PH response hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biopolymer materials, and particularly discloses pH response hydrogel as well as a preparation method and application thereof. The pH response hydrogel comprises a hydrogel matrix and a pH response material, wherein the hydrogel matrix comprises carboxymethyl chitosan N-hydroxymethyl acrylamide and 3-aminophenylboronic acid covalently connected with the carboxymethyl chitosan N-hydroxymethyl acrylamide; the loading particle comprises a polydopamine particle and an insulin-like growth factor 1 loaded on the polydopamine particle; wherein the covalently connected covalent bond is a boric acid ester bond, and the loaded particles are uniformly dispersed in the hydrogel matrix. According to the invention, carboxymethyl chitosan N-hydroxymethacrylamide and 3-aminophenylboronic acid are covalently connected through a borate bond, so that the pH response hydrogel is rapidly cured under ultraviolet irradiation and is attached to a specific periodontal tissue inflammation area, and the borate bond is decomposed in an inflammation acid environment; the insulin-like growth factor 1 is quickly released in an inflammation region, so that periodontitis is relieved, and periodontal bone tissue regeneration is promoted.

Description

Technical Field

[0001] The present application relates to the technical field of biopolymer materials, and in particular to a pH-responsive hydrogel and a preparation method and application thereof. Background Art

[0002] Periodontal disease is an inflammatory, destructive disease that affects the periodontal supporting tissues. The prevalence of periodontal disease in my country is as high as 90%, making it one of the most common chronic infectious diseases. Periodontal disease usually causes damage to periodontal soft tissues (such as gums and periodontal ligament) and hard tissues (cementum and alveolar bone). Once the periodontal tissues are damaged, their regenerative capacity will also be limited. Therefore, how to regenerate periodontal tissues is currently a hot topic of research.

[0003] In recent years, domestic and foreign researchers have paid increasing attention to in situ polymerized hydrogel systems, which are attributed to their remarkable adhesion properties and ability to control drug release characteristics. Compared with traditional drug delivery mechanisms, they have obvious advantages and have shown significant therapeutic effects in periodontal disease applications. However, there are still challenges in selecting appropriate polymers, formulations, and achieving the desired long-term drug release characteristics using precise polymer concentrations. Therefore, the existing technology needs to be improved. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a pH-responsive hydrogel and a preparation method and application thereof, aiming to solve the problem of unstable drug release of the hydrogel.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect of the present application, a pH-responsive hydrogel is provided, wherein the pH-responsive hydrogel comprises: a hydrogel matrix, comprising carboxymethyl chitosan N-hydroxymethyl acrylamide and 3-aminophenylboronic acid covalently linked thereto; loaded particles, comprising polydopamine particles and insulin-like growth factor 1 loaded on the polydopamine particles; wherein the covalent bond of the covalent link is a borate ester bond, and the loaded particles are uniformly dispersed in the hydrogel matrix.

[0007] Preferably, the mass ratio of the hydrogel matrix to the loaded particles is 1:1.5-2:1.

[0008] The second aspect of the present application provides a method for preparing the pH responsive hydrogel of the first aspect of the present application, the preparation method comprising the steps of: mixing the hydrogel matrix, the loaded particles and a photoinitiator to obtain the pH responsive hydrogel.

[0009] Preferably, the mass ratio of the hydrogel matrix, the loaded particles and the photoinitiator is 10:5:1-10:15:1.

[0010] Preferably, the preparation method of the hydrogel matrix comprises the steps of: dissolving carboxymethyl chitosan N-hydroxymethyl acrylamide in 2-morpholineethanesulfonic acid buffer to obtain solution A; dissolving 3-aminophenylboronic acid in dimethyl sulfoxide solution to obtain solution B; mixing solution A and solution B, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring for reaction, centrifuging after the reaction to obtain the supernatant and dialyzing to obtain the hydrogel matrix.

[0011] Preferably, the mass ratio of the carboxymethyl chitosan N-hydroxymethyl acrylamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:0.5:0.7-5:1.5:2.1; the stirring reaction time is 24-48h, the centrifugal speed is 6000-8000rpm, and the time is 0.5-2h.

[0012] Preferably, the method for preparing the loaded particles comprises the steps of: dissolving dopamine hydrochloride in an ethanol solution of ammonia, then adding insulin-like growth factor 1 and mixing evenly, centrifuging the mixture to collect the precipitate and washing it to obtain the loaded particles.

[0013] Preferably, the pH value of the ammonia ethanol solution is 8.5, and the volume ratio of ethanol to water in the ammonia ethanol solution is 1:1-4:9.

[0014] Preferably, the mass ratio of dopamine hydrochloride to insulin-like growth factor 1 is 1:0.2-1:5.

[0015] The third aspect of the present application provides a use of the pH-responsive hydrogel of the first aspect of the present application in the preparation of drugs for treating periodontal diseases and products for repairing periodontal tissues.

[0016] Compared with the prior art, this application has the following advantages:

[0017] The carboxymethyl chitosan N-hydroxymethyl acrylamide in the pH-responsive hydrogel in this application has good biocompatibility and antibacterial properties, and is suitable for use in periodontal tissue locations. Carboxymethyl chitosan N-hydroxymethyl acrylamide is covalently linked to 3-aminophenylboronic acid through a borate bond, and the borate bond can make the hydrogel matrix quickly polymerize into a gel under ultraviolet light, and cooperate with the adhesion of polydopamine particles, so that the pH-responsive hydrogel enters the periodontal tissue, and quickly solidifies and adheres to the specific periodontal tissue inflammation area under ultraviolet light, and has injectability and remodeling properties. At the same time, the borate bond decomposes in the acidic environment produced by inflammation, and the insulin-like growth factor 1 is loaded on the polydopamine particles through π-π stacking and hydrogen bonding, so that the insulin-like growth factor 1 loaded in the pH-responsive hydrogel is quickly released in the inflammation area, while relieving periodontal inflammation and promoting periodontal bone tissue regeneration. The pH-responsive hydrogel in this application has good biocompatibility, antibacterial, anti-inflammatory and osteogenic properties, and can be used to prepare drugs for the treatment of periodontal diseases and products for repairing periodontal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for describing the embodiments are briefly introduced below.

[0019] Figure 1 FT-IR spectrum of the pH-responsive hydrogel provided in the examples of the present application;

[0020] Figure 2 This is a physical picture of the pH-responsive hydrogel before and after gelation provided in the examples of the present application;

[0021] Figure 3 This is a scanning electron microscope morphology of the pH-responsive hydrogel provided in the examples of the present application;

[0022] Figure 4 A diagram for verifying the injectability and remodeling properties of the pH-responsive hydrogel provided in the examples of the present application;

[0023] Figure 5 This is an oscillatory shear rheological analysis diagram of the pH-responsive hydrogel provided in the examples of the present application;

[0024] Figure 6 The drug release performance analysis diagram of the pH-responsive hydrogel provided in the examples of this application:

[0025] A is the analysis of drug release performance of the pH-responsive hydrogel prepared in Example 1 under different pH environments; B is the analysis of drug release performance of the pH-responsive hydrogel prepared in Examples 1-3 under the same pH environment;

[0026] Figure 7The in vitro antibacterial test diagram of the pH-responsive hydrogel provided in the embodiment of the present application is as follows:

[0027] A is a physical picture of the co-culture of the pH-responsive hydrogel prepared in Example 1-3 and bacteria; B is a colony count analysis picture after the co-culture of the pH-responsive hydrogel prepared in Example 1-3 and bacteria;

[0028] Figure 8 Biocompatibility test diagram of pH-responsive hydrogel provided in the examples of the present application:

[0029] A is a CCK-8 cell proliferation analysis chart; B is a cell live / dead staining chart;

[0030] Fig. 9 This is a test chart of the anti-inflammatory effect of the pH-responsive hydrogel provided in the examples of this application:

[0031] A is the fluorescence staining diagram of cell DPPH scavenging ability; B is the diagram of cell DPPH scavenging rate;

[0032] Fig.10 Gene expression analysis diagram of the osteogenic performance and anti-inflammatory effect of the pH-responsive hydrogel provided in the examples of this application:

[0033] A is the expression analysis of inflammation-related genes IL-6 and TNF-α; B is the expression analysis of mineralization-related genes OCN and RUNX2. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0035] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0036] The ultimate goal of periodontal treatment is to regenerate the structure of periodontal tissue and restore its function. Clinically, the treatment of periodontal disease (such as scaling, root planing and surgical treatment) is mainly aimed at plaque removal and local inflammation control. Although these treatments can relieve symptoms and control the progression of the disease to a certain extent, the clinical prognosis is usually unpredictable, especially once the periodontal bone tissue is defective, it is difficult to repair, which will eventually lead to tooth loss, affecting the patient's overall health and quality of life. Therefore, how to effectively and accurately repair the structure and function of periodontal bone tissue is needed now.

[0037] At present, a variety of bone transplant materials can be used to repair periodontal bone defects, including autologous bone, allogeneic bone, xenogeneic bone and synthetic biomaterials. Among them, although autologous bone transplantation is the "gold standard" for repairing bone defects, there is a possibility of complications such as insufficient bone mass, postoperative infection and bleeding; although allogeneic bone and xenogeneic bone are widely available, there are also inevitable risks such as immune rejection and disease transmission; although synthetic biomaterials have good biocompatibility and integration, they usually lack strong bone tissue regeneration induction and cannot achieve periodontal bone tissue regeneration well.

[0038] Although biomaterials have good biocompatibility and integration, they usually lack strong tissue regeneration induction and cannot achieve periodontal bone tissue regeneration. Combining biomaterials with bioactive substances can usually enhance the induction effect of biomaterials on cells, which is beneficial for repairing periodontal bone tissue defects. However, in the process of periodontal bone tissue regeneration, the introduction of exogenous mesenchymal stem cells (MSCs) has problems such as immune rejection and tumorigenic risks, and the clinical application of exogenous MSCs still faces huge challenges.

[0039] At present, scholars at home and abroad have prepared a variety of biomaterials to repair periodontal bone tissue defects. For example, Souto-Lopes et al. constructed a supercritical CO2 sterilized nanohydroxyapatite / chitosan bioscaffold, Yu et al. constructed a double-layer structure material composed of intra-fiber mineralized collagen and unmineralized parallel-arranged fibrils, and Ullas Mony et al. prepared a three-phase scaffold composed of chitin-polylactic acid-glycolic acid loaded nano-bioactive ceramics or growth factors. Although these biomaterials can regenerate structures similar to periodontal bone tissue to a certain extent, the regeneration of periodontal bone tissue is a complex physiological process that depends on the interaction of biomaterials, MSCs, cytokines, inflammation, immune microenvironment, etc. In addition, periodontal bone tissue defects are usually complex and irregular, and it is difficult to prepare biomaterials that precisely match the defect contour in advance.

[0040] Based on this, in the first aspect of the embodiment of the present application, a pH-responsive hydrogel is provided, comprising a hydrogel matrix and load particles dispersed in the hydrogel matrix. Wherein, the hydrogel matrix comprises carboxymethyl chitosan N-hydroxymethyl acrylamide (CMCSMA) and 3-aminophenylboronic acid (PBA) covalently linked thereto, and CMCSMA and PBA are linked via a borate ester bond. The load particles comprise polydopamine particles (PDA) and insulin-like growth factor 1 (IGF-1) loaded on PDA.

[0041] CMCSMA has certain photocuring properties as well as good biocompatibility and antibacterial properties. The surface of CMCSMA is positively charged, while the bacterial cell wall is usually negatively charged. The electrostatic attraction between opposite charges enables CMCSMA to contact the bacterial surface, resulting in the destruction of the bacterial membrane structure, leading to bacterial death. In addition, the CMCSMA molecule is small in size and can easily penetrate the bacterial cell membrane. After entering the bacterial cell membrane, the chitosan molecules interact with important biological molecules in the bacteria, disrupting their normal functions, thereby destroying the bacterial metabolism and enzyme system, and ultimately leading to bacterial death.

[0042] CMCSMA and PBA are covalently linked through borate bonds to form a hydrogel matrix (CMCSMA-PBA). Polydopa PDA has strong interactions with other materials through chemical and physical interactions, thereby showing high adhesion. At the same time, PDA can also load other drugs. IGF-1 is a type of growth factor that can promote the regeneration of dental tissue. PDA has rich functional groups. IGF-1 can be loaded on PDA through π-π stacking and hydrogen bonding to form loaded particles (PDA@IGF-1). CMCSMA-PBA can be quickly cured under ultraviolet light, and PDA@IGF-1 in the pH-responsive hydrogel is dispersed in it under the rapid curing of CMCSMA-PBA.

[0043] The borate bonds in CMCSMA-PBA decompose in the inflammatory acidic environment, and partially transform from a solid state to a liquid state, which accelerates the release of the loaded particles in the pH-responsive hydrogel, and then quickly releases IGF-1, which promotes the regeneration of tooth tissue.

[0044] In some embodiments, the mass ratio of the hydrogel matrix (CMCSMA-PBA) and the loaded particles (PDA@IGF-1) is 1:1.5-2:1. For example, the mass ratio of CMCSMA-PBA and PDA@IGF-1 is 1:1.5, 1:1 or 2:1, etc. The pH-responsive hydrogel at this ratio has good dynamic mechanical properties, anti-inflammatory effects, and the effect of promoting periodontal bone regeneration.

[0045] The second aspect of the embodiment of the present application provides a method for preparing the pH-responsive hydrogel in the first aspect of the embodiment of the present application, comprising the steps of:

[0046] The hydrogel matrix, the loaded particles and the photoinitiator are mixed to obtain a pH-responsive hydrogel.

[0047] In some embodiments, the mass ratio of the hydrogel matrix, the loaded particles, and the photoinitiator is 10:5:1-10:15:1. For example, the mass ratio of the hydrogel matrix, the loaded particles, and the photoinitiator is 10:5:1, 10:10:1, or 10:15:1.

[0048] In some embodiments, the photoinitiator is selected from one of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (Irgacure 2959) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0049] In some embodiments, the method for preparing the hydrogel matrix comprises the steps of:

[0050] S11, dissolving carboxymethyl chitosan N-hydroxymethyl acrylamide in 2-morpholineethanesulfonic acid (MES) buffer to obtain solution A;

[0051] S12, dissolving 3-aminophenylboronic acid in a dimethyl sulfoxide (DMSO) solution to obtain a solution B;

[0052] S13, mixing solution A and solution B, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), stirring for reaction, centrifuging after the reaction, taking the supernatant and dialyzing it to obtain the hydrogel matrix.

[0053] The carboxyl groups on CMCSMA were successfully activated by EDC and NHS, and the boronic acid groups were subsequently integrated into the CMCSMA framework as follows:

[0054]

[0055] In some embodiments, the mass ratio of CMCSMA, EDC, and NHS is 1:0.5:0.7-5:1.5:2.1. For example, the mass ratio of CMCSMA, EDC, and NHS is 1:0.5:0.7, 2:1:1.2, 4:1.2:1.4, 4:1.4:1.8, or 5:1.5:2.1. The stirring reaction time is 24-48h. For example, the stirring reaction time is 24h, 30h, 36h, 42h, or 48h. The speed of centrifugation after stirring the reaction is 6000-8000rpm. For example, the speed of centrifugation is 6000rpm, 6500rpm, 7000rpm, 7500rpm, or 8000rpm. The centrifugation time is 0.5-2h. For example, the centrifugation time is 0.5h, 1h, 1.5h, or 2h.

[0056] In some embodiments, the method for preparing the hydrogel matrix comprises the steps of:

[0057] S11', dissolving 1-10 g CMCSMA in 0.1-1 M MES buffer to obtain solution A;

[0058] S12', dissolving 5-50 mL 1% PBA (w / v) in DMSO to obtain solution B;

[0059] S13', take 1-10g solution A, 1-10g solution B, 0.5-5g EDC and 0.7-7g NHS, mix, and stir at room temperature for 24-48h. After the reaction is completed, the solution is centrifuged at 6000-8000rpm for 0.5-2h. Finally, the supernatant is dialyzed in deionized water using a MWCO3500 dialysis bag for 5-10 days and freeze-dried for storage.

[0060] In some embodiments, the method for preparing the loaded particles comprises the steps of:

[0061] S21. Dissolve dopamine hydrochloride in an ethanol solution of ammonia, then add insulin-like growth factor 1 and mix well, centrifuge the mixture to collect the precipitate and wash it to obtain the loaded particles.

[0062] The pH value of the ethanol solution of ammonia is 8.5, and the volume ratio of ethanol to water in the ethanol solution of ammonia is 1:1-4:9. The centrifugal speed is 5000-12500rpm. For example, the centrifugal speed is 5000rpm, 6000rpm, 7000rpm, 8000rpm, 9000rpm, 10000rpm, 11000rpm, 12000rpm or 12500rpm. The centrifugal time is 5-10min. For example, the centrifugal time is 5min, 6min, 7min, 8min, 9min or 10min.

[0063] In some embodiments, the mass ratio of dopamine hydrochloride to insulin-like growth factor 1 is 1:0.2-1:5.

[0064] In some embodiments, the method for preparing the loaded particles comprises the steps of:

[0065] S21', 40-400mL of anhydrous ethanol and 90-900mL of deionized water are mixed to form solution C. Ammonia water is added to solution C, the pH value is adjusted to 8.5, and stirred at room temperature for 0.5-2h. Subsequently, 0.5-5g of dopamine hydrochloride is added and stirred to dissolve. Then 0.1-1g of IGF-1 is added to obtain a mixture. The obtained mixture is centrifuged at 5000-12500rpm for 5-10min, the precipitate is collected and rinsed with ultrapure water for 3-4 times, and the precipitate is freeze-dried for 24-48h to obtain loaded particles.

[0066] A third aspect of the embodiments of the present application provides a use of the pH-responsive hydrogel of the first aspect of the embodiments of the present application in the preparation of drugs for treating periodontal diseases and products for repairing periodontal tissues.

[0067] In some embodiments, the pH responsive hydrogel is stored in a syringe in liquid form. When needed, the pH responsive hydrogel is injected into the periodontal inflammation area or a specific location using a syringe and irradiated with ultraviolet light for 10-20 seconds to quickly polymerize into a gel and adhere to the periodontal inflammation area or a specific area.

[0068] The following will provide further explanation through specific examples.

[0069] Example 1

[0070] (1) Dissolve 1 g of CMCSMA in 0.1 M MES buffer to obtain 1% (w / v) solution A. Dissolve 5 mL of 1% PBA (w / v) in dimethyl sulfoxide (DMSO) to obtain solution B. After mixing solution A and solution B, add 0.5 g of EDC and 0.7 g of NHS, and stir at room temperature for 24 hours. After the reaction, centrifuge the solution at 7500 rpm for 0.5 h to remove unreacted PBA. Finally, dialyze the supernatant in deionized water for 5 days using a MWCO 3500 dialysis bag, freeze-dry, and obtain a hydrogel matrix for later use.

[0071] (2) 40 mL of anhydrous ethanol and 90 mL of deionized water were mixed to prepare a solution. Ammonia water was added to the solution to adjust the pH value to 8.5. The resulting solution was stirred at room temperature for 2 h. Subsequently, 0.5 g of dopamine hydrochloride was added to dissolve it. After dissolution, 0.1 g of IGF-1 was added. Subsequently, the mixture was centrifuged at 12500 rpm for 5 minutes, and the precipitate was collected and rinsed with ultrapure water three to four times. Finally, the precipitate was freeze-dried for 48 hours to obtain loaded particles for use.

[0072] (3) 10 g of the hydrogel matrix prepared in (1), 15 g of the loaded particles prepared in (2), and 1 g of photoinitiator 2959 (Irgacure 2959, CAS#: 106797-53-9) were mixed to prepare a pH-responsive hydrogel.

[0073] Example 2

[0074] (1) Dissolve 1 g of CMCSMA in 0.1 M MES buffer to obtain 1% (w / v) solution A. Dissolve 5 mL of 1% PBA (w / v) in dimethyl sulfoxide (DMSO) to obtain solution B. After mixing solution A and solution B, add 0.5 g of EDC and 0.7 g of NHS, and stir at room temperature for 24 hours. After the reaction, centrifuge the solution at 7500 rpm for 0.5 h to remove unreacted PBA. Finally, dialyze the supernatant in deionized water for 5 days using a MWCO 3500 dialysis bag, freeze-dry, and obtain a hydrogel matrix for later use.

[0075] (2) 40 mL of anhydrous ethanol and 90 mL of deionized water were mixed to prepare a solution. Ammonia water was added to the solution to adjust the pH value to 8.5. The resulting solution was stirred at room temperature for 0.5-2 h. Subsequently, 0.5 g of dopamine hydrochloride was added to dissolve it. After dissolution, 0.1 g of IGF-1 was added. Subsequently, the mixture was centrifuged at 12500 rpm for 5 minutes, and the precipitate was collected and rinsed with ultrapure water three to four times. Finally, the precipitate was freeze-dried for 48 hours to obtain loaded particles for use.

[0076] (3) 10 g of the hydrogel matrix prepared in (1), 10 g of the loaded particles prepared in (2), and 1 g of Irgacure 2959 were mixed to prepare a pH-responsive hydrogel.

[0077] Example 3

[0078] (1) Dissolve 1 g of CMCSMA in 0.1 M MES buffer to obtain 1% (w / v) solution A. Dissolve 5 mL of 1% PBA (w / v) in dimethyl sulfoxide (DMSO) to obtain solution B. After mixing solution A and solution B, add 0.5 g of EDC and 0.7 g of NHS, and stir at room temperature for 24 hours. After the reaction, centrifuge the solution at 7500 rpm for 0.5 h to remove unreacted PBA. Finally, dialyze the supernatant in deionized water for 5 days using a MWCO 3500 dialysis bag, freeze-dry, and obtain a hydrogel matrix for later use.

[0079] (2) 40 mL of anhydrous ethanol and 90 mL of deionized water were mixed to prepare a solution. Ammonia water was added to the solution to adjust the pH value to 8.5. The resulting solution was stirred at room temperature for 0.5-2 h. Subsequently, 0.5 g of dopamine hydrochloride was added to dissolve it. After dissolution, 0.1 g of IGF-1 was added. Subsequently, the mixture was centrifuged at 12500 rpm for 5 minutes, and the precipitate was collected and rinsed with ultrapure water three to four times. Finally, the precipitate was freeze-dried for 48 hours to obtain loaded particles for use.

[0080] (3) 10 g of the hydrogel matrix prepared in (1), 5 g of the loaded particles prepared in (2), and 1 g of Irgacure 2959 were mixed to prepare a pH-responsive hydrogel.

[0081] Test Example 1

[0082] The chemical structure of the pH-responsive hydrogel prepared in Example 1 was analyzed using Fourier transform infrared spectroscopy (FT-IR). -1 FT-IR spectra were recorded in the range of Figure 1 As shown. Figure 1 It can be seen that at 1670cm -1 The stretching vibration peak corresponding to the amide bond C=O (carbonyl) appears at 1500-1600 cm -1 The bending vibration at 1340 cm represents the carbon-carbon double bond in the benzene ring, and the stretching vibration of BO occurs at 1340 cm -1 In addition, at 700cm -1 The carbon-hydrogen bond bending vibration of the BA phenyl group was detected, which confirmed the synthesis of the polymer (pH-responsive hydrogel) and successfully loaded IGF-1-rich PDA particles using this structure.

[0083] Test Example 2

[0084] (1) In order to explore the gel-forming ability of the pH-responsive hydrogel prepared in Example 1, the polymer solution was irradiated with ultraviolet light, and the hydrogel was formed within 10 seconds. The states before and after formation are as follows: Figure 2shown.

[0085] (2) The porosity and surface structure of the hydrogel were examined using a scanning electron microscope (SEM). Figure 3 As shown. Figure 3 It can be seen that the pH-responsive hydrogel presents a uniform porous structure, which is conducive to the growth and colonization of cells.

[0086] (3) The injectability and remodeling properties of the pH-responsive hydrogel prepared in the example were further verified. The pH-responsive hydrogel was drawn into the shape of the number "1" and the letter "A" by a syringe, and then irradiated with ultraviolet light, and its obvious gelation effect was seen. The results are as follows: Figure 4 As shown. It is further shown that the pH-responsive hydrogel shows dynamic bond rearrangement at the molecular level, which is different from traditional rigid covalent network hydrogels. Due to its dynamic bond formation and breaking cycle, the pH-responsive hydrogel can self-heal.

[0087] Test Example 3

[0088] (1) In order to study the dynamic mechanical properties of pH-responsive hydrogels, the storage modulus (G') and loss modulus (G") of the pH-responsive hydrogels prepared in Examples 1-3 were tested by oscillatory shear rheology under different frequency and strain test models. The results are as follows: Figure 5 As shown, from Figure 5 It can be seen that G' exceeds G" in all groups, confirming the formation of hydrogel.

[0089] (2) The drug release performance of the pH-responsive hydrogel under different pH conditions was tested. The drug release performance of the pH-responsive hydrogel prepared in Example 1 under pH 4.5 and pH 7.2 was tested, and the drug release performance of the pH-responsive hydrogel prepared in Examples 1-3 under the same pH conditions was tested. The results are shown in FIG. Figure 6 shown.

[0090] from Figure 6 As can be seen in Figure A, by using hydrochloric acid solution to adjust the pH value of the solution to 4.5, the hydrogel will rupture due to the pH-dependent dissociation of the boron ester bonds in the polymer chain. Specifically, within the initial 24 hours at a pH of 4.5, the release rate of IGF-1 was as high as 45.6%, and it stabilized within the next 24 hours. In contrast, at a pH of 7.2, less than 40% of IGF-1 was released within 48 hours. Since the boron ester bonds in the hydrogel are easily decomposed in an acidic environment, as the pH value decreases, the amount of IGF-1 released by the hydrogel also increases accordingly, which is beneficial to the release of drugs in the acidic environment of periodontal inflammation sites. Figure 6In B, CPPI-1, CPPI-2 and CPPI-3 represent pH-responsive hydrogels prepared in Examples 1-3, respectively. Figure 6 As can be seen in Figure B, at the same time point, IGF-1 release decreases as PDA content decreases, indicating that in the high ROS environment at the inflammatory site, PDA is oxidized and degraded by ROS, thereby achieving accurate and controllable release of drugs in the inflammatory environment.

[0091] Test Example 4

[0092] Periodontal disease is a chronic inflammatory disease associated with bacteria. The long-term persistence of bacteria not only destroys the periodontal microenvironment, but also damages the patient's overall health. Porphyromonas gingivalis (P.gingivalis) is a representative periodontal pathogen that causes adult periodontitis and is also the most common pathogen of periodontal disease. The in vitro antibacterial activity of the pH-responsive hydrogel prepared in Example 1 against Staphylococcus aureus (S.aureus), Escherichia coli (E.coli) and Porphyromonas gingivalis (P.gingivalis) was detected by the dilution plate method. The results are as follows Figure 7 As shown, Figure 7 CCPI-1, CPPI-2, and CPPI-3 represent the pH-responsive hydrogels prepared in Examples 1-3, respectively. Figure 7 A is a physical picture of the co-culture of the pH-responsive hydrogel prepared in Example 1-3 and bacteria. Figure 7 Figure B is a colony count analysis diagram of the pH-responsive hydrogel prepared in Example 1-3 after co-culturing with bacteria. Figure 7 As can be seen from Figures A and B, the pH-responsive hydrogels prepared in Examples 1-3 have significant antibacterial effects on Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Porphyromonas gingivalis (P. gingivalis).

[0093] Test Example 5

[0094] The biocompatibility of the pH-responsive hydrogels prepared in Examples 1-3 was tested by CCK-8 cell proliferation assay and live / dead cell staining. Figure 8 As shown, Figure 8 CCPI-1, CPPI-2, and CPPI-3 represent the pH-responsive hydrogels prepared in Examples 1-3, respectively. Figure 8 A in the middle is the analysis chart of cell proliferation ability detected by CCK-8. Figure 8 B is a live / dead cell immunofluorescence staining image, CPPI-1, CPPI-2 and CPPI-3 represent the pH-responsive hydrogels prepared in Examples 1-3, respectively. Figure 8As can be seen from Figures A and B, the pH-responsive hydrogels prepared in Examples 1-3 have no inhibitory effect on cell growth, indicating that they have good biocompatibility and can be used in the medical field.

[0095] Test Example 6

[0096] In the microenvironment of periodontal inflammation, the massive generation of reactive oxygen species (ROS) will hinder the healing of inflammation. The anti-inflammatory effect of the pH-responsive hydrogel prepared in Examples 1-3 was tested by free radical scavenging experiments. The results are as follows: Fig. 9 As shown, Fig. 9 CCPI-1, CPPI-2, and CPPI-3 represent the pH-responsive hydrogels prepared in Examples 1 to 3, respectively. Fig. 9 As can be seen from Figure A, the green fluorescence signal of the pH-responsive hydrogel group is weakened, indicating that the ROS in the cells can be effectively removed, which further indicates that the pH-responsive hydrogel prepared in Examples 1-3 has a good anti-inflammatory effect.

[0097] In addition, the expression of IL-6 and TNF-α, the genes related to cell inflammation, was analyzed by qRT-PCR. Fig.10 As shown in A, Fig.10 CCPI-1, CPPI-2, and CPPI-3 represent the pH-responsive hydrogels prepared in Examples 1 to 3, respectively. Fig.10 It can be seen from Figure A that the pH-responsive hydrogel prepared in Examples 1-3 can effectively reduce the expression levels of inflammation-related genes IL-6 and TNF-α in cells after LPS stimulation. Combined with the aforementioned free radical scavenging experiment, it can be shown that the pH-responsive hydrogel in the present application has good anti-inflammatory effect.

[0098] Test Example 7

[0099] After the pH-responsive hydrogel prepared in Example 1-3 was co-cultured with cells, mineralization was induced, and the expression levels of mineralization-related genes OCN and RUNX2 in the cells were analyzed by qRT-PCR. The results are as follows: Fig.10 As shown in B. Fig.10 As can be seen in Figure B, the expression levels of the mineralization-related genes OCN and RUNX2 in the cells also increased, indicating that the pH-responsive hydrogel prepared in Examples 1-3 has the effect of promoting bone formation, which is closely related to the ability of the pH-responsive hydrogel to sustainably release IGF-1.

[0100] In summary, the carboxymethyl chitosan N-hydroxymethyl acrylamide in the pH-responsive hydrogel of the present application has good biocompatibility and antibacterial properties, and is suitable for use in periodontal tissue positions. Carboxymethyl chitosan N-hydroxymethyl acrylamide is covalently linked to 3-aminophenylboronic acid through a borate bond, and the borate bond can make the hydrogel matrix quickly polymerize into a gel under ultraviolet light, and cooperate with the adhesion of polydopamine particles, so that the pH-responsive hydrogel enters the periodontal tissue, and quickly solidifies and adheres to the specific periodontal tissue inflammation area under ultraviolet light, and has injectability and remodeling properties. At the same time, the borate bond decomposes in the acidic environment generated by inflammation, and the insulin-like growth factor 1 is loaded on the polydopamine particles through π-π stacking and hydrogen bonding, so that the insulin-like growth factor 1 loaded in the pH-responsive hydrogel is quickly released in the inflammation area, while relieving periodontal inflammation and promoting periodontal bone tissue regeneration. The pH-responsive hydrogel in the present application has good biocompatibility, antibacterial, anti-inflammatory and osteogenic properties, and can be used to prepare drugs for the treatment of periodontal diseases and products for repairing periodontal tissues.

[0101] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A pH-responsive hydrogel, characterized in that The pH responsive hydrogel is prepared by comprising the following components: A hydrogel matrix comprising carboxymethyl chitosan N-hydroxymethyl acrylamide and 3-aminophenylboronic acid covalently linked thereto; Loaded particles, including polydopamine particles and insulin-like growth factor 1 loaded on the polydopamine particles; Wherein, the covalent bond of the covalent connection is a borate ester bond, and the loaded particles are dispersed in the hydrogel matrix.

2. The pH-responsive hydrogel according to claim 1, characterized in that The mass ratio of the hydrogel matrix to the loaded particles is 1:1.5-2:

1.

3. A method for preparing the pH responsive hydrogel according to claim 1 or 2, characterized in that: The preparation method comprises the steps of: The hydrogel matrix, the loaded particles and a photoinitiator are mixed to obtain a pH-responsive hydrogel.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the hydrogel matrix, the loaded particles and the photoinitiator is 10:5:1-10:15:

1.

5. The preparation method according to claim 3, characterized in that: The preparation method of the hydrogel matrix comprises the steps of: Dissolving carboxymethyl chitosan N-hydroxymethyl acrylamide in 2-morpholineethanesulfonic acid buffer to obtain solution A; Dissolving 3-aminophenylboronic acid in dimethyl sulfoxide solution to obtain solution B; Solution A and solution B are mixed, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added, stirred for reaction, and after the reaction, the supernatant is centrifuged and dialyzed to obtain the hydrogel matrix.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the carboxymethyl chitosan N-hydroxymethyl acrylamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:0.5:0.7-5:1.5:2.1; The stirring reaction time is 24-48 hours, the centrifugal speed is 6000-8000 rpm, and the time is 0.5-2 hours.

7. The preparation method according to claim 3, characterized in that: The method for preparing the loaded particles comprises the steps of: Dopamine hydrochloride is dissolved in an ethanol solution of ammonia, and then insulin-like growth factor 1 is added and mixed evenly. The mixture is centrifuged, and the precipitate is collected and washed to obtain the loaded particles.

8. The preparation method according to claim 7, characterized in that: The pH value of the ammonia ethanol solution is 8.5, and the volume ratio of ethanol to water in the ammonia ethanol solution is 1:1-4:

9.

9. The preparation method according to claim 7, characterized in that: The mass ratio of dopamine hydrochloride to insulin-like growth factor 1 is 1:0.2-1:

5.

10. Use of the pH-responsive hydrogel according to claim 1 or 2 in preparing drugs for treating periodontal diseases and products for repairing periodontal tissues.

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