess of hydroxylamine compound. D2O was added to prepare a 20 mg / mL γCD-COOH solution. The CMP-ADH test solution and the γCD-COOH solution were detected by 1H NMR spectroscopy, and the corresponding degrees of substitution were calculated.

[0085] The NMR spectrum of CMP-ADH is as follows: Figure 1 As shown, the NMR spectrum of γCD-COOH is as follows: Figure 2 As shown. NMR spectroscopy confirmed that the degree of ADH substitution X of CMP-ADH in Example 1 was 0.16 and the degree of aldehyde substitution Y of γCD-CHO was 3.07.

[0086] 2. Determination of ICA content: 10 mg of ICA was dissolved in 10 mL of DMSO solution to prepare a 1 mg / mL ICA solution. The solution was then successively diluted with DMSO to prepare ICA solutions with concentrations of 0.9 mg / mL, 0.8 mg / mL, 0.7 mg / mL, 0.6 mg / mL, 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL and 0.1 mg / mL. The absorbance at 270 nm was measured using an ELISA reader to establish a standard curve.

[0087] 4 mg of γCD-CHO@ICA from Example 1 was redissolved in 2 mL of DMSO to obtain a γCD-CHO@ICA solution. The absorbance of the γCD-CHO@ICA solution at a wavelength of 270 nm was measured, and the encapsulation efficiency and drug loading of ICA were calculated based on the above standard curve.

[0088] The standard curve of ICA is as follows Figure 3 As shown. The results show that the equation of the ICA standard curve is y = 0.7068x - 0.0095, R0 2 =0.9976. Based on the standard curve, the drug loading of ICA in Example 1 was calculated to be 16.1% and the encapsulation efficiency to be 26.2%. The preparation process of Gel@ICA is as follows... Figure 4 As shown.

[0089] 3. Fourier transform infrared spectra of CMP-ADH, γCD-CHO, and Gel@ICA: Dry CMP-ADH, γCD-CHO, and Gel@ICA were mixed with KBr powder at a mass ratio of 1:100, ground evenly, and then compressed into tablets. Fourier transform infrared spectroscopy was used to analyze the samples at 500 cm⁻¹.-1 ~4000cm -1 The wavenumber range is used to detect the characteristic peaks of the sample.

[0090] Fourier transform infrared spectra of CMP-ADH, γCD-CHO, and Gel@ICA are shown below. Figure 5 As shown, γCD-CHO at 1707 cm⁻¹ -1 The appearance of a C=O stretching vibration peak at 1281 cm⁻¹ indicates that γCD is oxidized to form an aldehyde group; CMP-ADH shows a peak at 1281 cm⁻¹. -1 A stretching vibration signal corresponding to the CN bond on the ADH functional group was observed at 1650 cm⁻¹. -1 and 1535cm -1 The peak at 1610 cm⁻¹ also confirms the presence of the ADH hydrazide group; relative to γCD-CHO and CMP-ADH, the peak at 1610 cm⁻¹ in Gel is [missing information]. -1 A new C=N stretching vibration peak appeared, indicating that Gel@ICA is formed by cross-linking of hydrazone bonds, which gives the Gel@ICA of this invention its pH-responsive properties.

[0091] 4. Rheological characterization of Gel@ICA: Rheological tests were performed on the hydrogel in Example 1 using a Kinexus Lab and a rotational rheometer. After rapid mixing of the hydrogel precursor solution on a plate at room temperature, a time-scan experiment was conducted using an 8 mm parallel plate at a frequency of 1 Hz and a strain of 1%, acquiring storage modulus G′ and loss modulus G″ data. Subsequently, a frequency scan was performed at 1% strain, ranging from 0.1 Hz to 10 Hz, to examine the viscoelastic properties of the hydrogel. At a frequency of 1 Hz and a strain of 1%, the time-scan experiment was completed within 0.1 s... -1 ~40s -1 Shear tests were performed at shear rates of 1 Hz to evaluate the shear thinning properties of the hydrogel. Amplitude strain scanning was conducted at a frequency of 1 Hz, ranging from 0.1% to 300%, to evaluate the linear viscoelastic region of the hydrogel. Finally, alternating strain scans of 1% low strain and 120% high strain were performed to evaluate the self-healing ability of the hydrogel.

[0092] The rheological time-series scan results of Gel@ICA are as follows: Figure 6 As shown in the figure. The results show that the storage modulus G′ and loss modulus G″ of Gel@ICA increase significantly with the increase of solid content. When the solid content is fixed at 3wt%, the hydrogel exhibits a moderate storage modulus G′, which is about 502.7 Pa.

[0093] The rheological frequency scan results of Gel@ICA are as follows: Figure 7 As shown in the figure, the results indicate that Gel@ICA exhibits a significant frequency dependence.

[0094] The rheological amplitude scan and high / low strain cyclic scan results of Gel@ICA are as follows: Figure 8 As shown in the figure. The results show that when the shear strain is greater than 100%, the storage modulus G′ of Gel@ICA is less than the loss modulus G″, indicating that the gel network of Gel@ICA dissociates under high strain. In the high and low strain cyclic scanning, when the shear strain recovers from 120% to 1%, the storage modulus of the hydrogel instantly recovers to its initial value, indicating that the gel network can recover rapidly after being damaged.

[0095] Viscosity scans of pH-responsive injectable drug-loaded hydrogels at different shear rates are shown in the figure below. Figure 9 As shown in the figure. The results show that Gel@ICA exhibits significant shear-thinning properties and has injection potential.

[0096] Injection demonstration of pH-responsive injectable drug-loaded hydrogels, as shown below. Figure 10 As shown in the figure. The results show that it can be successfully injected through a 22G needle, and the ICA contained in the injected Gel@ICA remains stable and does not precipitate out. This ensures the stability of ICA during the injection process and lays the foundation for stable drug release in subsequent treatment of SCI.

[0097] Example 7 Performance testing of pH-responsive injectable drug-loaded hydrogel: 1. Adhesion test of pH-responsive injectable drug-loaded hydrogel: After staining the Gel@ICA precursor solution from Example 1 with methylene blue, it was placed on rectangular pigskin and muscle tissue, respectively. After the hydrogel was fully cured, the tissues were inverted, folded, twisted, and rinsed with water to test the adhesion of Gel@ICA. Furthermore, the methylene blue-stained hydrogel precursor solution was placed on plastic, glass, metal, and wood surfaces, respectively. After the Gel@ICA was fully cured, the various materials were inverted to evaluate the adhesion of Gel@ICA.

[0098] Gel@ICA adhesion test, such as Figure 11 As shown in the image, test results indicate that Gel@ICA exhibits good tissue adhesion on materials such as pigskin, pork, plastic, glass, metal, and wood. This suggests that Gel@ICA does not easily disperse in tissue and can concentrate in one location. Therefore, Gel@ICA can precisely and continuously release ICA at the lesion site during SCI treatment.

[0099] 2. Mass degradation of Gel@ICA: 500 μL of Gel@ICA from Example 1 was placed in a 10 mL centrifuge tube and allowed to mature completely. 5 mL of PBS buffer (pH 6.5 and 7.4) was added, respectively. The tubes were then placed in a shaker at 120 rpm and 37°C. Gel@ICA was removed from the centrifuge tubes at 1 h, 3 h, 7 h, 12 h, 1 d, 2 d, 3 d, 5 d, and 7 d, and the remaining mass of Gel@ICA was recorded. Each experiment was repeated three times.

[0100] The mass degradation curve of Gel@ICA is as follows: Figure 12 As shown in the results, Gel@ICA was almost completely degraded within 7 days at pH 6.5, while at pH 7.4, the swelling rate remained around 200% on day 7. This demonstrates that Gel@ICA exhibits good responsiveness to slightly acidic environments. Therefore, the degradation of Gel@ICA in the slightly acidic environment of SCI can release ICA, achieving a therapeutic effect on SCI.

[0101] 3. Releasing ICA in Gel@ICA: 500 μL of Gel@ICA from Example 1 was placed in a 10 mL centrifuge tube and allowed to mature completely. 5 mL of PBS buffer at pH 6.5 and 7.4 were added, respectively. The tubes were then placed in a shaker at 120 rpm and 37°C. At 1 h, 3 h, 7 h, 12 h, 1 d, 2 d, 3 d, 5 d, and 7 d, 2 mL of supernatant was collected from the centrifuge tube, and 2 mL of PBS buffer at the corresponding pH was added. The absorbance of the supernatant at 270 nm was measured, and the cumulative release was calculated based on the ICA standard curve. Each experiment was repeated three times.

[0102] The release curve of ICA in Gel@ICA is as follows Figure 13 As shown in the figure, the results indicated that ICA release also exhibited a slightly acidic environment responsiveness. At pH 6.5, 53.1% of ICA was released on day 1, 10.8% on day 2, 11.1% on day 3, 8.7% on day 5, and 7.2% on day 7, for a total release of 90.9% of ICA over the first 7 days. In contrast, the group at pH 7.4 released only 42.0% of ICA over 7 days. This demonstrates that Gel@ICA releases ICA in the slightly acidic environment of SCI with a rapid initial release followed by a slower release, and releases over 90% of the ICA within 7 days. Therefore, Gel@ICA can rapidly release ICA to treat SCI in the early stages of injection and can also prolong the duration of its therapeutic effect by continuously releasing ICA after injection.

[0103] 4. Blood compatibility assessment of Gel@ICA: 500 μL of mouse whole blood was collected in an anticoagulant tube and then dispersed in 5 mL of 10 mM PBS buffer (pH 7.4). The mixture was centrifuged at 2500 rpm for 5 min, and red blood cells were collected. This centrifugation was repeated three times. At the end of the last centrifugation, the red blood cells were collected and resuspended in 10 mL of physiological saline. 500 μL of the red blood cell suspension was mixed with 500 μL of the hydrogel extract from Example 1 at concentrations of 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, and 6.25 mg / mL, respectively. A mixture of 500 μL of red blood cell suspension and 500 μL of physiological saline served as a negative control; a mixture of 500 μL of red blood cell suspension and 500 μL of ultrapure water served as a positive control. The mixtures were incubated at 37°C and 120 rpm for 1 h, and then centrifuged at 10000 rpm for 10 min. Hemolysis images of different groups were collected. Take the supernatant of the centrifuged sample after incubation and measure its absorbance at a wavelength of 540 nm to calculate the hemolysis rate.

[0104] The blood compatibility assessment results of Gel@ICA are as follows: Figure 14 As shown in the figure. The results showed that red blood cells from different sample groups at all concentrations aggregated at the bottom of the centrifuge tubes, and no hemolysis occurred. The hemolysis rate determination results of Gel@ICA in this embodiment of the invention are as follows. Figure 15 As shown in the figure. The results showed that the hemolysis rate in each group was less than 5%, indicating that Gel@ICA has good blood compatibility and meets the standards for safe biomaterials.

[0105] 5. Total antioxidant capacity of Gel@ICA: First, mix 40 μL of ABTS solution with 40 μL of oxidant solution thoroughly and let stand at room temperature in the dark for 12 h. Dilute it 30-50 times with 10 mM PBS solution (pH 7.4) to obtain an absorbance of 0.7 ± 0.05 at 734 nm, thus obtaining the ABTS working solution. Add 200 μL of ABTS working solution to a 96-well plate, followed by adding 10 μL of 100 mg / mL Gel, γCD-CHO@ICA, and Gel@ICA from Example 1 to the wells, respectively. Add 10 μL of 10 mM PBS buffer (pH 7.4) to the blank control wells. Establish a Trolox standard curve: Add 10 μL of Trolox standard solution at concentrations of 0.15 mM, 0.3 mM, 0.6 mM, 0.9 mM, 1.2 mM, and 1.5 mM to the 96-well plate, respectively. After incubation at room temperature for 5 minutes, the absorbance at 734 nm was measured. A standard curve was constructed based on the concentration and absorbance of the Trolox standard solution. The total antioxidant capacity of the hydrogel extract was calculated based on the standard curve.

[0106] The total antioxidant capacity results of Gel@ICA are as follows: Figure 16 As shown. The Trolox standard curve is as follows. Figure 17 As shown, the equation of the curve is y = -0.5133x + 0.643, R. 2 =0.9921. The total antioxidant capacity calculated from the standard curve shows that the total antioxidant capacity of the Gel group was 0.0048 mM / g, while the total antioxidant capacities of γCD-CHO@ICA and Gel@ICA reached 0.1055 mM / g and 0.1076 mM / g, respectively. This indicates that ICA endows Gel@ICA with good antioxidant capacity, which is ideal for mitigating oxidative stress damage caused by SCI injury.

[0107] Example 8: Effect of pH-responsive injectable drug-loaded hydrogel on promoting SCI repair in mice: The steps for establishing a mouse SCI model are as follows: Eight-week-old C57BL / 6 mice, weighing approximately 20 grams, were selected. The mice were anesthetized with isoflurane. The mice were fixed to the operating table, their back hair was shaved, and the surgical area was disinfected with povidone-iodine. The spine was exposed: the skin was incised along the midline of the back, the muscles were separated, and the T9 lamina was exposed. The T9 lamina was carefully removed, taking care not to damage the spinal cord. The dura mater was incised, and approximately 2 mm of spinal cord tissue was aspirated. Gel@ICA was implanted into the SCI area. The muscles and skin were sutured.

[0108] Experimental grouping: Mice were randomly divided into three groups: Control group received only SCI without additional intervention. Comparative example 1 group received the hydrogel from Comparative example 1, which was transplanted to the center of the lesion, denoted as Gel+ICA. Example 1 group received the Gel@ICA from Example 1, which was transplanted to the center of the lesion, denoted as Gel@ICA.

[0109] Postoperatively, the Basso Mouse Motor Function Rating Scale was used to assess the hind limb motor function of mice, with assessments conducted weekly for 4 weeks.

[0110] Histological evaluation: Seven days post-surgery, mice were fixed by cardiac perfusion with 4% paraformaldehyde, and spinal cord tissue was taken for frozen sections, DCF immunofluorescence staining, and the ability of hydrogel to clear ROS was evaluated.

[0111] Twenty-eight days post-surgery, mice underwent cardiac fixation with 4% paraformaldehyde. Spinal cord tissue was harvested for frozen sectioning, and immunofluorescence staining was performed for microtubule-associated protein (GFAP) and glial fibrillary acidic protein (GAPI). Cell nuclei were labeled with 4',6-diamino-2-phenylindole (DAPI) to observe nerve regeneration. GFAP was observed as red, TAPI as green, and DAPI as blue.

[0112] Twenty-eight days post-surgery, internal organs were harvested from the animals and stained with hematoxylin and eosin (HE) to assess the biocompatibility of the hydrogel in mice.

[0113] Immunofluorescence detection of ROS expression in mouse spinal cord tissue 7 days post-surgery is as follows: Figure 18 As shown, DCF is displayed in red, and DAPI-labeled cell nuclei are displayed in blue. Results showed that, compared to the Control group and Comparative Example 1, Example 1 group significantly reduced ROS expression levels in the SCI region.

[0114] BMS scores of mice from day 1 to day 28 after SCI are as follows: Figure 19 As shown, the results indicated that on day 28 post-surgery, the functional recovery of mice in Group 1 after SCI was superior to the other two groups. This suggests that pH-responsive injectable drug-loaded hydrogels can promote the recovery of motor function in mice.

[0115] The results of immunofluorescence staining of spinal cord tissue in mice 28 days after SCI in each group are as follows: Figure 20 As shown in the figure, GFAP is displayed in red, Tuj1 in green, and DAPI-labeled cell nuclei in blue. The results showed that the fluorescence intensity of Tuj1 in the spinal cord of mice in Example 1 group was significantly higher than that in the other two groups. This result indicates that Gel@ICA can significantly promote neurogenesis after SCI in mice.

[0116] On day 28 post-surgery, the mice were euthanized, and their major organs, including the heart, liver, spleen, lungs, and kidneys, were removed. These organs were then stained with hematoxylin and eosin to assess the safety of the hydrogel treatment. [Further details about the procedure are needed for accurate translation.] Figure 21 Observations revealed that no toxic effects of Gel@ICA treatment were observed in HE-stained sections of major organs of mice treated with Gel@ICA, further confirming the safety of Gel@ICA treatment.

[0117] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0118] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pH-responsive injectable drug-loaded hydrogel, characterized in that, Including carriers loaded with icariin; The carrier is formed by grafting carboxymethyl poria cocos polysaccharide with adipic acid dihydrazide and aldehyde-modified γ-cyclodextrin via a hydrazone coupling reaction.

2. The pH-responsive injectable drug-loaded hydrogel according to claim 1, characterized in that, The concentration of icariin is 0.05~3.4 mg / mL.

3. The pH-responsive injectable drug-loaded hydrogel according to claim 1, characterized in that, The weight-average molecular weight of the carboxymethyl poria polysaccharide grafted with adipate dihydrazide is 206 kDa to 224 kDa. The degree of carboxymethyl substitution is 0.88, based on the number of moles of carboxymethyl groups per mole of repeating unit. The degree of substitution of adipic dihydrazide is 0.16 to 0.31, based on the number of moles of adipic dihydrazide groups per mole of repeating unit.

4. The pH-responsive injectable drug-loaded hydrogel according to claim 1, characterized in that, The aldehyde-substituted γ-cyclodextrin has a degree of aldehyde substitution of 2.02 to 3.07, based on the number of aldehyde groups on each γ-cyclodextrin molecule.

5. A method for preparing a pH-responsive injectable drug-loaded hydrogel according to any one of claims 1-4, characterized in that, include: S1: The carboxymethyl poria cocos polysaccharide grafted with adipate dihydrazide was prepared by reacting carboxymethyl poria cocos polysaccharide with adipate dihydrazide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. S2: The aldehyde-modified γ-cyclodextrin was prepared by reacting γ-cyclodextrin with Dess-Martin reagent; S3: The aldehyde-modified γ-cyclodextrin was mixed with icariin to prepare an aldehyde-modified γ-cyclodextrin inclusion complex of icariin. S4: The aldehyde-modified γ-cyclodextrin inclusion complex of the icariin was mixed with carboxymethyl poria cocos polysaccharide grafted with adipic acid dihydrazide to obtain the pH-responsive injectable drug-loaded hydrogel.

6. The preparation method according to claim 5, characterized in that, The molar ratio of carboxymethyl poria cocos polysaccharide, adipic acid dihydrazide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide in S1 is 1:5:0.21~0.4:0.21~0.

4.

7. The preparation method according to claim 5, characterized in that, The molar ratio of aldehyde-modified γ-cyclodextrin to icariin in S3 is 1:0.5~2; preferably, the molar ratio is 1:

1.

8. The preparation method according to claim 5, characterized in that, The molar ratio of the aldehyde group in S4 to the hydrazine group in carboxymethyl poria cocos polysaccharide grafted adipic acid dihydrazide is 1:0.5~2; preferably, the molar ratio is 1:

1.

9. A pH-responsive injectable drug-loaded hydrogel, characterized in that, It is obtained by the preparation method described in any one of claims 5-8.

10. The use of the pH-responsive injectable drug-loaded hydrogel of any one of claims 1-4 or 9 in the preparation of drugs for the prevention and / or treatment of spinal cord injury.

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