Customized analgesic hydrogel and preparation method and application thereof

Abstract

The application discloses a kind of customized analgesic hydrogel and its preparation and application.Specifically, the application discloses a new analgesic target, screens suitable drugs, and according to the characteristics of drug molecules, two-step method is customized to obtain sustained-release analgesic hydrogel.The two-step method is specifically divided into the following: first, according to the structure of drug molecules, aldehyde-based hyaluronic acid is synthesized to realize chemical drug loading;Second, adjust the concentration of Schiff base to form a double grid environment to realize injectability.Load the inhibitory drug TAPI-1 for the new target ADAM17 of stomatognathic inflammatory pain, construct a drug delivery system with targeted delivery, sustained-release analgesia, local injection and long-acting effect.Compared with the prior art, the drug delivery system obtained by the application has excellent sustained-release performance, the prolonged drug release is consistent with the hyperalgesia window period of stomatognathic inflammatory pain, precise and effective pain management is achieved, and the operation is simple, and has good conversion application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of gel production, and particularly relates to a customized analgesic hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] The global incidence of orofacial inflammatory pain (OFP) is high, and has complex etiology and pathophysiological mechanisms, which can cause persistent pain and seriously affect the quality of life of patients. Dental OFP is mainly treated by dental treatment, and non-dental OFP mainly adopts a treatment method combining drug and non-drug treatment. Drug treatment mainly adopts non-steroidal anti-inflammatory drugs, opioid drugs, anti-anxiety drugs and the like for systemic administration, and long-term use of these drugs can cause systemic side effects, including gastrointestinal disorders, addiction risk and off-target effects. Therefore, it is urgent to explore new analgesic targets and preferentially develop local drug delivery systems to reduce adverse reactions related to systemic treatment.

[0003] ADAM17 is also known as TNF-alpha convertase (TACE) or recombinant protease active domain protein, and is an important member of the disintegrin-metalloproteinase (ADAMs) family. ADAM17 is a membrane-bound protease composed of multiple domains, and the key part that plays an enzymatic activity is a metalloenzyme active domain. The enzymatic activity of ADAM17 can hydrolyze various cytokines and cell surface receptors, such as TNF-alpha, IL-6, notch, etc., and is therefore also known as an "inflammatory molecule switch" and plays an important role in the inflammatory process. Abnormal expression or activity of ADAM17 is closely related to the occurrence and development of various diseases. Studies have shown that ADAM17 is highly expressed in human glioma cells, and it can activate the EGFR-related signaling pathway (such as PI3K / AKT and MEK / ERK) by shedding and activating various epidermal growth factor receptor (EGFR) ligands, thereby promoting the proliferation, migration and invasion of glioma cells. Diabetic cardiomyopathy is a serious complication of diabetes, and studies have found that in DCM, the protein expression and activity of ADAM17 are significantly increased, while the ACE2 protein level is significantly decreased. Cardiomyocyte-specific knockout of ADAM17 can significantly reduce myocardial cell apoptosis and cardiac fibrosis in DCM mice, and improve pathological cardiac remodeling and heart failure. In addition, studies have confirmed that ADAM17 is a key protease required for calcium channel proteins, making it a potential drug target for neuropathic pain. In summary, given the key role of ADAM17 in inflammatory signaling and regulation and pathological pain, it is expected to become a new target for treating OFP.

[0004] Precise drug delivery is an important part of achieving maximum efficacy and minimizing drug side effects. Currently, OFP administration methods include systemic administration and local administration. Systemic administration includes oral administration and intravenous injection, which can achieve extensive analgesia, but has high secondary risk and often has low efficacy due to drug metabolism and degradation. Local administration such as transdermal patch, implantable device and nerve block provides more direct analgesia by directly targeting the pain site, but due to incomplete tissue penetration and other reasons, their action time is short and repeated administration is required. In recent years, with the development of materials science and bioengineering, hydrogel as a new type of drug-loaded matrix is gradually being developed and utilized. As a highly hydrophilic three-dimensional network polymer, hydrogel has good biocompatibility, adjustability and other advantages, and can be used as an ideal drug delivery system. However, the traditional hydrogel system cannot effectively achieve drug release, and there are defects such as loose embedding with the loaded drug. By modifying the hydrogel, changing its three-dimensional structure provides a solution to overcome the limitations of traditional methods. Developing new drug delivery strategies not only protects the drug from degradation, but also matches different composition, size and morphology of drug molecules through carrier modification, achieving drug release and controlled release, and providing the possibility of customized drug delivery.

[0005] Therefore, it is of great significance to develop a customized hydrogel drug delivery system that can be precisely delivered, released, injected subcutaneously in the maxillofacial region, and has good biocompatibility. SUMMARY

[0006] For the design of hydrogel release performance, it is necessary to form an effective chemical bond with the loaded drug, which not only maintains the stability of the drug and prevents degradation, but also realizes reversible combination and release. Therefore, the aldehyde-modified hyaluronic acid (HAALD) is synthesized by modifying hyaluronic acid, which can react with the drug molecule TAPI-1 to form a chemical bond. However, the three-dimensional structure of HAALD under scanning electron microscopy is more ordered and regular than that of pure hydrogel, so the injectability is not strong. Therefore, the present application adds hyaluronic acid and gelatin to the drug-loaded system, and synthesizes amine-modified gelatin by modifying gelatin, so as to combine with HAALD and have both stability and fluidity.

[0007] For the efficacy of the hydrogel drug delivery system, it is necessary to ensure its biocompatibility and as much as possible to make the drug release period consistent with the peak period of pain and allergy, so as to effectively exert the drug effect. Therefore, under the premise of determining the key window period of oral and maxillofacial inflammatory pain (OFP) in the early experiment, the present application continuously adjusts the proportion of each component of the hydrogel, and then adjusts the release period, to ensure the effective exertion of the drug efficacy.

[0008] For the analgesic effect of drugs, the existing systemic drugs often lead to many adverse reactions due to the wide range of action targets. Therefore, targeted drug is very meaningful. However, there is no study to reveal the specific target of oral and maxillofacial inflammatory pain, so the present application verifies the molecular target ADAM17 related to the inflammatory response pathway and the pain signal transduction pathway through a series of biomedical tests, and verifies the analgesic treatment effect of its molecular inhibitor TAPI-1, so as to find the targeted drug for oral and maxillofacial inflammatory pain. The development and application of this new target, combined with the innovative sustained-release drug system, can greatly change the use of analgesic drugs in clinical practice.

[0009] The application provides application of ADAM17 as a drug target in preparation of a drug for preventing, inhibiting, relieving, reducing or treating oral and maxillofacial inflammatory pain.

[0010] The ADAM17 is also called TNF-alpha convertase (TACE) or recombinant protease active domain protein, and is an important member of the disintegrin-metalloproteinase (ADAMs) family. ADAM17 is a membrane-bound protease composed of multiple domains, wherein the key part for enzyme activity is a metalloenzyme active domain. The enzyme activity of ADAM17 can hydrolyze various cytokines and cell surface receptors, such as TNF-alpha, IL-6, notch, etc., and thus is also called “inflammatory molecule switch”, and plays an important role in the inflammatory process. In addition, researches have proved that ADAM17 is a key protease required for calcium channel protein, and thus becomes a potential drug target for neuropathic pain. In summary, in view of the key role of ADAM17 in inflammatory signal transduction and regulation and pathological pain, it is expected to become a new target for treating OFP.

[0011] The drug is used for inhibiting the activity of ADAM17, so as to block the release of cytokines and receptors mediated by ADAM17, and then regulate the inflammatory response process.

[0012] The application further provides an ADAM17 modulator, which comprises an inhibitor or an antagonist, and can regulate or inhibit or antagonize the enzyme activity of ADAM17.

[0013] The inhibitor is TAPI-1.

[0014] The TAPI-1 refers to a tumor necrosis factor convertase inhibitor, and the molecular formula is C26H37N5O, and the structural formula is shown in formula 1.

[0015]

[0016] The application also provides the use of the ADAM17 modulator in the preparation of a medicament for preventing / inhibiting / alleviating / reducing / treating oral and maxillofacial inflammatory pain.

[0017] The application also provides a customized analgesic hydrogel, which comprises aldehyde-hyaluronic acid (HAALD), AGel, TAPI-1, gelatin, hyaluronic acid and silica microspheres.

[0018] Further, the customized analgesic hydrogel further comprises the loaded drug TAPI-1.

[0019] In the application, the term "customized" refers to a drug loading system for TAPI-1 molecules. Further, "customized" can also refer to the sustained analgesic effect of OFP. Specifically, the "dual customization" proposed in the application refers to: first, synthesizing aldehyde-hyaluronic acid according to the structure of the drug molecules to realize chemical drug loading; and second, adjusting the concentration of Schiff base to form a double grid environment to realize injectability.

[0020] The application also provides a preparation method of a customized double sustained analgesic hydrogel, which comprises the following steps:

[0021] Step 1: Synthesis of HAALD and AGel

[0022] HA is dissolved in deionized water, and then NaIO4 solution is added for reaction. After the reaction is completed, dialysis is performed to obtain HAALD.

[0023] Gelatin and ADH are dissolved in deionized water, and EDC and HOBt dissolved in a first solvent are added for reaction. Dialysis and freeze-drying are performed to obtain AGel.

[0024] Step 2: Preparation of HHGA hydrogel

[0025] First, TAPI-1 is dissolved in a second solvent; gelatin is added to deionized water, and then AGel is added, and stirring is performed to form solution A; HA is added to deionized water, stirring is performed, and then HAALD is added, and stirring is performed to form solution B; then TAPI-1, solution A, solution B and silica microspheres are stirred and mixed to obtain the customized analgesic hydrogel, i.e., HHGA hydrogel.

[0026] In the synthesis process of HAALD,

[0027] The deionized water has a pH of 5.5-7; preferably, 6.0.

[0028] The amount ratio of the HA, deionized water, NaIO4 solution is 0.5-2g: 50-200mL: 5.5-22mL; preferably, 1g: 100mL: 11mL.

[0029] The concentration of the NaIO4 solution is 0.25mol / L.

[0030] The temperature of the stirring reaction is 35-45℃; preferably, 40℃.

[0031] The time of the stirring reaction is 2.5-3.5h; preferably, 3h.

[0032] The ethylene glycol is added during the stirring reaction to terminate the reaction.

[0033] The time of the dialysis is 2-5 days; preferably, 3 days.

[0034] The molecular weight of the HAALD is 14000.

[0035] In the synthesis process of the AGel,

[0036] The amount ratio of the gelatin, ADH, deionized water, EDC, HOBt and the first solvent is 0.5-2g: 1.18-4.72g: 50-200mL: 1.40-5.58g: 0.92-3.66g: 6.5-26mL; preferably, 1g: 2.36g: 100mL: 2.79g: 1.83g: 13mL.

[0037] The first solvent is selected from one or mixed solution of DMSO, H2O. The volume ratio of the DMSO, H2O is 1:1.

[0038] The temperature of the reaction is 20-30℃; preferably, 25℃.

[0039] The time of the reaction is 20-28h; preferably, 24h.

[0040] The time of the dialysis is 2-5 days; preferably, 3 days.

[0041] The gel strength of the AGel is 250g Bloom.

[0042] In the preparation process of the HHGA hydrogel,

[0043] The amount ratio of the TAPI-1, second solvent is 0.5-2mg: 0.5-2mL; preferably, 1mg: 1mL.

[0044] The diameter of the TAPI-1 molecule is 1.5nm.

[0045] The second solvent is selected from one or more of DMSO, alcohol, physiological saline; preferably, DMSO.

[0046] The use amount ratio of the gelatin, AGel, HA, HAALD, deionized water is 0.3-1.2g: 0.125-0.5g: 0.05-0.2g: 0.125-0.5g: 10-40mL; preferably, 0.6g: 0.25g: 0.1g: 0.25g: 20mL.

[0047] In one specific embodiment, the preparation of the customized analgesic hydrogel comprises the following steps:

[0048] Synthesis of HAALD and AGel: (1) HAALD: first adjust the pH of deionized water to 6.0 with dilute HCl; weigh 1g HA, dissolve it in 100mL deionized water with pH=6.0; and stir for one day to make it uniform. Then add 11mL 0.25mol / L NaIO4 solution, stir for 3h in a 40℃ water bath. After that, add 30mL ethylene glycol to terminate the reaction; after dialysis for 3 days, HAALD is obtained (molecular weight: 14000, change water once a day). (2) AGel: dissolve 1g gelatin and 2.36g ADH in 100mL deionized water; dissolve 2.79g EDC and 1.83g HOBt in 13mL DMSO and H2O (volume ratio 1:1); after dissolving uniformly, mix the two. Then adjust the pH of the mixed solution to 5.0 with dilute HCl. Perform the reaction in a 25℃ water bath for 24h; dialyze with deionized water for 3 days, change water every day. Finally, freeze-drying AGel is obtained.

[0049] Preparation of HHGA hydrogel: first dissolve TAPI-1 in a small amount of DMSO. Add 0.6g gelatin to 10mL deionized water, wait for it to dissolve, then add 0.25g AGel, stir to form A solution. Add 0.1g HA to 10mL deionized water, stir for one day, then add 0.25g HAALD, stir to form B solution. Then mix TAPI-1, A solution, B solution, and silica microspheres to obtain HHGA hydrogel. And stand for 15 minutes.

[0050] The application also provides a customized analgesic hydrogel prepared by the above method.

[0051] The application also provides a drug / medicinal composition comprising an ADAM17 modulator as described above, or the customized analgesic hydrogel, and a pharmaceutically acceptable carrier.

[0052] Further, the drug / medicinal composition is used alone.

[0053] Preferably, the pharmaceutically acceptable carrier means that when the drug is properly administered to an animal or human, they do not produce adverse, allergic or other adverse reactions. As a pharmaceutically acceptable carrier, it can include but is not limited to: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa oil; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solution; and phosphate buffer, etc. These substances are used as needed to help the stability of the formula or to help improve the activity or its bioavailability or in the case of oral to produce an acceptable taste or smell.

[0054] Specifically, the drug / drug composition can also contain a physiologically compatible auxiliary material, including buffers, diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc.

[0055] Specifically, the drug / drug composition can be made into injections, sterile powders for injection, tablets, pills, capsules, lozenges, elixirs, powders, granules, syrups, solutions, tinctures, aerosols, powder sprays, or suppositories, etc. The above various dosage forms of the drug / drug composition can be prepared according to the conventional method in the pharmaceutical field.

[0056] Specifically, the drug / drug composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nose drops, eye drops, penetration, absorption, physical or chemical mediated methods; or mixed or wrapped with other substances before being introduced into the body. Preferably, it is administered by injection. The drug / drug composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, targeted therapy.

[0057] The present application also provides a reagent / kit, wherein the reagent / kit comprises the ADAM17 modulator as described above, or the customized dual sustained-release analgesic hydrogel, or the drug / drug composition.

[0058] The present application also provides the use of the customized dual sustained-release analgesic hydrogel in the preparation of a drug for preventing / inhibiting / alleviating / reducing / treating stomatognathic inflammatory pain.

[0059] The application also provides the use of the drug / medicinal composition in the preparation of a drug for preventing / inhibiting / alleviating / reducing / treating stomatognathic inflammatory pain.

[0060] The application also provides the use of the reagent / kit for diagnosing the onset and / or cure and / or recurrence of stomatognathic inflammatory pain.

[0061] The application also provides a method for preventing / inhibiting / alleviating / reducing / treating stomatognathic inflammatory pain, wherein an ADAM17 modulator, or the customized dual-release analgesic hydrogel, or the drug / medicinal composition is administered to a subject.

[0062] The application also provides a method for detecting the onset, cure and recurrence of stomatognathic inflammatory pain, wherein the expression of ADAM17 in the stomatognathic region of a subject is detected using the ADAM17 modulator, or the customized dual-release analgesic hydrogel, or the drug / medicinal composition, or the reagent or kit.

[0063] The application also provides a method for screening a candidate drug for preventing / inhibiting / alleviating / reducing / treating stomatognathic inflammatory pain, wherein the method comprises detecting the effect of the candidate drug on the level of ADAM17 in a subject or a sample obtained from the subject, and wherein the biological function of ADAM17 is inhibited after the use of the candidate drug, indicating that the candidate drug has an effect of preventing / inhibiting / alleviating / reducing / treating stomatognathic inflammatory pain.

[0064] The beneficial effects of the application include but are not limited to the following:

[0065] The application constructs a customized hydrogel drug delivery system targeting molecular targets, has a biological basis for target verification, and realizes precise delivery and targeting of drug molecules. The innovation of this manufacturing concept can be used for more experimental research on the combination of targeted drugs and hydrogel drug delivery systems.

[0066] The application takes into account that the drug molecule TAPI-1 has abundant amino groups, and innovatively synthesizes HAALD by aldehyde group of hyaluronic acid, so as to form a reversible Schiff base combination with the drug molecule. This chemical loading system utilizes the natural combination potential between molecules to create a stable and effective drug delivery complex, and at the same time, the pores of the hydrogel structure formed by multiple components are beneficial to the slow release of the drug. Figure 5As shown, the release effect of the application is evaluated by near-infrared fluorescence imaging technology (NIRF), and the experimental compositions are as follows: C1 group (HA-Gelatin+TAPI-1), C2 group (HAALD-A Gel+TAPI-1), and T group (HHGA hydrogel). The results show that in the C1 group, more than half of the TAPI-1 is released within the first 12 hours, while in the C2 group, 50% of the drug concentration remains unchanged within 3 days. In the T group, fluorescence intensity analysis shows that the drug is gradually and continuously released within 7 days (n=3 / group, p<0.05). This indicates that the modified hydrogel can prolong the retention time of local drugs and has excellent sustained-release performance. The application prolongs the drug release time from 6 hours to 7 days in the prior art, and the effect is obvious. In addition, unmodified gelatin and hyaluronic acid are added to the hydrogel system constructed by the application to enhance the injectability of the system. The preparation process adopted by the application can achieve local sustained release and long-lasting analgesia, and has good practical value. For example Figure 6 As shown, the application is applied to OFP mice to evaluate the analgesic effect, and the behavior evaluation shows that a single injection of the customized hydrogel prepared by the application achieves significant analgesic effect within 30 minutes and effectively lasts to the 5th day (n=6 / group, p<0.05). This indicates that the customized hydrogel prepared by the application has better analgesic effect than the control group and has longer analgesic time within the same time.

[0067] The customized hydrogel drug delivery system proposed by the application has good biocompatibility and sustained-release performance, can achieve effective analgesia of stomatognathic inflammatory pain, and is a new and efficient analgesic method. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 The figure is a schematic diagram of the material composition and structure of the application;

[0069] Figure 2 The figure is an electron microscope structure characterization of various components of the hydrogel drug delivery system prepared by the application;

[0070] Figure 3 The figure is a chemical structure analysis of various components of the hydrogel drug delivery system prepared by the application;

[0071] Figure 4 The figure is a result graph of in vivo HE staining and immunohistochemical experiment, in vitro live and dead staining experiment to evaluate the biocompatibility of the hydrogel drug delivery system prepared by the application; wherein, Blank (blank group), Hydrogel (modified hydrogel group), TAPI-1 (drug group), Hydrogel+TAPI-1 (HHGA hydrogel);

[0072] Figure 5 The figure is a result graph of near-infrared fluorescence imaging technology to evaluate the sustained-release performance of the hydrogel drug delivery system prepared by the application;

[0073] Figure 6 Figure 1 shows the behavioral changes and analgesic effect of the hydrogel drug delivery system prepared in the present application after injection into the model mice; wherein, CFA (pain group), CFA + Hydrogel without TAPI-1 (pain + no drug-loaded hydrogel group), CFA + Hydrogel with TAPI-1 (pain + drug-loaded hydrogel group);

[0074] Figure 7 Figure 2 shows the analgesic effect of TAPI-1 for the pain experiment of systemic injection of TAPI-1; wherein, Shame (blank control group), CFA (positive control group), CFA + TAPI-1 (pain + drug group). DETAILED DESCRIPTION

[0075] The present application will be further described in conjunction with the following specific examples. The process, conditions, experimental methods, etc. for implementing the present application, except for the following specifically mentioned content, are the general knowledge and common sense in the art, and the present application has no special limitation.

[0076] The following description of at least one example embodiment is merely illustrative in nature and is in no way intended to limit the application or its application or uses. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

[0077] In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, not as a limitation. Therefore, other examples of the example embodiments can have different values.

[0078] The present application uses raw materials: hyaluronic acid (HA, molecular weight: 800000-1500000), gelatin, hydrochloric acid (HCl), NaIO4, ethylene glycol, adipic dihydrazide (ADH), 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC), 1-hydroxybenzotriazole hydrate (HOBt), dimethyl sulfoxide (DMSO) 。

[0079] This invention comprises the discovery of novel analgesic targets, screening of suitable drugs, and the development and application of a two-step customized sustained-release analgesic system based on the characteristics of the drug molecules. Specifically, the customization involves the following two steps: First, synthesizing aldehyde-modified hyaluronic acid based on the drug molecule structure to achieve chemical drug loading; second, adjusting the Schiff base concentration to create a dual-grid environment to achieve injectability. The invention utilizes TAPI-1, an inhibitor of the novel target ADAM17 for oromandibular inflammatory pain, to construct a drug loading system with targeted delivery, sustained-release analgesia, local injection, and long-lasting effects. Compared with existing technologies, the drug loading system obtained in this invention exhibits sustained-release properties, with the prolonged drug release coinciding with the hyperalgesic window period of oromandibular inflammatory pain. In previous studies, a mouse oromandibular inflammatory pain model induced by complete Freund's adjuvant was used, and mechanical abnormal pain was detected using the von Frey wire method. The results showed that pain was most pronounced 3-7 days after injection of complete Freund's adjuvant. This aligns with the innovative sustained-release time of over 7 days proposed in this invention. This invention achieves precise and effective pain management (peripheral injection ensures precise site of action, and sustained release prolongs the duration of drug action to ensure effective treatment) and is easy to operate, and has good prospects for translational application.

[0080] Example 1

[0081] 1. The composition and structure of the customized dual-release analgesic hydrogel are as follows: Figure 1 The preparation method includes the following steps:

[0082] Synthesis of HAALD and AGel: (1) HAALD: First, adjust the pH of deionized water to 6.0 with diluted HCl; weigh 1g of HA and dissolve it in 100mL of deionized water with pH=6.0; stir for one day to make it uniform. Then add 11mL of 0.25mol / L NaIO4 solution and stir the reaction in a 40℃ water bath for 3h. After that, add 30mL of ethylene glycol to terminate the reaction; after dialysis for 3 days, obtain HAALD (molecular weight: 14000, water changed once a day). (2) AGel: Dissolve 1g of gelatin and 2.36g of ADH in 100mL of deionized water; dissolve 2.79g of EDC and 1.83g of HOBt in 13mL of DMSO and H2O (volume ratio 1:1); after dissolving evenly, mix the two. Then adjust the pH of the mixed solution to 5.0 with dilute HCl. The reaction is carried out in a 25℃ water bath for 24 hours; dialyze with deionized water for 3 days, changing the water every day. Finally, AGel is obtained by freeze-drying.

[0083] Preparation of HHGA hydrogel: TAPI-1 was first dissolved in a small amount of DMSO. 0.6 g of gelatin was added to 10 mL of deionized water, and it was dissolved, and then 0.25 g of AGel was added, and stirred to form solution A. 0.1 g of HA was added to 10 mL of deionized water, and stirred for one day, and then 0.25 g of HAALD was added, and stirred to form solution B. TAPI-1, solution A, solution B and silica microspheres were mixed by stirring to obtain HHGA hydrogel. And stand for 15 minutes.

[0084] 2. Electron microscope structure and chemical structure characterization analysis of each component of the hydrogel

[0085] ① Electron microscope structure characterization: as shown in Figure 2 , respectively, are the SEM images of HA-Gel hydrogel, HAALD-AGel hydrogel, and HHGA hydrogel (A, B, C). The SEM images were obtained by VEGA 3 (TESCAN). From the regularity of the network and the size of the pores, it can be inferred that the network formed based on the Schiff base bond in HAALD-AGel is more robust than the network formed based on hydrogen bonds in HA-Gel.

[0086] ② Chemical structure analysis: Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance hydrogen spectrum (HNMR) were used to analyze the chemical structure of TAPI-1, HAALD and TAPI-1-HAALD. The FT-IR spectra of monomers and composites were obtained by Nicolet 6700 (Thermo Fisher). 1 HNMR spectra were obtained by Avance III (Bruker). As shown in Figure 3 , due to the stretching vibration of -NH2, an absorption peak will appear at about 3055 cm -1 . In the FT-IR of TAPI-1-HAALD, this peak was not clearly observed, indicating that the generation of Schiff base bond consumed the amino group in TAPI. In addition, in TAPI-1-HAALD and HHGA hydrogel, a new peak appeared at 1550 cm -1 , which is attributed to the C=N double bond. 1 The numbers in the H nuclear magnetic resonance spectrum correspond to the number of chemical structures, which can also confirm the chemical structure of the three molecules. In the spectrum of TPAI-1-HAALD, the -NH2 peak (δ = 1.5 ppm, 21 in TAPI-1) disappeared, indicating that the -NH2 in TAPI-1 was consumed in the Schiff base reaction. The position of the aldehyde group proton changed to δ = 8.3 ppm, indicating the reduction of the aldehyde group.

[0087] 3. Performance analysis experiment of the material

[0088] In vitro drug release: The UV absorption curve of TAPI-1 confirmed that it had a significant absorption peak at 225 nm. The UV curves of different concentrations of TAPI-1 were determined, and the standard curve of TAPI-1 concentration and absorption peak intensity was fitted. The TAPI-1-containing HHGA hydrogel was soaked with 10 mL of normal saline and placed in a 37°C constant temperature incubator. At the corresponding time, 0.5 mL of solution was taken to determine the UV absorbance at 225 nm, and then 0.5 mL of normal saline was added. The 225 nm UV absorbance was converted to the concentration of TAPI-1 to obtain the in vitro release curve of the drug.

[0089] Rheological test: The hydrogel samples (diameter 20 mm, thickness 2 mm) were tested by a rotary rheometer (Discovery HR-10, USA). The storage modulus and loss modulus were measured as a function of angular velocity under constant temperature conditions (37°C). The results showed that the storage modulus of the HHGA hydrogel increased with the increase of the content of HAALD and AGel, while the loss modulus did not change significantly. Finally, the hydrogel system of the present application selected a concentration of 2.5%, so that it could not only undergo Schiff base reaction, but also maintain injectability.

[0090] 4、HHGA hydrogel biocompatibility test results

[0091] Cell culture: At 4 weeks after birth, the bilateral trigeminal ganglion (TG) of mice was collected and separated in ice-cold serum-free medium. The TG was incubated in a mixture of collagenase and trypsin at 37°C for 60 minutes to digest the tissue. After digestion, the TG neurons were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS). Then the neurons were purified by Percoll gradient screening to ensure a high concentration of live neurons. The purified neurons were inoculated in a culture dish containing DMEM / F12 (1:1) medium, and the culture dish was added with 5% FBS, 1% B27 and nerve growth factor (NGF) at a concentration of 50 ng / mL. The cells were stored in a 37°C incubator with 95% air and 5% CO2.

[0092] In vitro live and dead staining experiment: The live cell / dead cell double staining kit (Calcein-AM / PI) was used for staining. The experimental group was the HHGA hydrogel prepared by the present application, and the control group was blank and did not contain drug (Hydrogel (modified hydrogel group)), TAPI-1 (drug group), as shown in Figure 4 There was no significant difference in cell survival rate among all experimental groups, which confirmed the high biological safety of the drug-loaded system of the HHGA hydrogel of the present application (n=3 / group, p<0.05).

[0093] In vivo HE staining and immunohistochemical detection (IHC): Since the HHGA hydrogel material is administered by subcutaneous injection, skin and subcutive tissue samples are collected from the injection site for histological analysis. On the 5th day after modeling, the mice were anesthetized with 1% sodium pentobarbital, and 0.9% saline was injected intracardially, followed by 4% paraformaldehyde. The isolated TG and brain were fixed in 4% paraformaldehyde at 4°C overnight. Then, the tissues were dehydrated in a gradient of 20% phosphate buffered sucrose (containing 0.1M PBS) and 30% phosphate buffered sucrose (containing 0.1M PBS) for 48 hours until the tissues sank to the bottom. The tissues were embedded in an optimal cutting temperature (OCT) compound (Sakura Finetek) and stored at -80°C. TG sections (15μm) or Sp5c sections (25μm) were prepared using a cryostat and stored at -20°C for later use. As shown in FIG. 8, HE staining results showed that there was no obvious inflammatory reaction in the epidermis and connective tissue on days 0, 7 and 14. IHC analysis of immune cell infiltration focused on T cells (CD3), B cells (CD19), macrophages (F4 / 80) and dendritic cells (MHC II), and there was no significant difference in immune cell infiltration before and after injection (n=6 / group, p<0.05), indicating that the HHGA hydrogel material had little immune response and good material biocompatibility. Figure 5

[0094] 5. Evaluation of sustained-release performance by near-infrared fluorescence imaging technology

[0095] Using near-infrared fluorescence imaging technology (NIRF), the fluorescent dye Rhodamine B was incorporated into the hydrogel matrix along with TAPI-1 mixed into the hydrogel matrix. Specifically, the composition of the experimental groups of the present application were as follows: C1 group (HA-Gelatin+TAPI-1), C2 group (HAALD-AGel+TAPI-1), and T group (HHGA hydrogel). Each dosage form was injected into the preauricular region of mice in equal amounts, and the fluorescence intensity at each time point was monitored to track the in vivo drug release kinetics. As shown in FIG. 9, in the C1 group, more than half of the TAPI-1 was released within the first 12 hours, while in the C2 group, 50% of the drug concentration remained unchanged for 3 days. In the T group, the hydrogel combined with TAPI-1 loaded with silica nanoparticles (i.e., the HHGA hydrogel prepared by the present application), fluorescence intensity analysis showed that the drug was gradually and continuously released within 7 days (n=3 / group, p<0.05). The results showed that the dual sustained-release ability of the modified hydrogel and inorganic nanospheres can prolong the retention time of local drugs, and the sustained-release performance is excellent. Figure 5

[0096] 6. Evaluation of analgesic effect of customized hydrogel on OFP mice

[0097] ​​The mouse oromaxillofacial pain model was constructed using complete Freund's adjuvant (CFA), and the customized hydrogel drug delivery system of the present application was injected on the day of model induction. The test group (injection of the customized HHGA hydrogel of the present application) was compared with the pain model group (blank control) and the model + no drug hydrogel group (negative control). As shown in Figure 6 behavioral assessment showed that a single injection of the customized hydrogel achieved significant analgesic effect within 30 minutes, and effectively lasted to the 5th day (n = 6 / group, p < 0.05). The results showed that the customized hydrogel developed by the present application had better analgesic effect and longer analgesic time than the control group within the same time.

[0098] 7. Analgesic effect of systemic TAPI-1 on oromaxillofacial pain

[0099] After the establishment of the oromaxillofacial inflammatory pain model, the experimental group mice were injected with TAPI-1 intraperitoneally every day for 5 consecutive days (10 mg / kg, once a day for 5 consecutive days). The control group was divided into a negative control group (injection of saline) and a pain positive group (injection of complete Freund's adjuvant without injection of TAPI-1). As shown in Figure 7 behavioral experiment results showed that the experimental group mice had no obvious pain response on the 3rd to 5th day, and abnormal pain response reappeared on the 7th day after drug withdrawal, indicating that systemic injection of TAPI-1 had analgesic effect (n = 6 / group, p < 0.05). However, daily injection is a necessary but tedious task to maintain efficacy, and continuous injection caused the death of one mouse in the experimental group.

[0100] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0101] As used in the present application, the terms "include", "contain" are open expressions, i.e. including the contents indicated by the present application, but not excluding other aspects.

[0102] As used in the present application, the term "and / or" includes any one and all combinations of the related listed items.

[0103] The protection scope of the present application is not limited to the above embodiments. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. A customized analgesic hydrogel, characterized in that, The customized analgesic hydrogel comprises aldehyde-hyaluronic acid (HAALD), AGel, gelatin and hyaluronic acid, silica microspheres and drug-loaded TAPI-1; and the preparation method of the customized analgesic hydrogel comprises the following steps: Step 1: synthesis of HAALD and AGel dissolve HA in deionized water, then add NaIO4 solution for reaction, the reaction temperature is 35-45 DEG C, the reaction time is 2.5h-3.5h, after the reaction is finished, dialysis is carried out, and HAALD is obtained; the dosage ratio of HA, deionized water, NaIO4 is 0.5-2g: 50-200mL: 5.5-22mL; the molecular weight of HAALD is 14000; dissolve gelatin and ADH in deionized water, add EDC and HOBt dissolved in the first solvent for reaction, the reaction temperature is 20-30 DEG C, the reaction time is 20-28h, dialysis, freeze-drying to obtain AGel; the dosage ratio of gelatin, ADH, deionized water, EDC, HOBt and the first solvent is 0.5-2g: 1.18-4.72g: 50-200mL: 1.40-5.58g: 0.92-3.66g: 6.5-26mL; Step 2: preparation of HHGA hydrogel firstly, dissolve TAPI-1 in the second solvent to obtain a solution containing TAPI-1; add gelatin to deionized water, then add AGel, stir to form A solution; add HA to deionized water, stir, then add HAALD, stir to form B solution; then stir and mix the solution containing TAPI-1, A solution, B solution and silica microspheres to obtain the customized analgesic hydrogel, namely HHGA hydrogel; the dosage ratio of TAPI-1 and the second solvent is 0.5-2mg: 0.5-2mL; the dosage ratio of gelatin, AGel, HA, HAALD and deionized water is 0.3-1.2g: 0.125-0.5g: 0.05-0.2g: 0.125-0.5g: 10-40mL.

2. A method of preparing a customized analgesic hydrogel, characterized in that, The method comprises the following steps: Step 1: synthesis of HAALD and AGel dissolve HA in deionized water, then add NaIO4 solution for reaction, the reaction temperature is 35-45 DEG C, the reaction time is 2.5h-3.5h, after the reaction is finished, dialysis is carried out, and HAALD is obtained; the dosage ratio of HA, deionized water, NaIO4 is 0.5-2g: 50-200mL: 5.5-22mL; the molecular weight of HAALD is 14000; dissolving gelatin and ADH in deionized water, adding EDC and HOBt dissolved in a first solvent to react, the reaction temperature is 20-30℃, the reaction time is 20-28h, dialysis, freeze-drying to obtain AGel; the amount ratio of the gelatin, ADH, deionized water, EDC, HOBt and the first solvent is 0.5-2g:1.18-4.72g:50-200mL:1.40-5.58g:0.92-3.66g:6.5-26mL; Step 2: Preparation of HHGA hydrogel Firstly, dissolving TAPI-1 in a second solvent to obtain a solution containing TAPI-1; adding gelatin into deionized water, then adding AGel to form A solution by stirring; adding HA into deionized water, stirring, then adding HAALD to form B solution by stirring; then mixing the solution containing TAPI-1, A solution, B solution and silica microspheres to obtain the customized analgesic hydrogel, i.e. HHGA hydrogel; the amount ratio of TAPI-1 and the second solvent is 0.5-2mg:0.5-2mL; the amount ratio of gelatin, AGel, HA, HAALD, deionized water is 0.3-1.2g:0.125-0.5g:0.05-0.2g:0.125-0.5g:10-40mL.

3. The method of claim 2, wherein, In the step 1, the first solvent is selected from one or mixed solution of DMSO, H2O; and / or, the gel strength of AGel is 250g Bloom.

4. The method of claim 2, wherein, In the step 2, the second solvent is selected from one or more of DMSO, alcohol, physiological saline.

5. The method of claim 2, wherein, The diameter of TAPI-1 molecule is 1.5nm.

6. The customized analgesic hydrogel prepared by the method of any one of claims 2-5.

7. A pharmaceutical composition, characterized by, It comprises the customized analgesic hydrogel of claim 1 or 6, and a pharmaceutically acceptable carrier.

8. The use of the customized analgesic hydrogel of claim 1 or 6 or the pharmaceutical composition of claim 7 in the preparation of a drug for preventing or treating stomatognathic inflammatory pain.

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