An active oxygen response type glycyrrhizin anti-inflammatory hydrogel and its application in the preparation of a drug for treating compression neuropathic pain

Abstract

This invention discloses a reactive oxygen species (ROS) responsive glycyrrhizic acid anti-inflammatory hydrogel and its application in the preparation of drugs for treating compressive neuropathic pain. Belonging to the field of biomedical materials technology, the hydrogel uses glycyrrhizic acid and 1,4-phenylboronic acid as raw materials. It is heated in an alkaline environment until a homogeneous mixed solution is formed, and then naturally cooled to room temperature to obtain the ROS responsive glycyrrhizic acid anti-inflammatory hydrogel. This hydrogel possesses good injectability, self-healing properties, biocompatibility, and biodegradability. It can specifically cleave borate ester bonds in the highly reactive oxygen species pathological microenvironment of diabetic patients with lumbar disc herniation, releasing glycyrrhizic acid on demand. By targeting and regulating the PI3K-AKT signaling pathway, it promotes the polarization of macrophages from a pro-inflammatory M1 phenotype to a reparative M2 phenotype, significantly inhibiting the inflammatory response of the dorsal root ganglion and the dorsal horn of the spinal cord, effectively relieving compressive neuropathic pain in the dorsal root ganglion caused by diabetic patients with lumbar disc herniation, and has good prospects for clinical translation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel and its application in the preparation of drugs for treating compressive neuropathic pain. Background Technology

[0002] Lumbar disc herniation is a common degenerative spinal disease in adults. Its pathological feature is the protrusion of the nucleus pulposus, annulus fibrosus, and endplate cartilage into the spinal canal or neural foramen, causing mechanical compression and chemical stimulation of adjacent nerve roots, triggering local and systemic inflammatory responses. Clinically, it manifests as radiating pain in the lower extremities (sciatica), severely impacting the patient's function and work ability. The dorsal root ganglia, as a key hub for sensory transmission, are highly sensitive to mechanical and chemical stimuli. Chronic compression can create a pro-inflammatory and nociceptive microenvironment, lowering the pain threshold and amplifying nociceptive signal transmission.

[0003] Metabolic diseases such as diabetes can lead to reactive oxygen species (ROS) overload and chronic oxidative stress, forming a synergistic pain mechanism of "mechanical first blow – metabolic second blow" with the mechanical compression of lumbar disc herniation. This pathological microenvironment causes macrophage polarization to tilt towards the pro-inflammatory M1 phenotype and inhibit the reparative M2 phenotype, resulting in a large release of pro-inflammatory mediators, exacerbating peripheral and central sensitization, and ultimately causing focal radicular pain to develop into widespread, refractory neuropathic pain. Diabetic lumbar disc herniation-induced compressive neuropathic pain has the dual pathological characteristics of "high ROS + mechanical compression," which is fundamentally different from simple spinal cord injury and ordinary neuropathic pain. Current treatment methods are difficult to adapt to this special microenvironment.

[0004] Currently, epidural steroid injections are a routine interventional method for relieving radicular inflammation and sciatica. By delivering medication locally, high drug concentrations are achieved around the affected nerve, rapidly suppressing inflammation. However, this method has a short-lived effect and requires repeated invasive injections, increasing medical costs and posing risks such as bleeding, infection, and nerve damage. Therefore, developing novel drug delivery systems that respond to the pathological microenvironment, prolong local therapeutic effects, and reduce the frequency of invasive administration has become an urgent need for translational clinical research on metabolic diseases complicated by nerve compression pain.

[0005] Boronate ester bonds exhibit high sensitivity to biomarkers such as reactive oxygen species, pH, and glucose, and phenylboronic acid and its derivatives have a high affinity for vicinal diols, making them widely used in stimulus-responsive drug delivery. Hydrogels, due to their excellent biocompatibility and tunable physicochemical properties, are ideal carriers for constructing dynamic boronate ester bonds, achieving intelligent responses to pathological microenvironments, and controlling drug release. Glycyrrhizic acid, as a natural anti-inflammatory small molecule, can target and regulate the PI3K-AKT signaling pathway, affecting macrophage polarization balance.

[0006] Existing hydrogel drug delivery systems generally employ a separate design of "carrier material + loaded drug," requiring multiple steps such as chemical grafting and physical embedding to load the drug. This results in problems such as low drug loading rate, uneven drug distribution, complex preparation processes, and residual organic solvents. The chemical grafting method, in particular, is cumbersome and requires harsh reaction conditions, making it difficult to scale up industrially. Furthermore, current technologies have not designed drug delivery systems specifically for the unique pathological microenvironment of "high reactive oxygen species + mechanical compression" in diabetic patients with lumbar disc herniation.

[0007] Therefore, developing a drug delivery system that combines the functions of a scaffold and a drug, employs a green and simplified preparation process, is adapted to the pathological microenvironment of highly reactive oxygen species, and can achieve multiple effects such as macrophage polarization regulation, peripheral inflammation inhibition, central sensitization relief, and nerve function repair, is of great significance for solving the bottleneck problem in the current treatment of lumbar disc herniation compressive nerve pain in diabetic patients, improving analgesic effects, and reducing the side effects of invasive drug administration. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention proposes a reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel and its application in the preparation of drugs for treating compressive neuropathic pain. The hydrogel provided by this invention is a reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel that uses glycyrrhizic acid as both the gel framework and the sole active drug, and is prepared using a one-pot, green preparation method. The hydrogel provided by this invention can precisely treat compressive neuropathic pain in the dorsal root ganglion caused by lumbar disc herniation complicated by diabetes.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing a reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel. The method uses glycyrrhizic acid and 1,4-phenylboronic acid as raw materials, and heats them in a water bath under an alkaline environment until the glycyrrhizic acid is completely dissolved to form a homogeneous mixed solution. Then, the solution is naturally cooled to room temperature, and a three-dimensional network gel structure is formed by cross-linking of borate esters through dynamic covalent bonds to obtain the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel. The mass ratio of glycyrrhizic acid to 1,4-phenylboronic acid is (7.5~12.5):1.

[0010] This invention uses glycyrrhizic acid and 1,4-phenylboronic acid as core raw materials. Glycyrrhizic acid serves as both a building block of the hydrogel framework and the sole active anti-inflammatory and analgesic drug. Under mild, alkaline conditions, it is dissolved in a water bath and cross-linked with borate esters via dynamic covalent bonds to form a three-dimensional network gel structure, thus producing a reactive oxygen species (ROS) responsive glycyrrhizic acid anti-inflammatory hydrogel. This invention breaks away from the traditional technical bias of "carrier and drug separation." By using glycyrrhizic acid as both a building block of the hydrogel framework and the sole active anti-inflammatory and analgesic drug, the hydrogel is synthesized in a one-pot, green process. The resulting hydrogel exhibits ROS responsiveness, injectability, self-healing properties, high drug loading capacity, and good biocompatibility. It can precisely cleave borate ester bonds and release glycyrrhizic acid on demand in the highly reactive oxygen species-rich microenvironment of diabetes, effectively relieving neuropathic pain by regulating macrophage polarization. This provides a minimally invasive, intelligent, and long-lasting local treatment strategy for this type of disease, achieving a synergistic effect of precise response, long-lasting analgesia, and nerve repair.

[0011] Traditional hydrogels require the independent synthesis of polymer carriers, followed by drug loading through physical embedding or chemical grafting, resulting in low drug loading rates and complex processes. In this invention, glycyrrhizic acid combines structural support and pharmacological activity, eliminating the need for additional carrier skeletons and post-drug loading steps, achieving 100% in-situ drug loading, with the drug uniformly distributed within the gel skeleton, and eliminating the risk of drug leakage and burst release.

[0012] The method of this invention is compared with the existing chemical grafting method as follows: Traditional stimulus-responsive hydrogels require multiple reaction steps, organic solvents, purification and separation, which are cumbersome and difficult to scale up; This invention uses a one-pot green synthesis of hydrogels, with alkaline solution (dilute alkaline aqueous solution) as the only solvent. The reaction conditions are mild, and no organic solvents, catalysts, or purification steps are required. The operation is simple, the cost is low, and it is environmentally friendly, with significant advantages for industrial applications.

[0013] Furthermore, the mass ratio of glycyrrhizic acid to 1,4-phenylboronic acid is 7.5:1, 10:1, or 12.5:1.

[0014] Furthermore, an alkaline environment is created using an alkaline solution, wherein the alkaline solution is a KOH solution.

[0015] Furthermore, the concentration of the KOH solution is 0.05 mol / L.

[0016] Furthermore, the water bath heating temperature is 90~100℃, preferably 95℃.

[0017] The present invention also provides a reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel prepared according to the above method.

[0018] The present invention also provides the application of the above-mentioned reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel in the preparation of a drug for treating compressive neuropathic pain.

[0019] Furthermore, the medication for treating compressive nerve pain is a medication for treating compressive nerve pain in the dorsal root ganglion caused by lumbar disc herniation complicated by diabetes.

[0020] The present invention also provides a medicament for treating compressive neuralgia, the active ingredient of which is the above-mentioned reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel.

[0021] Furthermore, the medication for treating compressive neuralgia also includes pharmaceutically acceptable excipients.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects: This invention crosslinks glycyrrhizic acid with 1,4-phenylboronic acid via dynamic borate ester bonds to construct a reactive oxygen species (ROS)-responsive anti-inflammatory hydrogel, achieving precise local delivery of glycyrrhizic acid. Compared to existing neuropathic pain treatments such as epidural steroid injections and conventional drug delivery systems, the ROS-responsive glycyrrhizic acid anti-inflammatory hydrogel prepared by this invention possesses ideal characteristics, including being green and easy to prepare, having a 100% ultra-high drug loading capacity, precise controlled release dependent on ROS, adjusting the release rate to match the pathological microenvironment, avoiding drug waste, significantly improving analgesic efficacy, and greatly reducing the adverse side effects of invasive repeated administration. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 Comparative images show the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogels prepared in Examples 1-3, the glycyrrhizic acid-1,4-phenylboronic acid system prepared in Comparative Examples 1-2, and the glycyrrhizic acid system without 1,4-phenylboronic acid prepared in Comparative Examples 3-7.

[0024] Figure 2 The results of the reactive oxygen species responsiveness verification experiment of the glycyrrhizic acid anti-inflammatory hydrogel prepared in Example 1 are shown in (a) as the release curve (drug / solute release) of the hydrogel in different solutions (PBS, 0.1mM H2O2, 1mM H2O2, 10mM H2O2), and (b) as the remaining mass of the hydrogel on day 1 and day 5 after soaking in different solutions (PBS, 0.1mM H2O2, 1mM H2O2, 10mM H2O2). Figure 3The experimental results of the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel prepared in Example 1 in terms of anti-inflammatory, biocompatibility and in vivo tissue repair are shown in (a) cell viability / proliferation fluorescence staining results, (b) CCK-8 analysis results, (c) hemolysis test results (scale bar is 1 cm), and (d) representative images of subcutaneous implantation of hydrogel in rats at different time points, as well as H&E staining, inducible nitric oxide synthase and arginase-1 immunofluorescence results.

[0025] Figure 4 The expression of pro-inflammatory / anti-inflammatory related gene mRNAs in the control group, lipopolysaccharide group, glycyrrhizic acid aqueous solution group, and glycyrrhizic acid hydrogel group during the regulation of macrophage polarization (from pro-inflammatory M1 type to anti-inflammatory repair M2 type).

[0026] Figure 5 Gene expression heatmaps for the control group, lipopolysaccharide group, glycyrrhizic acid aqueous solution group, and glycyrrhizic acid hydrogel group during the regulation of macrophage polarization (from pro-inflammatory M1 type to anti-inflammatory repair M2 type).

[0027] Figure 6 This study presents the immunoblotting results of the blank group, lipopolysaccharide group, glycyrrhizic acid aqueous solution group, and glycyrrhizic acid hydrogel group in regulating macrophage polarization (from pro-inflammatory M1 type to anti-inflammatory repair M2 type).

[0028] Figure 7 The study quantitatively detected M1 markers in the cell matrix of different treatment groups (blank group, lipopolysaccharide group, glycyrrhizic acid aqueous solution group, and glycyrrhizic acid hydrogel group) when regulating macrophage polarization (from pro-inflammatory M1 type to anti-inflammatory repair M2 type).

[0029] Figure 8 The results of immunofluorescence staining for inducible nitric oxide synthase and arginase-1 are shown in the blank group, lipopolysaccharide group, glycyrrhizic acid aqueous solution group, and glycyrrhizic acid hydrogel group.

[0030] Figure 9 To analyze the expression of M1 (CD86) and M2 (CD206) markers in the cell matrix of different treatment groups (blank group, lipopolysaccharide group, glycyrrhizic acid aqueous solution group, and glycyrrhizic acid hydrogel group) by flow cytometry.

[0031] Figure 10 This study aimed to quantify the immunofluorescence intensity of inducible nitric oxide synthase and arginase-1, as well as the results of M1 (CD86) and M2 (CD206) markers in the blank group, lipopolysaccharide group, glycyrrhizic acid aqueous solution group, and glycyrrhizic acid hydrogel group using flow-through cytometry.

[0032] Figure 11The functional recovery of rats in different groups (blank group, sham operation group, glycyrrhizic acid aqueous solution group, and glycyrrhizic acid hydrogel group) is shown in (a)-(d), where (a)-(d) are the assessment results of motor function, mechanosensory function and thermal sensation function of rats in different groups at specific time points after surgery; (e)-(f) are representative images of paw prints of different groups one week after surgery and their quantitative analysis results; and (g)-(h) are the electromyography results and quantitative analysis results of action potential amplitude of different groups one week after surgery.

[0033] Figure 12 The histological evaluation results of the dorsal root ganglion and spinal cord dorsal horn in different groups (blank group, sham operation group, glycyrrhizic acid aqueous solution group, and glycyrrhizic acid hydrogel group) are shown in (a) for immunofluorescence labeling of macrophage polarization markers in the dorsal root ganglion, (b) for immunofluorescence labeling of inflammatory cytokines in the spinal cord dorsal horn, (c) for quantitative analysis of immunofluorescence signal intensity, and (d) for detection of pain-related genes in the dorsal root ganglion by reverse transcription-quantitative polymerase chain reaction. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] The present invention provides a method for preparing a reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel. The method uses glycyrrhizic acid and 1,4-phenylboronic acid as raw materials, and heats them in a water bath under an alkaline environment until the glycyrrhizic acid is completely dissolved to form a homogeneous mixed solution. Then, the solution is naturally cooled to room temperature, and a three-dimensional network gel structure is formed by cross-linking of borate esters through dynamic covalent bonds to obtain a reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel. The mass ratio of glycyrrhizic acid to 1,4-phenylboronic acid is (7.5~12.5):1.

[0040] This invention provides a simple, mild, one-pot, green method for preparing hydrogels, achieving in-situ loading of glycyrrhizic acid during gel formation without subsequent drug loading steps. The drug loading efficiency reaches 100%, and the drug is uniformly distributed. The resulting hydrogel exhibits reactive oxygen species (ROS) responsive drug release, injectability, and self-healing properties. This invention utilizes the covalent bonding between the vicinal diol structure of glycyrrhizic acid and the borate group of 1,4-phenylboronic acid to form dynamic borate ester crosslinks, constructing a stable three-dimensional hydrogel network. These borate ester bonds can specifically cleave in the pathological microenvironment rich in ROS, achieving on-demand controlled release of glycyrrhizic acid. Simultaneously, glycyrrhizic acid, as a crucial component of the gel framework, serves both drug activity and structural support functions.

[0041] In a preferred embodiment of the present invention, in a homogeneous mixed solution, the concentration of 1,4-phenylboronic acid is 4 mg / mL, and the concentration of glycyrrhizic acid is 30-50 mg / mL, with an optimal concentration of 30 mg / mL. This ratio ensures that the boric acid groups and the vicinal diol groups of glycyrrhizic acid fully combine to form crosslinking points of suitable density. This guarantees both the mechanical stability and gelation efficiency of the hydrogel, and also ensures efficient bond cleavage and drug release in an active oxygen environment, avoiding gelation failure or insufficient responsiveness caused by imbalance in the ratio.

[0042] In a preferred embodiment of the present invention, the mass ratio of glycyrrhizic acid to 1,4-phenylboronic acid is 7.5:1, 10:1, or 12.5:1.

[0043] In a preferred embodiment of the present invention, an alkaline environment is created using an alkaline solution, which is a KOH solution.

[0044] In a preferred embodiment of the present invention, the concentration of the KOH solution is 0.05 mol / L, and the KOH solution at this concentration... + The content is far below the normal intracellular level, posing no risk of cytotoxicity, and can provide a mild alkaline reaction environment for the formation of borate ester bonds, avoiding the destruction of the activity of raw materials by strongly alkaline conditions.

[0045] In a preferred embodiment of the invention, the water bath heating temperature is 90~100℃, preferably 95℃. Heating in the water bath is sufficient until the glycyrrhizic acid is completely dissolved; there is no fixed heating time. After dissolution, the mixture is allowed to cool and gel naturally. The heating temperature of 95℃ rapidly promotes the dissolution of glycyrrhizic acid, ensuring thorough mixing of the raw materials. Furthermore, this temperature does not impair the formation of borate ester bonds. Natural cooling allows the cross-linking reaction to proceed slowly, resulting in a uniformly structured gel network.

[0046] An exemplary method for preparing a reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel according to an embodiment of the present invention includes the following steps: (1) Take 1 mL of 0.05 mol / L KOH solution, mix it with 4 mg of 1,4-phenylboronic acid, and heat it in a 95℃ constant temperature water bath until the solution is completely clear to obtain a 1,4-phenylboronic acid alkaline solution; (2) Take 10~50mg of glycyrrhizic acid and add it to the 1,4-phenylboronic acid alkaline solution obtained in step (1). Continue to heat it in a 95℃ constant temperature water bath until the glycyrrhizic acid is completely dissolved and a homogeneous mixed solution is formed. (3) Remove the mixed solution from the constant temperature water bath in step (2) and let it cool naturally to room temperature. During the cooling process, the mixed solution forms a three-dimensional network gel structure through dynamic cross-linking of borate ester bonds, thus obtaining reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel.

[0047] The raw materials used in this invention are all commercially available conventional reagents, which are easy to obtain and have high biosafety.

[0048] Embodiments of the present invention also provide a reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel prepared according to the above method.

[0049] The reactive oxygen species (ROS) responsive glycyrrhizic acid anti-inflammatory hydrogel of the present invention is a smart hydrogel with ROS responsiveness, 100% high drug loading capacity and multifunctional anti-inflammatory and analgesic properties. It can achieve on-demand controlled release and targeted therapy of glycyrrhizic acid in the pathological microenvironment. The gel network is constructed based on glycyrrhizic acid-1,4-phenylboronic acid borate ester bond and has ROS-sensitive cleavage characteristics (accelerated release in the pathological microenvironment of high oxidative stress in diabetes). Glycyrrhizic acid targets and inhibits the PI3K-AKT signaling pathway, achieving synergistic therapeutic effects of macrophage polarization regulation, inflammation suppression and nerve function repair.

[0050] Embodiments of the present invention also provide the application of the above-mentioned reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel in the preparation of a drug for treating compressive neuropathic pain.

[0051] In a preferred embodiment of the present invention, the drug for treating compressive nerve pain is a drug for treating compressive nerve pain of the dorsal root ganglion caused by lumbar disc herniation complicated by diabetes.

[0052] The reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel prepared in this invention possesses excellent injectability, self-healing properties, biocompatibility, and biodegradability. It can specifically cleave borate ester bonds in the highly reactive oxygen species-rich pathological microenvironment of diabetic lumbar disc herniation, releasing glycyrrhizic acid on demand. By targeting and regulating the PI3K-AKT signaling pathway, it promotes the polarization of macrophages from a pro-inflammatory M1 phenotype to a reparative M2 phenotype, significantly inhibiting the inflammatory response of the dorsal root ganglion and the dorsal horn of the spinal cord, effectively relieving neuropathic pain caused by dorsal root ganglion compression due to diabetic lumbar disc herniation. This invention solves the technical problems of existing epidural steroid injections, such as short efficacy, the need for repeated invasive administration, and the risk of puncture. It provides a minimally invasive, intelligent, and long-lasting local treatment option for metabolic diseases complicated by neuropathic pain, and has good prospects for clinical translation.

[0053] Embodiments of the present invention also provide a medicament for treating compressive neuralgia, the active ingredient of which is the above-mentioned reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel.

[0054] In a preferred embodiment of the invention, the medicament for treating compressive neuralgia further includes pharmaceutically acceptable excipients.

[0055] This invention prepares a reactive oxygen species (ROS)-responsive glycyrrhizic acid anti-inflammatory hydrogel that overcomes the traditional technical bias of "carrier-drug separation." Glycyrrhizic acid simultaneously serves as both the hydrogel's structural unit and the sole anti-inflammatory active drug, eliminating the need for additional carriers and post-loading, achieving 100% in-situ drug loading. The drug and matrix work synergistically, avoiding problems such as carrier waste, drug leakage, and burst release. Compared to existing complex processes like chemical grafting and multi-step loading, this invention employs a one-pot green synthesis method using dilute alkaline aqueous solution as a solvent. The process is mild, free of organic solvents and cumbersome purification steps, significantly simplifying the preparation process, reducing costs, and improving biocompatibility, making it suitable for industrial production. This invention uses glycyrrhizic acid as a natural anti-inflammatory ligand, synthesizing it with 1,4-phenylboronic acid to obtain a high-drug-loading sustained-release system dependent on ROS through green synthesis. Glycyrrhizic acid can target and bind to PI3K protein, inhibiting excessive activation of the PI3K-AKT signaling pathway, effectively regulating macrophage polarization balance, and promoting the conversion of the pro-inflammatory M1 phenotype to the reparative M2 phenotype. Designed specifically for the unique pathological microenvironment of "high reactive oxygen species + mechanical compression" in diabetic patients with lumbar disc herniation, the borate ester bonds can specifically cleave under high reactive oxygen species conditions. This modulates the drug release rate according to the reactive oxygen species level at the lesion site. The active ingredient and responsive carrier synergistically relieve nerve compression pain, achieving on-demand controlled release driven by the pathological microenvironment. This is fundamentally different from conventional neuropathic pain treatment materials, making the switch less obvious. Compared to epidural steroid injections, this method achieves minimally invasive single-dose administration, long-lasting analgesia, and nerve repair, avoiding the risks of repeated punctures and systemic side effects, thus improving the risks of repeated punctures and systemic side effects caused by traditional invasive drug delivery. The injectability and self-healing properties of the hydrogel further enhance the minimally invasive nature of local drug delivery, achieving a dual therapeutic effect of analgesia and nerve function repair.

[0056] Unless otherwise specified, the room temperature in this invention is 25±2℃.

[0057] All raw materials used in the embodiments of this invention were purchased commercially. Glycyrrhizic acid, 1,4-phenylboronic acid, and potassium hydroxide (KOH) were purchased from conventional chemical reagent suppliers, and phosphate buffered saline (PBS) was either routinely prepared in the laboratory or a commercially available finished product.

[0058] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0059] The technical solution of the present invention will be further illustrated by the following embodiments.

[0060] Example 1 A method for preparing a reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel, comprising the following steps: (1) Take 1 mL of 0.05 mol / L KOH solution, mix it with 4 mg of 1,4-phenylboronic acid, and heat it in a 95℃ constant temperature water bath until the solution is completely clear to obtain a 1,4-phenylboronic acid alkaline solution; (2) Take 30 mg of glycyrrhizic acid and add it to 1 mL of the 1,4-phenylboronic acid alkaline solution obtained in step (1). Continue to heat it in a 95°C constant temperature water bath until it is completely dissolved, forming a homogeneous mixed clear solution. There is no drug residue loss during the preparation process. (3) Remove the mixed solution from the constant temperature water bath in step (2) and let it cool naturally to room temperature. The mixed solution forms a three-dimensional network gel structure through dynamic cross-linking of borate ester bonds, thus obtaining reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel.

[0061] Example 2 A method for preparing a reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel is the same as in Example 1, except that the amount of glycyrrhizic acid added in step (2) is 40 mg.

[0062] Example 3 A method for preparing a reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel is the same as in Example 1, except that the amount of glycyrrhizic acid added in step (2) is 50 mg.

[0063] As can be seen from the above embodiments, the preparation method of the reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel of the present invention only requires heating to dissolve and then cooling, which is time-saving. The preparation process uses only mild alkaline solvents, without organic solvents, catalysts, or purification. The process is simple and can be scaled up industrially.

[0064] Comparative Example 1 A method for preparing a glycyrrhizic acid-1,4-phenylboronic acid system, comprising the following steps: (1) Take 1 mL of 0.05 mol / L KOH solution, mix it with 4 mg of 1,4-phenylboronic acid, and heat it in a 95℃ constant temperature water bath until the solution is completely clear to obtain a 1,4-phenylboronic acid alkaline solution; (2) Take 10 mg of glycyrrhizic acid and add it to 1 mL of the 1,4-phenylboronic acid alkaline solution obtained in step (1). Continue to heat it in a 95°C constant temperature water bath until it is completely dissolved to form a homogeneous mixed solution. (3) Remove the mixed solution from the constant temperature water bath in step (2) and allow it to cool naturally to room temperature to obtain the glycyrrhizic acid-1,4-phenylboronic acid system.

[0065] Comparative Example 2 A method for preparing a glycyrrhizic acid-1,4-phenylboronic acid system, comprising the following steps: (1) Take 1 mL of 0.05 mol / L KOH solution, mix it with 4 mg of 1,4-phenylboronic acid, and heat it in a 95℃ constant temperature water bath until the solution is completely clear to obtain a 1,4-phenylboronic acid alkaline solution; (2) Take 20 mg of glycyrrhizic acid and add it to 1 mL of the 1,4-phenylboronic acid alkaline solution obtained in step (1). Continue to heat it in a 95°C constant temperature water bath until it is completely dissolved to form a homogeneous mixed solution. (3) Remove the mixed solution from the constant temperature water bath in step (2) and allow it to cool naturally to room temperature to obtain the glycyrrhizic acid-1,4-phenylboronic acid system.

[0066] Comparative Example 3 A method for preparing a glycyrrhizic acid system without 1,4-phenylboronic acid includes the following steps: (1) Take 1 mL of 0.05 mol / L KOH solution and preheat the solution in a 95°C constant temperature water bath (ensure that the preheating time in this comparative example is the same as the heating time in step (1) of Example 1). (2) Take 10 mg of glycyrrhizic acid and add it to 1 mL of KOH solution that has been preheated in step (1). Continue to heat it in a 95°C constant temperature water bath until the glycyrrhizic acid is completely dissolved and a homogeneous glycyrrhizic acid solution is formed. (3) Remove the glycyrrhizic acid solution from step (2) from the constant temperature water bath and allow it to cool naturally to room temperature to obtain a glycyrrhizic acid system that does not contain 1,4-phenylboronic acid.

[0067] Comparative Example 4 A method for preparing a glycyrrhizic acid system without 1,4-phenylboronic acid includes the following steps: (1) Take 1 mL of 0.05 mol / L KOH solution and preheat the solution in a 95°C constant temperature water bath (ensure that the preheating time in this comparative example is the same as the heating time in step (1) of Example 1). (2) Take 20 mg of glycyrrhizic acid, add 1 mL of KOH dilute alkaline solution preheated in step (1), and continue to heat in a 95°C constant temperature water bath until the glycyrrhizic acid is completely dissolved to form a homogeneous glycyrrhizic acid solution; (3) Remove the glycyrrhizic acid solution from step (2) from the constant temperature water bath and allow it to cool naturally to room temperature to obtain a glycyrrhizic acid system that does not contain 1,4-phenylboronic acid.

[0068] Comparative Example 5 A method for preparing a glycyrrhizic acid system without 1,4-phenylboronic acid includes the following steps: (1) Take 1 mL of 0.05 mol / L KOH solution and preheat the solution in a 95°C constant temperature water bath (ensure that the preheating time in this comparative example is the same as the heating time in step (1) of Example 1). (2) Take 30 mg of glycyrrhizic acid, add 1 mL of KOH dilute alkaline solution preheated in step (1), and continue to heat in a 95°C constant temperature water bath until the glycyrrhizic acid is completely dissolved to form a homogeneous glycyrrhizic acid solution; (3) Remove the glycyrrhizic acid solution from step (2) from the constant temperature water bath and allow it to cool naturally to room temperature to obtain a glycyrrhizic acid system that does not contain 1,4-phenylboronic acid.

[0069] Comparative Example 6 A method for preparing a glycyrrhizic acid system without 1,4-phenylboronic acid includes the following steps: (1) Take 1 mL of 0.05 mol / L KOH solution and preheat the solution in a 95°C constant temperature water bath (ensure that the preheating time in this comparative example is the same as the heating time in step (1) of Example 1). (2) Take 40 mg of glycyrrhizic acid, add 1 mL of KOH dilute alkaline solution that was preheated in step (1), and continue to heat in a 95°C constant temperature water bath until the glycyrrhizic acid is completely dissolved to form a homogeneous glycyrrhizic acid solution; (3) Remove the glycyrrhizic acid solution from step (2) from the constant temperature water bath and allow it to cool naturally to room temperature to obtain a glycyrrhizic acid system that does not contain 1,4-phenylboronic acid.

[0070] Comparative Example 7 A method for preparing a glycyrrhizic acid system without 1,4-phenylboronic acid includes the following steps: (1) Take 1 mL of 0.05 mol / L KOH solution and preheat the solution in a 95°C constant temperature water bath (ensure that the preheating time in this comparative example is the same as the heating time in step (1) of Example 1). (2) Take 50 mg of glycyrrhizic acid, add 1 mL of KOH dilute alkaline solution preheated in step (1), and continue to heat in a 95°C constant temperature water bath until the glycyrrhizic acid is completely dissolved to form a homogeneous glycyrrhizic acid solution; (3) Remove the glycyrrhizic acid solution from step (2) from the constant temperature water bath and allow it to cool naturally to room temperature to obtain a glycyrrhizic acid system that does not contain 1,4-phenylboronic acid.

[0071] The room-temperature gelling properties of the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogels prepared in Examples 1-3, the glycyrrhizic acid-1,4-phenylboronic acid systems prepared in Comparative Examples 1-2, and the glycyrrhizic acid systems without 1,4-phenylboronic acid prepared in Comparative Examples 3-7 were observed. The results are as follows: Figure 1As shown, the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogels prepared in Examples 1-3 rapidly formed a uniform three-dimensional network gel structure after cooling at room temperature, exhibiting good morphological stability and injectability; while the systems of Comparative Examples 1-7 remained liquid after cooling at room temperature, without the formation of a gel structure.

[0072] To verify the reactive oxygen species (ROS) responsiveness, biosafety, anti-inflammatory properties, and therapeutic effect on dorsal root ganglion compression-induced neuralgia caused by lumbar disc herniation in diabetic patients, an in vitro drug release experiment, biocompatibility test, macrophage polarization regulation experiment, and in vivo animal behavioral and histological tests were conducted to systematically clarify the physicochemical functions and in vivo therapeutic effects of the ROS-responsive glycyrrhizic acid anti-inflammatory hydrogel (the ROS-responsive glycyrrhizic acid anti-inflammatory hydrogel prepared in Example 1 is used as an example in the following experiments). The experiment used bone marrow-derived macrophages (BMDMs) from SD rats and mice as the research subjects, glycyrrhizic acid aqueous solution (Comparative Example 5) as a non-gel control, lipopolysaccharide (LPS) as an inflammation inducer, and epidural steroid injection (ESI) as a routine clinical treatment control. A rat model of neuralgia caused by chronic compression of the dorsal root ganglion in diabetic patients was constructed (STZ-induced type 2 diabetes + L4-L5 dorsal root ganglion ligation). A series of verification experiments were carried out, with specific grouping and testing as follows: I. Experimental Verification of the Reactive Oxygen Response of Glycyrrhizic Acid Anti-inflammatory Hydrogel To clarify the reactive oxygen species (ROS) responsive drug release characteristics of glycyrrhizic acid anti-inflammatory hydrogels, in vitro drug release and gel mass loss experiments were conducted to detect the release efficiency of glycyrrhizic acid and the stability of the gel structure under different ROS concentrations. The glycyrrhizic acid anti-inflammatory hydrogels were placed in PBS (pH=7.2), 0.1 mM H2O2, 1 mM H2O2, and 10 mM H2O2 solutions, respectively. The PBS group served as a blank control group, and each group had three replicates. The gel system was placed in a 37℃ incubator, and samples were taken at different time points to detect the cumulative release of glycyrrhizic acid in the supernatant. Simultaneously, the remaining mass of the gel was weighed at 1 day and 5 days to observe changes in gel morphology and perform quantitative analysis. The results showed that the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel had good stability in H2O2-free PBS with no obvious burst release phenomenon. However, the cumulative release of glycyrrhizic acid increased significantly with increasing H2O2 concentration. The gel mass loss was consistent with the drug release trend, confirming that the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel has highly efficient reactive oxygen species-responsive drug release characteristics. Figure 2 ).

[0073] II. Biosafety Verification Experiment of Reactive Oxygen Response Glycyrrhizic Acid Anti-inflammatory Hydrogel To clarify the cell compatibility, blood compatibility, in vivo biodegradability, and tissue safety of reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel, in vitro cell proliferation experiments, hemolysis experiments, and in vivo subcutaneous implantation and major organ toxicity experiments were conducted.

[0074] Cell compatibility assay: BMDMs were prepared at a concentration of 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and, after adhesion, were divided into a blank control group (Con), a lipopolysaccharide group, a glycyrrhizic acid aqueous solution treatment group, and a glycyrrhizic acid hydrogel treatment group, with three replicates in each group. After co-culturing for 1 day and 3 days, cell survival and proliferation morphology were observed using the live / dead staining method, and cell proliferation activity was detected by the CCK-8 assay to quantitatively analyze the effects of different treatments on cell viability.

[0075] Blood compatibility testing: PBS was used as a negative control, 0.1% Triton X-100 as a positive control, and reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel was used as the experimental group, with three replicates per group. After co-incubating the gel with fresh rat whole blood, the color change of the supernatant was observed, the hemolysis rate was detected, and the hemolytic activity of the gel was evaluated.

[0076] In vivo biodegradation and tissue safety testing: Glycyrrhizic acid anti-inflammatory hydrogel was subcutaneously implanted into the back of SD rats. Samples were collected on days 1, 3, and 7 post-operation to macroscopically observe the amount of residual gel and the morphology of surrounding tissues. Hematoxylin-eosin (H&E) staining was used to observe the inflammatory response of the implantation site, and immunofluorescence staining was used to detect the expression of inducible nitric oxide synthase (M1) and arginase-1 (M2). At the same time, H&E staining was performed on the main organs of the rats (heart, liver, spleen, lung, and kidney) to assess the in vivo systemic toxicity of the hydrogel.

[0077] Figure 3 The results showed that the cell proliferation activity of the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel group was significantly higher than that of the glycyrrhizic acid aqueous solution group. Live / dead staining showed that it could significantly promote the proliferation of BMDMs. The hemolysis rate of the glycyrrhizic acid anti-inflammatory hydrogel group was extremely low, and the supernatant was only light red, with no obvious hemolytic effect. After subcutaneous implantation, the gel could be gradually biodegraded without obvious tissue fibrosis. The inflammatory reaction of the surrounding tissues was mild, and there was no obvious pathological damage to the major organs. This confirmed that the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel has good cell compatibility, blood compatibility, in vivo biodegradability, and systemic tissue safety.

[0078] III. Experimental Verification of Anti-inflammatory Properties of Reactive Oxygen Response Glycyrrhizic Acid Anti-inflammatory Hydrogel To clarify the anti-inflammatory effect of reactive oxygen species-responsive glycyrrhizic acid (GMAA) hydrogels and their regulatory role in macrophage polarization, an in vitro inflammation model was constructed using lipopolysaccharide (LPS)-induced macrophage membrane dendritic cells (BMDMs). The regulatory ability of the gel on the phenotypic transformation of M1 / M2 macrophages was then examined. BMDMs were seeded in 6-well plates and divided into a blank control group (blank group), a LPS group, a glycyrrhizic acid aqueous solution treatment group (glycyrrhizic acid aqueous solution group), and a glycyrrhizic acid hydrogel treatment group (glycyrrhizic acid hydrogel group), with three replicates in each group. LPS was added to the LPS group to induce M1 polarization. The corresponding gel extracts were added to the glycyrrhizic acid aqueous solution group and the glycyrrhizic acid hydrogel group. After co-culturing, the expression of macrophage polarization markers was detected using multiple techniques. At the gene level: the mRNA expression levels of M1 markers (interleukin-1β, inducible nitric oxide synthase, tumor necrosis factor-α, CD86) and M2 markers (interleukin-10, transforming growth factor-β, CD206, arginase-1) were detected by qRT-PCR. Protein levels: The protein expression levels of interleukin-1β, inducible nitric oxide synthase (M1), CD206, and arginase-1 (M2) were quantitatively detected by Western blot (WB). At the cellular level: the distribution and fluorescence intensity of inducible nitric oxide synthase and arginase-1 positive cells were observed by immunofluorescence (IF) staining, and the proportion of CD86 (M1) and CD206 (M2) positive cells were quantitatively detected by flow cytometry.

[0079] Figures 3-10 The results showed that, compared with the lipopolysaccharide inflammation group, the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel group significantly downregulated the gene and protein expression of M1 markers, upregulated the expression of M2 markers, significantly reduced the proportion of inducible nitric oxide synthase-positive cells, and significantly increased the proportion of arginase-1-positive cells. This confirmed that the glycyrrhizic acid anti-inflammatory hydrogel can effectively inhibit macrophage M1 polarization and promote M2 repair polarization, and has significant in vitro anti-inflammatory properties.

[0080] IV. In vivo experimental study of reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel for the treatment of diabetic patients with dorsal root ganglion compression pain To clarify the in vivo therapeutic effect of reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel on dorsal root ganglion compression neuropathic pain caused by lumbar disc herniation in diabetic patients, a chronic compression model of dorsal root ganglion in diabetic rats was constructed, and behavioral, electrophysiological, and histological tests were conducted.

[0081] Model building SD rats were fed a high-fat, high-sugar diet and received intraperitoneal injections of 1% STZ (70 mg / kg). After 14-28 days, random blood glucose levels >16.7 mmol / L were used to establish a diabetic model. Subsequently, the right dorsal root ganglia of L4-L5 were surgically exposed and ligated to simulate the mechanical compression of lumbar disc herniation, thus establishing a diabetic model with dorsal root ganglion compression pain.

[0082] Experimental Groups Rats that successfully developed the model were divided into a model control group (also known as a blank group, which received only local injection of physiological saline), a glycyrrhizic acid aqueous solution treatment group (local injection of glycyrrhizic acid aqueous solution), a glycyrrhizic acid hydrogel treatment group (local injection of reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel into the dorsal root ganglion), and a sham surgery group (only surgical exposure of the dorsal root ganglion without ligation). Each group had 3 replicates, and the drugs were precisely injected locally around the dorsal root ganglion.

[0083] detection indicators Behavioral testing: At different time points after surgery, the mechanical claw retraction threshold was detected by the plantar tactile test, and the thermal claw retraction latency was detected by the plantar thermal tingling test. The sensitivity of rats to noxious thermal stimuli was assessed by the tail-flick test and the hot plate test. Gait trajectory analysis was used to quantitatively detect gait parameters such as the imprint area, average optical density, and standing time of the rat's right hind paw to assess the recovery of motor function.

[0084] Electrophysiological testing: One week after the operation, stimulating electrodes were placed above the lesion site in rats, and recording electrodes were placed in the target muscle to detect the amplitude of the complex muscle action potential (CMAP) of the gastrocnemius muscle, which indirectly assessed the recovery status of nerve function in the dorsal root ganglion.

[0085] Histological and molecular detection: Tissues from the dorsal root ganglion and dorsal horn of the spinal cord were collected from rats at 1 and 3 weeks post-surgery. Immunofluorescence staining was used to detect the expression of macrophage M1 / M2 polarization markers (inducible nitric oxide synthase / arginase-1) in the dorsal root ganglion, and the expression of microglia marker CD68 and inflammatory cytokine interleukin-1β (IL-1β) in the dorsal horn of the spinal cord. The mRNA expression levels of pain-related calcitonin gene-related peptide and neurotrophic factor brain-derived neurotrophic factor in the dorsal root ganglion tissue were detected by qRT-PCR to quantitatively analyze the changes in the expression of tissue inflammation and pain-related molecules.

[0086] Figure 11 , Figure 12The results showed that, compared with the model control group, the mechanical claw retraction threshold and thermal claw retraction latency of rats treated with reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel were significantly increased, the pain threshold of tail flick and hot plate tests was significantly improved, and gait parameters were significantly improved, approaching the level of the sham-operated group; the CMAP amplitude of the gastrocnemius muscle was significantly higher than that of the model group, and the neurophysiological function was well restored; the proportion of M1 macrophages in the dorsal root ganglion tissue was significantly reduced, and the proportion of M2 macrophages was significantly increased; the expression of CD68 and interleukin-1β in the dorsal horn of the spinal cord was significantly downregulated; and the expression of calcitonin gene-related peptide and brain-derived neurotrophic factor in the dorsal root ganglion was significantly inhibited. These results confirm that the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel can effectively relieve diabetic dorsal root ganglion compression pain, inhibit peripheral and central nerve inflammation, and promote the recovery of nerve function.

[0087] In summary, the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel possesses highly efficient reactive oxygen species responsiveness, good biocompatibility, significant anti-inflammatory and macrophage polarization regulation capabilities, and can effectively treat dorsal root ganglion compression pain caused by diabetic lumbar disc herniation by regulating the local immune microenvironment of the dorsal root ganglion and inhibiting central sensitization, providing a novel candidate for minimally invasive treatment of this type of disease in clinical practice.

[0088] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel, characterized in that, Using glycyrrhizic acid and 1,4-phenylboronic acid as raw materials, the mixture was heated in a water bath under an alkaline environment until a homogeneous mixed solution was formed, and then naturally cooled to room temperature to obtain the reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel. The mass ratio of glycyrrhizic acid to 1,4-phenylboronic acid is (7.5~12.5):

1.

2. The method for preparing the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel according to claim 1, characterized in that, The mass ratio of glycyrrhizic acid to 1,4-phenylboronic acid is 7.5:1, 10:1, or 12.5:

1.

3. The method for preparing the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel according to claim 1, characterized in that, An alkaline environment is created using an alkaline solution, which is a KOH solution.

4. The method for preparing the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel according to claim 3, characterized in that, The concentration of the KOH solution is 0.05 mol / L.

5. The method for preparing the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel according to claim 1, characterized in that, The water bath heating temperature is 90~100℃.

6. A reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel, characterized in that, It is prepared according to any one of claims 1 to 5.

7. The use of the reactive oxygen species-responsive glycyrrhizic acid anti-inflammatory hydrogel as described in claim 6 in the preparation of a drug for treating compressive neuralgia.

8. The application according to claim 7, characterized in that, The medication mentioned is for treating compressive nerve pain caused by lumbar disc herniation in patients with diabetes.

9. A drug for treating compressive neuralgia, characterized in that, The active ingredient is the reactive oxygen species responsive glycyrrhizic acid anti-inflammatory hydrogel as described in claim 6.

10. The medicament for treating compressive neuralgia according to claim 9, characterized in that, It also includes pharmaceutically acceptable excipients.

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