Oxygen-releasing hydrogel with hydrogen sulfide response and preparation method thereof

Abstract

The application discloses an oxygen-releasing hydrogel with hydrogen sulfide response and a preparation method thereof. The gel comprises hemoglobin-polydopamine nanoparticles, aldehyde sodium alginate, carboxymethyl chitosan and four-arm azide polyethylene glycol. In the application, the aldehyde sodium alginate and the carboxymethyl chitosan are crosslinked into a gel through Schiff base reaction; the hemoglobin-polydopamine nanoparticles in the gel can release oxygen quickly in a low-oxygen environment to inhibit the growth and reproduction of anaerobic bacteria, thereby realizing rapid control of early-stage periodontitis infection; the carboxymethyl chitosan has good antibacterial performance and can realize sustained antibacterial effect; the four-arm azide polyethylene glycol can be entangled with the polymer chains of the sodium alginate and the chitosan to enhance the mechanical properties of the gel, and can also react with hydrogen sulfide, thereby playing a role in removing oral odor on one hand and forming amino groups through reaction of azide on the other hand, so as to further enhance the sterilization effect of the gel; and the polydopamine can also recruit stem cells to promote osteogenic differentiation and play a role in bone induction.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a hydrogen sulfide-responsive oxygen-releasing hydrogel that regulates the periodontal microenvironment and promotes periodontal tissue regeneration, and its preparation method. Background Technology

[0002] Periodontitis is a chronic infectious disease caused by dental plaque biofilm and is listed by the World Health Organization as the sixth leading cause of global epidemic. As the leading cause of tooth loss in adults, its pathological characteristics are the progressive destruction of periodontal supporting tissues (including the gingiva, alveolar bone, periodontal ligament, and cementum), ultimately leading to tooth loosening and loss. Studies have shown that dental plaque biofilm is the initiating factor for inflammation and destruction of periodontal tissues. It triggers an excessive immune response in the host through the colonization of the microbial community and its metabolites (such as lipopolysaccharides and proteolytic enzymes), forming a chronic inflammatory microenvironment characterized by neutrophil infiltration and the release of pro-inflammatory factors. Notably, the hypoxic microenvironment (oxygen partial pressure <5%) formed during the progression of chronic periodontitis further exacerbates disease progression. This microenvironment has a dual pathological effect: at the microbial level, hypoxia significantly promotes the proliferation of anaerobic bacteria, leading to periodontitis manifesting as a mixed infection dominated by anaerobic bacteria and enhancing the secretion of their virulence factors; at the host cell level, hypoxia activates the HIF-1α / NF-κB pathway, inducing apoptosis of periodontal ligament stem cells while simultaneously promoting osteoclast differentiation, resulting in an imbalance between periodontal tissue repair and destruction. Currently, clinical treatments for periodontitis are mainly divided into non-surgical and surgical treatments. Non-surgical treatment alone is unlikely to completely eliminate pathogens in deep periodontal pockets, resulting in limited therapeutic effects; surgical treatment, due to the influence of the periodontal inflammatory microenvironment, cannot fully achieve efficient periodontal tissue regeneration. Furthermore, its indications are narrow, and it increases the risk of infection. The overall goals of periodontitis treatment are: first, to control infection, eliminate inflammation, and halt disease progression; and second, to achieve periodontal tissue regeneration, restoring periodontal tissue function and physiological morphology. Therefore, developing a treatment strategy that can efficiently control infection and achieve efficient tissue regeneration has become a core challenge and a difficult problem in current research.

[0003] To achieve rapid infection control, the bioactive substance hemoglobin can rapidly release oxygen in a hypoxic environment, improving the hypoxic microenvironment within the periodontal pocket while inhibiting the growth and reproduction of anaerobic bacteria. Throughout the treatment process, we continuously release chitosan, which has excellent antibacterial properties, to maintain antibacterial activity. When the material is injected into the periodontal pocket, the four-armed azidopolyethylene glycol reacts with hydrogen sulfide, effectively removing oral odor, generating amino groups, increasing the cross-linking density of the hydrogel, forming a hydrogel with stronger mechanical properties and compressive strength, occupying a certain space, and playing a bone-guiding role. Polydopamine can also recruit stem cells, promote osteogenic differentiation, exert a bone-inducing effect, and ultimately achieve periodontal tissue regeneration.

[0004] To achieve functional regeneration of periodontal tissues, ideal scaffold materials must meet the following key conditions: Space maintenance and bone guidance: providing a three-dimensional porous structure to support new tissue growth and guide the directional migration of host cells; Bone induction: inducing mesenchymal stem cells (MSCs) to differentiate into osteoblasts through bioactive factors (such as BMP-2 and PDGF) or the material's inherent properties (such as biomimetic mineral coatings); Microenvironment regulation: addressing the hypoxic characteristics of periodontal pockets, integrating oxygen-controlled release carriers or anti-anaerobic components to synergistically resolve the conflict between infection and regeneration. Current materials used for periodontal regeneration (such as collagen membranes and β-tricalcium phosphate scaffolds) possess some of the above functions, but still suffer from the following problems: most materials only provide physical support and lack the ability to dynamically regulate the microenvironment; rapid degradation leads to failure of space maintenance, or excessive degradation triggers inflammatory responses; and complex manufacturing processes make large-scale clinical production difficult. Therefore, developing a new material that can continuously inhibit bacteria during the treatment phase, rapidly correct the hypoxic inflammatory environment within the pocket, prevent disease progression, and simultaneously have bone guiding and inducing effects to promote efficient regeneration of periodontal tissues would provide new insights for the clinical treatment of periodontitis. Summary of the Invention

[0005] To address the shortcomings mentioned above, the purpose of this invention is to provide a hydrogen sulfide-responsive oxygen-releasing hydrogel and its preparation method that regulates the periodontal microenvironment and promotes periodontal tissue regeneration.

[0006] To solve the above-mentioned technical problems, one technical solution adopted by the present invention is: a hydrogen sulfide-responsive oxygen-releasing hydrogel, comprising hemoglobin-polydopamine nanoparticles, sodium aldehyde alginate, carboxymethyl chitosan, and tetra-armed polyethylene glycol azido. The hemoglobin-polydopamine nanoparticles are obtained by mixing and stirring hemoglobin and dopamine in an alkaline environment; Sodium aldehyde alginate is prepared by oxidizing sodium alginate with sodium periodate.

[0007] Furthermore, the content of each component in the hydrogel is as follows: hemoglobin-polydopamine nanoparticles concentration 1-20 mg / mL, carboxymethyl chitosan concentration 10-50 mg / mL, sodium aldehyde alginate concentration 10-50 mg / mL, and tetra-armed azido polyethylene glycol 1-100 mg / mL.

[0008] Furthermore, the hemoglobin-polydopamine nanoparticles have a particle size of 1-200 nm.

[0009] This invention also discloses a method for preparing the hydrogen sulfide-responsive oxygen-releasing hydrogel, comprising the following steps: (1) Synthesis of hemoglobin-polydopamine core-shell structured nanoparticles: Hemoglobin solution and dopamine solution were mixed in an alkaline environment, and hemoglobin-polydopamine core-shell structured nanoparticles were synthesized by stirring oxidation method and then prepared into a hemoglobin-polydopamine nanoparticle solution. (2) Preparation of aldehyde-modified sodium alginate: Sodium alginate was synthesized by oxidizing sodium alginate with sodium periodate; (3) Synthesis of oxygen-releasing hydrogel: Prepare a four-armed azido polyethylene glycol solution and an aldehyde-modified sodium alginate solution; prepare a carboxymethyl chitosan solution and add the hemoglobin-polydopamine nanoparticle solution obtained in step (1) and stir slowly to obtain solution A; mix the four-armed azido polyethylene glycol solution, the aldehyde-modified sodium alginate solution and solution A and stir to obtain an oxygen-releasing hydrogel with hydrogen sulfide response.

[0010] Further, in step (1), the hemoglobin concentration is 1-100 mg / mL, the dopamine concentration is 1-20 mg / mL, and the hemoglobin solution and the dopamine solution are mixed in equal volumes.

[0011] Further, in step (1), the alkaline conditions are a Tris-HCl buffer environment with pH=8.0-9.0, and the stirring time is 3-4 hours.

[0012] Furthermore, in step (2), the molar ratio of sodium periodate to sodium alginate sugar unit is 0.1:1 to 10:1.

[0013] Further, in step (2), the preparation of aldehyde-modified sodium alginate is as follows: sodium alginate is dissolved in water, sodium periodate solution is added under light-protected conditions, and the reaction is carried out at 20-50℃ for 6-24 hours. The reaction is terminated by ethylene glycol, purified by dialysis, and then freeze-dried to obtain aldehyde-modified sodium alginate.

[0014] Finally, this invention discloses the application of the hydrogen sulfide-responsive oxygen-releasing hydrogel in the preparation of periodontitis treatment materials.

[0015] The beneficial effects of this invention are: 1. Multifunctional oxygen release and hydrogen sulfide (H2S) removal: The intelligent oxygen-releasing hydrogel developed in this invention constructs nanoparticles by encapsulating hemoglobin with polydopamine, which precisely releases oxygen in the periodontal inflammatory microenvironment to relieve local hypoxia. At the same time, by introducing azide groups, it efficiently removes H2S, improves oral odor and inhibits the growth of anaerobic bacteria, breaking through the limitation of single function of traditional treatment materials.

[0016] 2. Dynamic cross-linking and injectability: Based on the dynamic cross-linking mechanism of sodium alginate / chitosan, the hydrogel forms an injectable dual-network structure that adapts to complex periodontal anatomy and is convenient for clinical operation; its self-healing properties ensure the stability of the material in the dynamic oral environment and have a low conversion threshold.

[0017] 3. Synergistic antibacterial and bone regeneration promotion: The hydrogel has both antibacterial (especially against anaerobic bacteria) and osteogenic functions. By continuously releasing oxygen, it optimizes the local microenvironment, inhibits the progression of inflammation, and provides favorable conditions for periodontal tissue repair, thus achieving integrated treatment and function.

[0018] 4. Process and cost advantages: The one-step crosslinking process simplifies the production process, and the material stability is significantly better than traditional oxygen-releasing agents (such as CaO2). This reduces production costs while improving product performance, giving it market competitiveness.

[0019] 5. Microenvironment regulation and biocompatibility: Hydrogels regulate the periodontal immune microenvironment through the dual effects of improving hypoxia and clearing H2S. They have excellent biocompatibility and can promote periodontal tissue regeneration, providing an innovative solution for the treatment of periodontitis. Attached Figure Description

[0020] The accompanying drawings are provided to further explain the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Yes: A morphological diagram of the hemoglobin-polydopamine nanoparticles of this invention; Figure 2 Yes: the zeta potential of the hemoglobin-polydopamine nanoparticles of the present invention at 4° and 37°; Figure 3 Yes: Scanning electron microscope image of the hydrogen sulfide-responsive oxygen-releasing hydrogel of this invention; Figure 4 Yes: Characterization of the hydrogen sulfide-responsive oxygen-releasing hydrogel of the present invention; Figure 5 Yes: A diagram showing the results of an in vitro antibacterial experiment on the hydrogen sulfide-responsive oxygen-releasing hydrogel of this invention; Figure 6 Yes: This is a diagram showing the results of an in vitro osteogenic experiment using the hydrogen sulfide-responsive oxygen-releasing hydrogel of this invention. Detailed Implementation

[0021] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0022] Example 1: Synthesis and characterization of hemoglobin-polydopamine nanoparticles: (1) Synthesis of hemoglobin-polydopamine nanoparticles Mix equal volumes of 10 mg / ml hemoglobin solution and 1 mg / ml dopamine hydrochloride solution. Dissolve hemoglobin powder in ultrapure water and dopamine hydrochloride powder in 10 mM Tris-HCl solution (pH 8.5). Stir for 210 minutes, then dialysis to purify the mixture and prepare a hemoglobin-polydopamine nanoparticle solution.

[0023] (2) Characterization of hemoglobin-polydopamine nanoparticles The morphology of nanoparticles was observed using a transmission electron microscope; the particle size of nanoparticles was analyzed using a nanoparticle size analyzer; and the potential of nanoparticles was analyzed and measured using a zeta potential analyzer.

[0024] Transmission electron microscopy revealed that the hemoglobin-polydopamine nanoparticles were uniformly distributed spherical with a particle size of approximately 30-50 nm (e.g., ...). Figure 1 As shown), the zeta potential was measured to be negative at 4° and 37° (e.g. Figure 2 (As shown). In summary, the hemoglobin-polydopamine nanoparticles meet the basic characteristics of nanoparticles.

[0025] Example 2: Synthesis and characterization of hydrogen sulfide-responsive oxygen-releasing hydrogels: (1) Preparation of aldehyde-modified sodium alginate Sodium alginate was dissolved in water, and sodium periodate solution was added under light-protected conditions. The reaction was carried out at 25°C for 12 hours. The reaction was terminated by ethylene glycol. After purification by dialysis, the product was freeze-dried to obtain aldehyde-modified sodium alginate. The molar ratio of sodium periodate to sodium alginate sugar units was 0.8:1.

[0026] (2) Synthesis of hydrogen sulfide-responsive oxygen-releasing hydrogels A carboxymethyl chitosan solution was prepared and a hemoglobin-polydopamine nanoparticle solution was added. The mixture was stirred slowly to obtain solution A. An azide PEG solution was prepared. An aldehyde sodium alginate aqueous solution was prepared. The four-arm azide polyethylene glycol solution, the aldehyde sodium alginate solution, and solution A were mixed. The final concentrations of hemoglobin-polydopamine nanoparticles, carboxymethyl chitosan, aldehyde sodium alginate, and four-arm azide polyethylene glycol in the mixed solution were 10 mg / mL, 30 mg / mL, 25 mg / mL, and 50 mg / mL, respectively. The mixture was stirred to form a hydrogen sulfide-responsive oxygen-releasing hydrogel.

[0027] (3) Characterization of hydrogen sulfide-responsive oxygen-releasing hydrogels The morphology of the hydrogel was observed using scanning electron microscopy; the characteristics of the hydrogel were analyzed using Fourier transform infrared spectroscopy, a portable oxygen transporter, and lead acetate solution.

[0028] Transmission electron microscopy showed that the hydrogel had a uniform porous structure (e.g., Figure 3 As shown); it can gel through Schiff base reaction in 5-10 minutes, has a certain ability to release oxygen and remove hydrogen sulfide, and can generate amino groups after the reaction, which enhances the mechanical crosslinking strength of the hydrogel (e.g. Figure 4 (As shown).

[0029] Test case 1. In vitro antibacterial experiment of hydrogen sulfide-responsive oxygen-releasing hydrogel The hydrogel's ability to resist free bacteria and inhibit biofilm formation was evaluated by conducting antibacterial, inhibition zone, and biofilm inhibition experiments on Escherichia coli, Staphylococcus aureus, and Porphyromonas gingivalis.

[0030] CFU plate counting, inhibition zone experiments, and biofilm experiments all demonstrate that the hydrogen sulfide-responsive oxygen-releasing hydrogel of this invention possesses excellent antibacterial properties (e.g., Figure 5 ).

[0031] 2. In vitro osteogenic experiment of hydrogen sulfide-responsive oxygen-releasing hydrogel like Figure 6 As shown, human periodontal ligament mesenchymal stem cells were cultured and divided into five groups. The control group received 1 mL of osteogenic induction solution per well; the LPS group received 1 mL of osteogenic induction solution containing 10 ng / mL LPS per well; and the remaining experimental groups received 1 mL of osteogenic induction solution containing nanoparticles or extracts and 10 ng / mL LPS. ALP and ARS staining was performed after 7 and 21 days for analysis.

[0032] ALP and ARS staining results showed that after 7 and 21 days, both the nanoparticle group and the nanoparticle hydrogel group had the effect of promoting bone growth under inflammatory conditions, indicating that the hydrogen sulfide-responsive oxygen-releasing hydrogel of the present invention can improve the periodontal inflammatory microenvironment and promote periodontal tissue regeneration under inflammatory conditions.

[0033] In this invention, sodium alginate and carboxymethyl chitosan are cross-linked into a gel via a Schiff base reaction. Hemoglobin-polydopamine nanoparticles in the gel can rapidly release oxygen in a low-oxygen environment to inhibit the growth and reproduction of anaerobic bacteria, achieving rapid control of early-stage periodontitis infection. Carboxymethyl chitosan has excellent antibacterial properties, enabling sustained antibacterial action. Furthermore, tetra-armed azidopolyethylene glycol can entangle with the polymer chains of sodium alginate and carboxymethyl chitosan, further enhancing the gel's mechanical properties. It can also react with hydrogen sulfide, serving to eliminate oral odor, while the azido group reacts to form amino groups, further enhancing the gel's bactericidal effect. Polydopamine can also recruit stem cells, promote osteogenic differentiation, and exert a bone-inducing effect. The resulting hydrogel can be injected into periodontal pockets, adapting to irregular bone defect areas in periodontitis, thereby regulating the periodontal inflammatory microenvironment and promoting bone repair in periodontal tissues. This gel can rapidly control infection in the early stages, correct the hypoxic inflammatory microenvironment in periodontal pockets, and continuously inhibit bacteria during the treatment phase. It also has bone guiding and bone induction effects, exhibiting good biocompatibility and bioactivity. It provides new ideas for the clinical diagnosis and treatment of periodontitis and has promising application prospects.

[0034] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A hydrogen sulfide-responsive oxygen-releasing hydrogel for preparing periodontal disease treatment materials, characterized in that: Oxygen-releasing hydrogel is composed of hemoglobin-polydopamine nanoparticles, sodium aldehyde alginate, carboxymethyl chitosan, and tetra-armed azido polyethylene glycol. The hemoglobin-polydopamine nanoparticles are obtained by mixing and stirring hemoglobin and dopamine in an alkaline environment; The sodium aldehyde alginate is prepared by oxidizing sodium alginate with sodium periodate; the concentrations of each component in the hydrogel are as follows: hemoglobin-polydopamine nanoparticles: 1-20 mg / mL; carboxymethyl chitosan: 10-50 mg / mL; sodium aldehyde alginate: 10-50 mg / mL; and tetra-armed azido polyethylene glycol: 1-100 mg / mL.

2. The oxygen-releasing hydrogel according to claim 1, characterized in that: The hemoglobin-polydopamine nanoparticles have a particle size of 1-200 nm.

3. A method for preparing an oxygen-releasing hydrogel as described in claim 1 or 2, characterized in that: Includes the following steps: (1) Synthesis of hemoglobin-polydopamine nanoparticles: Hemoglobin solution and dopamine solution were mixed in an alkaline environment and hemoglobin-polydopamine nanoparticles were synthesized by stirring oxidation method and then prepared into a hemoglobin-polydopamine nanoparticle solution. (2) Preparation of aldehyde-modified sodium alginate: Sodium alginate was synthesized by oxidizing sodium alginate with sodium periodate; (3) Synthesis of oxygen-releasing hydrogel: Prepare a four-arm azidopolyethylene glycol solution and an aldehyde-modified sodium alginate solution; Prepare a carboxymethyl chitosan solution, add the hemoglobin-polydopamine nanoparticle solution obtained in step (1), and stir slowly to obtain solution A; The four-arm azidopolyethylene glycol solution, the aldehyde-modified sodium alginate solution, and solution A are mixed and stirred to obtain an oxygen-releasing hydrogel with hydrogen sulfide responsiveness.

4. The preparation method according to claim 3, characterized in that: In step (1), the hemoglobin concentration is 1-100 mg / mL, the dopamine concentration is 1-20 mg / mL, and the hemoglobin solution and the dopamine solution are mixed in equal volumes.

5. The preparation method according to claim 3, characterized in that: In step (1), the alkaline conditions are a Tris-HCl buffer environment with pH=8.0-9.0, and the stirring time is 3-4 hours.

6. The preparation method according to claim 4, characterized in that: In step (2), the molar ratio of sodium periodate to sodium alginate sugar units is 0.1:1 to 10:

1.

7. The preparation method according to claim 4, characterized in that: In step (2), the preparation of aldehyde-modified sodium alginate is as follows: sodium alginate is dissolved in water, sodium periodate solution is added under light-protected conditions, and the reaction is carried out at 20-50℃ for 6-24 hours. The reaction is terminated by ethylene glycol, purified by dialysis, and then freeze-dried to obtain aldehyde-modified sodium alginate.

8. The use of an oxygen-releasing hydrogel as described in claim 1 or 2 in the preparation of periodontitis treatment materials.

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