Glucose-responsive injectable antibacterial hydrogel, its preparation method and applications

Abstract

This invention discloses a glucose-responsive injectable antibacterial hydrogel, its preparation method, and its uses. It mainly consists of arginine-modified chitosan, a hypoglycemic drug, and a glucose-responsive ring brush polymer (c-PHODB), with the hypoglycemic drug loaded onto the c-PHODB. The injectable hydrogel is formed by hydrogen bonding, hydrophobic interaction, and electrostatic interaction between L-arginine-modified chitosan and metformin-loaded c-PHODB. Under in vivo metabolism, L-arginine generates the neurotransmitter nitric oxide (NO), promoting angiogenesis, and simultaneously exhibits a strong synergistic bactericidal effect with chitosan. As the hydrogel degrades, the glucose-responsive ring brush polymer releases the hypoglycemic drug (metformin), regulating blood glucose, restoring diabetic microenvironment homeostasis, and promoting bone healing. This strategy provides a novel approach for treating diabetic alveolar bone defects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymers and relates to the preparation of a glucose-responsive injectable antibacterial hydrogel. Background Technology

[0002] Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, which leads to excessive production of reactive oxygen species (ROS), triggering inflammation and damaging various tissues in the body. Simultaneously, diabetes is a metabolic disease that can cause disorders in bone metabolism, calcium and phosphorus metabolism. Factors such as absolute insulin deficiency / resistance and hyperglycemia can prevent the body from producing sufficient secondary bone tissue, leading to diabetic bone diseases such as osteoporosis. Diabetes-related complications are becoming increasingly common, and diabetic bone disease has become a major challenge in current medical research.

[0003] Periodontitis is a widespread oral disease affecting nearly half of the world's adults. The relationship between diabetes and oral health is close; the academic community has clearly established that diabetes is a significant risk factor for periodontitis. Numerous studies have demonstrated that the incidence of periodontal disease in diabetic patients is significantly higher than in those with normal blood sugar levels. Furthermore, diabetes affects the implementation of various oral treatments, including implant restorations. Periodontal disease complicated by diabetes can continuously worsen periodontal tissue inflammation, leading to irreversible periodontal bone resorption and even tooth loss. Diabetes is more common in middle-aged and elderly individuals, who are also a high-risk group for periodontal disease. Periodontal disease can lead to alveolar bone loss, and diabetes can exacerbate periodontitis and impair the healing of alveolar bone defects. The pathogenesis of diabetic alveolar bone regeneration disorders remains unclear, posing a challenge in clinical oral treatment. Therefore, effective periodontal bone regeneration in patients with type 2 diabetes mellitus (T2DM) requires a combined approach of anti-inflammatory, angiogenesis-promoting, antibacterial, and osteogenic effects. Although many biomaterials for periodontal tissue regeneration have been designed, few can simultaneously address the issues of periodontal vascular regeneration, antibacterial and anti-inflammatory effects, and simultaneous osteogenic regeneration in diabetic patients. Summary of the Invention

[0004] The technical problem to be solved by this invention is: how to construct a multifunctional hydrogel that integrates antibacterial, anti-inflammatory, angiogenesis and bone regeneration through a simple method for the treatment of alveolar bone defects in diabetic patients.

[0005] The technical solution of this invention is: a glucose-responsive ring brush polymer. c- PHODB, its structure is shown in Equation I:

[0006] Formula I.

[0007] A glucose-responsive injectable antibacterial hydrogel is mainly composed of arginine-modified chitosan, a hypoglycemic drug, and the aforementioned glucose-responsive ring brush polymer.c- PHODB composition, the hypoglycemic drug loaded in a glucose-responsive ring brush polymer c- On PHODB.

[0008] Furthermore, the hypoglycemic drug is metformin.

[0009] Furthermore, the arginine-modified chitosan has the structure shown in Formula II:

[0010] Formula II.

[0011] The method for preparing the glucose-responsive injectable antibacterial hydrogel described above includes the following steps: (1) L-arginine and chitosan undergo an amide reaction under EDC / NHS catalysis to generate arginine-modified chitosan; (2) with c -P(HEMA 50 Cyclic brush polymers were synthesized via ATRP using β-Br as a macromolecular initiator, 2,2′-bipyridine / cuprous bromide as ligands and catalysts, and N,N-dimethylaminomethyl methacrylate and polyethylene glycol methacrylate as monomers. c -PHOD; then through c -PHOD reacts with 4-bromomethylphenylboronic acid to synthesize glucose-responsive ring brush polymers. c- PHODB; (3) Loading hypoglycemic drugs onto glucose-responsive ring brush polymers c- On PHODB, the drug delivery system was obtained. c- PHODB; (4) Combine the arginine-modified chitosan obtained in step (1) and the drug-loaded material obtained in step (3). c- PHODB is dissolved in an aqueous solution and mixed to obtain a glucose-responsive injectable antibacterial hydrogel.

[0012] Furthermore, the macromolecular initiator c -P(HEMA 50 The structural formula of -Br) is shown in Formula III:

[0013] Formula III.

[0014] Furthermore, the hypoglycemic drug is metformin, which is loaded onto a glucose-responsive ring brush polymer via BN coordination. c- On PHODB.

[0015] The above-described glucose-responsive injectable antibacterial hydrogel is used in the preparation of products for treating diabetic alveolar bone defects.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention enables the formation of injectable hydrogels without the addition of the cross-linking agent sodium β-glycerophosphate. Specifically, L-arginine-modified chitosan and a glucose-responsive ring brush polymer loaded with metformin are combined via hydrogen bonding, hydrophobic interactions, and electrostatic interactions to form the injectable hydrogel. In vivo metabolism allows L-arginine to produce the neurotransmitter nitric oxide (NO), promoting angiogenesis, and simultaneously exhibiting a strong synergistic bactericidal effect with chitosan. As the hydrogel degrades, the glucose-responsive ring brush polymer releases the hypoglycemic drug (metformin), regulating blood glucose, restoring diabetic microenvironment homeostasis, and promoting bone healing. This strategy provides a novel approach for treating diabetic alveolar bone defects. Attached Figure Description

[0017] Figure 1 A schematic diagram of the synthesis of arginine-modified chitosan CS-Arg; Figure 2 1H NMR spectrum of arginine-modified chitosan CS-Arg; Figure 3 A schematic diagram of the synthesis of glucose-responsive cyclic brush polymers; Figure 4 Cyclic brush polymers c -P(OEGMA-co-DMAEMA) (CPHOD) 1H NMR spectrum; Figure 5 Glucose-responsive cyclic brush polymers c -PHOD's 1H NMR spectrum; Figure 6 Cyclic macromolecular initiators c -P(HEMA-Br) 50 and c -PHOD SEC rinsing curve; Figure 7 DLS and zeta potential diagrams of glucose-responsive cyclic brush polymers; Figure 8 Rheological plots of CS-Arg and CS-Arg / CPHODB@Met; Figure 9 UV absorption spectrum and standard curve of Met; Figure 10 In vitro drug loading, encapsulation efficiency, and in vitro drug release curves of the drug-loaded hydrogel CS-Arg / CPHODB@Met. Detailed Implementation

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0019] Example 1

[0020] In this embodiment, a method for preparing arginine-modified chitosan (CS-Arg) is provided, and the synthesis steps are as follows: Synthesis of CS-Arg: First, 885 mg of 2-(N-morpholine) ethanesulfonic acid (MES) monohydrate was dissolved in 200 mL of deionized water. At room temperature, 975.53 mg of L-Arg was dissolved in 50 mL of MES solution. The pH was adjusted to 6-7 with hydrochloric acid. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 5.368 g) and N-hydroxysuccinimide (NHS, 1.956 g) were added to the solution in sequence. The pH was maintained at 6-7 by adding sodium hydroxide (NaOH) or HCl. The mixture was stirred at room temperature to generate activated L-arginine.

[0021] Add chitosan (750 mg) to 150 mL of fresh MES solution, dissolve it with an appropriate amount of HCl, and adjust the pH to 6 with 5 M NaOH solution to obtain CS solution.

[0022] The activated L-arginine solution was poured into CS solution and stirred at room temperature for two days. Finally, the product was collected by dialysis with deionized water and lyophilized.

[0023] The above chemical reaction process is as follows Figure 1 As shown, the 1H NMR spectrum of the synthesized CS-Arg is as follows. Figure 2 As shown.

[0024] Example 2

[0025] In this embodiment, a method for preparing a glucose-responsive ring brush polymer is provided, and the synthesis steps are as follows: (1) Synthesis of hydrophilic cyclic brush polymers c -P(OEGMA-co-DMAEMA), i.e. c -PHOD: macromolecular initiators c -P(HEMA 50 -Br) (3.5 mg), N,NDimethylaminomethyl methacrylate (DMAEMA, 135 µL), polyethylene glycol methacrylate (OEGMA, 142 µL), and 2,2′-bipyridine (bpy, 3.872 mg) were dissolved in DMF. After complete dissolution, the mixture was transferred to a 25 mL polymerization tube. Three freeze-evacuation-thawing cycles were performed. Under nitrogen protection, an equivalent amount of cuprous bromide (CuBr, 1.77 mg) to the initiator was rapidly added to the bottom of the polymerization tube, and three more freeze-evacuation cycles were performed. The polymerization tube was then placed in a 70°C container. o In a preheated oil bath, the reaction was stirred for 5 hours, then the reaction was terminated. The reaction mixture was added dropwise until excess ice-cold diethyl ether precipitated the crude product. This crude product was further dialyzed against water using a dialysis bag with a molecular weight cutoff of 3.5 kDa, and finally freeze-dried to obtain a gel-like product. c -PHOD.

[0026] The macromolecular initiator c -P(HEMA 50 The structure of -Br) is shown below: (2) Synthesis of glucose-responsive cyclic brush polymers Weigh 135 mg of ring brush polymer c -PHOD was activated with 20 mL of tetrahydrofuran (THF) for 30 min. 4-(bromomethyl)phenylboronic acid (160 mg) was then weighed, dissolved in 4 mL of THF, and added to the activated ring brush polymer solution. The solution was then placed at 50 °C. o In a preheated oil bath, the mixture was stirred and reacted. After about 30 minutes, the solution became viscous. After 5 hours of reaction, the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da. Finally, the product was freeze-dried to obtain a white, fluffy solid. c- PHODB.

[0027] The above chemical reaction process is as follows Figure 3 As shown, Figure 4 The image shows the 1H NMR spectrum of the synthesized ring brush polymer. Figure 5 The 1H NMR spectrum of the glucose-responsive ring brush polymer was obtained. Figure 6 CP (HEMA) is a cyclic macromolecular initiator. 50 SEC elution curves of the cyclic brush polymer CPHOD (-Br) and the cyclic brush polymer CPHOD were obtained. Compared with the cyclic macromolecular initiator, the SEC elution curve of CPHOD showed a significant left shift, tending towards a shorter retention time (higher MW), indicating the successful preparation of the cyclic brush polymer CPHOD.

[0028] Example 3

[0029] In this embodiment, a method for measuring the particle size and potential of different materials is provided, the steps of which are as follows: First, apply the ring brush polymer. c - PHOD is dissolved in DMF, dissolved, and dialyzed to remove DMF as completely as possible, followed by glucose-responsive ring brushing of the polymer. c -PHODB is dissolved in ultrapure water to prepare a solution with a concentration of 1.0 mg / mL for the testing of particle size (DLS) and zeta potential.

[0030] DLS testing: The average hydrodynamic particle size of polymer micelles was measured by dynamic light scattering (DLS).

[0031] Zeta potential determination: The zeta potential can be used to determine the solid-liquid interface electrical properties (ζ potential) of particulate matter in a dispersion system. 800 µL of a well-dispersed 1.0 mg / mL polymer micelle solution is placed in a zeta potential cell. The CS, Arg, and CS-Arg values ​​are determined through three repeated tests. c -PHOD and c -PHODB potential value.

[0032] Figure 7 for c -PHOD and c -PHODB's DLS and CS, Arg, CS-Arg, c -PHOD and c -PHODB's Zeta potential.

[0033] The changes in DLS particle size and zeta potential demonstrate that we have successfully synthesized arginine-modified chitosan CS-Arg, the ring brush polymer CPHOD, and the glucose-responsive polymer CPHODB.

[0034] Example 4

[0035] In this embodiment, a method for preparing a glucose-responsive injectable antibacterial hydrogel is provided, the steps of which are as follows: (1) Preparation of CS-Arg hydrogel: Weigh 20 mg of dry CS-Arg solid powder and dissolve it in 1.0 mL of 0.1 mol / L hydrochloric acid solution under stirring, and adjust the pH to about 8.

[0036] (2) Preparation of drug-loaded polymer CPHODB@Met: Metformin (Met, 6 mg) was dissolved in 1 mL of deionized water and stirred overnight in the dark. Then 30 mg of the polymer was added. c-PHODB was dissolved in deionized water and added to the above Met solution. The mixture was stirred for 1 h at room temperature in the dark to achieve drug encapsulation. Finally, the drug-loaded polymer solution was transferred to a dialysis bag and dialyzed with ultrapure water for 24 h. The drug-loaded micelles were then collected by lyophilization.

[0037] (3) Preparation of CS-Arg / CPHODB@Met hydrogel: Weigh 10 mg of drug-loaded polymer CPHODB@Met, dissolve it in 1 mL of ultrapure water, and add it to the solution in step (1) to obtain glucose-responsive injectable antibacterial hydrogel CS-Arg / CPHODB@Met.

[0038] Figure 8 Rheological plots for CS-Arg and CS-Arg / CPHODB@Met.

[0039] Rheological experiments showed that the storage modulus G' and loss modulus G" change with frequency. Within a wide frequency range (0.1~100 Hz), the storage modulus G' is greater than the loss modulus G", indicating that CS-Arg / CPHODB@Met mainly exhibits elastic deformation within this frequency range, i.e., it exists in the form of a hydrogel.

[0040] Example 5

[0041] In this embodiment, a glucose-responsive cyclic brush polymer is provided. c- The calculation of PHODB's in vitro drug loading and encapsulation efficiency, as well as the drug release method, are as follows: One mg of the lyophilized drug-loaded micelles obtained in Example 4 was dissolved in 1 mL of phosphate-buffered saline (PBS, pH 7.4, 150 mM). The absorbance of the drug-loaded micelles at the maximum absorption wavelength (232 nm) was measured using a UV-Vis spectrophotometer. The drug loading (DLC) and encapsulation efficiency (EE) of the polymeric drug-loaded micelles were calculated based on the standard curve of Met in the buffer solution at pH 7.4. The formulas for calculating DLC ​​and EE are as follows:

[0042]

[0043] In vitro drug release studies were conducted in phosphate (PBS, pH 7.4, 150 mM) and glucose solutions of 6.8 mmol / L and 16.6 mmol / L, respectively. CS-Arg / CPHODB@Met hydrogels were placed in centrifuge tubes containing the three release media, with three replicates for each medium. The tubes were incubated at 37°C and 120 rpm in a shaker. In the dark, 1 mL of the release solution was collected at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h, with an equal volume of fresh culture medium added each time. The absorbance of the solutions collected at different time points was measured using UV-Vis, and the release amount of Met at different time points was determined using a standard curve, thus plotting the Met release curves under different solution conditions.

[0044] Figure 9 The UV absorption spectrum and standard curve of Met are shown.

[0045] Figure 10 The in vitro drug loading, encapsulation efficiency, and in vitro drug release curves of the drug-loaded hydrogel CS-Arg / CPHODB@Met are shown.

[0046] The calculated drug loading capacity (DLC) of CPHODB@Met was 9.6%, and the encapsulation efficiency (EE) was 48%, indicating successful loading of the hypoglycemic drug metformin. In vitro drug release curves showed that, compared to PBS solution, the drug-loaded hydrogel exhibited glucose-responsive release behavior, and at a high glucose concentration of 16.6 mmol / L, the release rate was close to 90% after 72 h, further demonstrating the glucose concentration-dependent release behavior of the drug-loaded hydrogel, which can effectively alleviate hyperglycemia in diabetic alveolar bone defects.

Claims

1. A glucose-responsive injectable antibacterial hydrogel, characterized in that, It is mainly composed of arginine-modified chitosan, hypoglycemic drugs, and glucose-responsive ring brush polymer c-PHODB, wherein the hypoglycemic drugs are loaded onto the glucose-responsive ring brush polymer c-PHODB; the structural formula of the glucose-responsive ring brush polymer c-PHODB is shown in Formula I: ， Formula I.

2. The glucose-responsive injectable antibacterial hydrogel according to claim 1, characterized in that, The hypoglycemic drug is metformin.

3. The glucose-responsive injectable antibacterial hydrogel according to claim 1, characterized in that, The arginine-modified chitosan has the structure shown in Formula II: ， Formula II.

4. A method for preparing a glucose-responsive injectable antibacterial hydrogel according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Arginine and chitosan undergo an amide reaction under EDC / NHS catalysis to generate arginine-modified chitosan; (2) Using cP(HEMA) 50 Using 2,2′-bipyridine / cuprous bromide as a ligand and catalyst, and N,N-dimethylaminomethyl methacrylate and methyl methacrylate as monomers, the ring brush polymer c-PHOD was synthesized by ATRP. Then, the glucose-responsive ring brush polymer c-PHODB was synthesized by reacting c-PHOD with 4-bromomethylphenylboronic acid. (3) Loading hypoglycemic drugs onto glucose-responsive ring brush polymer c-PHODB to obtain drug-loaded c-PHODB; (4) Dissolve the arginine-modified chitosan obtained in step (1) and the drug-loaded c-PHODB obtained in step (3) in an aqueous solution and mix them to obtain a glucose-responsive injectable antibacterial hydrogel.

5. The method for a glucose-responsive injectable antibacterial hydrogel according to claim 4, characterized in that, The macromolecular initiator cP (HEMA) 50 The structural formula of -Br) is shown in Formula III: ， Formula III.

6. The method for a glucose-responsive injectable antibacterial hydrogel according to claim 4, characterized in that, The hypoglycemic drug is metformin, which is loaded onto the glucose-responsive ring brush polymer c-PHODB via BN coordination.

7. Use of the glucose-responsive injectable antibacterial hydrogel according to any one of claims 1-3 in the preparation of products for treating diabetic alveolar bone defects.

Images