Preparation method of self-cascade active oxygen capturing hydrogel system based on hexagonal boron nitride
By using a self-cascaded capture reactive oxygen species hydrogel system based on hexagonal boron nitride in the periodontitis microenvironment, the problem of difficulty in periodontal tissue regeneration is solved, and the effect of periodontitis treatment is significantly improved.
Patent Information
- Application Number
- CN202411255725.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-09
- Publication Date
- 2024-12-17
AI Technical Summary
The elevated oxidative stress level in the microenvironment of periodontitis leads to difficulty in regeneration of periodontal tissue. The existing hydrogel system is damaged under oxidative stress and loses the regeneration niche.
Design a self-cascaded capture reactive oxygen species hydrogel system based on hexagonal boron nitride, place antioxidants at crosslinking sites as traps to capture excess ROS, and pass a two-dimensional sheet of hexagonal boron nitride and oxidized pectin. Synergistically, reverse the oxidative stress state and regulate the redox balance inside and outside the cell.
Effectively capture and remove ROS, improve the mechanical properties of hydrogels, promote bone differentiation, significantly improve the regeneration ability of periodontal tissue, and prove its biosafety and application value.
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Abstract
Description
Technical Field
[0001] The present application relates to the field of biomedicine technology, and more specifically, to a method for preparing a self-cascading reactive oxygen capture hydrogel system based on hexagonal boron nitride. Background Art
[0002] In recent years, periodontitis, as a chronic inflammatory disease, has attracted widespread attention in the field of oral health. Periodontitis is a chronic inflammation caused by pathogenic microorganisms in subgingival plaque, which is often accompanied by symptoms such as periodontal inflammation, periodontal pocket formation, and alveolar bone loss. Under the pathological condition of periodontitis, immune cells continuously produce reactive oxygen species (ROS) to fight against bacterial invasion. ROS has a "double-edged sword" effect. The appropriate amount of ROS released by the mitochondrial respiratory chain and most enzymatic reactions is essential for intracellular signal transduction. However, excessive production of ROS will exceed the clearance capacity of the endogenous antioxidant defense system, thereby causing oxidative stress (OS). In addition, elevated ROS levels will inhibit the osteogenic differentiation of bone marrow stem cells (BMSCs), promote RANKL-mediated osteoclast activation, and ultimately destroy the dynamic balance of bone regeneration. Therefore, changing the extracellular oxygen-rich environment and reversing the intracellular redox imbalance are crucial for periodontal regeneration.
[0003] In the field of tissue engineering, ROS-responsive hydrogels are designed and used to remove ROS and repair tissue defects to achieve comprehensive diagnosis and treatment under oxidative stress microenvironment. However, the OS microenvironment can damage the hydrogel cross-linking network, leading to the breakage of polymer segments, the collapse of the repair system, and ultimately the loss of the regenerative niche.
[0004] Therefore, the difficulty in periodontal tissue regeneration caused by increased oxidative stress levels in the periodontitis microenvironment is a technical problem that needs to be urgently solved in this field. Summary of the invention
[0005] The purpose of the present invention is to solve the problem of difficulty in periodontal tissue regeneration caused by increased oxidative stress levels in the periodontitis microenvironment.
[0006] In order to achieve the above invention purpose, the present invention strategically proposes to place antioxidants at the cross-linking sites. When the hydrogel system is oxidatively degraded in the OS state, the antioxidants at the cross-linking sites are "opened" as traps to capture ROS and balance their levels; the two-dimensional hexagonal boron nitride (hBN) with antioxidant and osteogenic differentiation activity takes over and removes the captured ROS while improving the mechanical properties of the hydrogel system; the self-cascading product of the hydrogel system, the oxidation product of pectin, further drives the reduction reaction, and finally cooperates with the oxidized hBN to reshape the oxidative stress state inside and outside the cell. In summary, the present invention designs a self-cascading reactive oxygen capture hydrogel system based on hexagonal boron nitride, and its biosafety and the effect of promoting periodontal tissue regeneration prove that it has good research prospects and application value.
[0007] In a first aspect, the present application provides a method for preparing methacrylated pectin, using the following technical solution:
[0008] The preparation method comprises the following steps: adding pectin to a 2-morpholineethanesulfonic acid (MES) buffer solution containing sodium chloride (NaCl), stirring at room temperature until completely dissolved, adding N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), stirring for 10 minutes, adding N-(2-aminoethyl) methacrylamide hydrochloride (AEMA) to the solution, and reacting in a dark place at room temperature for 24 hours;
[0009] An excess of acetone was added to the precipitation solution, and the obtained polymer was dried under a fume hood. The obtained polymer was dissolved in deionized water for purification, dialyzed with deionized water at room temperature for 72 hours, and freeze-dried to obtain a methacrylated pectin freeze-dried sample.
[0010] Preferably, the ratio of NHS:EDC:AEMA is 90mM:180mM:90mM to prepare methacrylated pectin with a high degree of substitution.
[0011] Further preferably, the raw material composition and ratio involved in the preparation method are: pectin 1g, NaCl 2.925g, MES buffer 100mL, NHS1.035g, EDC 3.45g and AEMA 1.491g.
[0012] In a second aspect, the present application provides a method for preparing glutathione-modified silk protein, using the following technical solution:
[0013] The preparation method comprises the following steps: using MES buffer to dissolve natural silk protein into a solution, adding EDC and NHS, reacting at room temperature for 15 minutes under magnetic stirring, adding reduced glutathione (GSH) to the solution, reacting at room temperature for 15 minutes under magnetic stirring, dialyzing the reaction solution with deionized water at room temperature for 24 hours, and obtaining a glutathione-modified silk protein freeze-dried sample after freeze-drying.
[0014] Preferably, the raw material composition and ratio involved in the preparation method are: 0.8 g natural silk protein, 10 mL MES buffer, 0.7 mg NHS, 0.5 mg EDC and 0.02 g GSH.
[0015] Preferably, the concentration of the prepared glutathione-modified silk protein is 8% (w / v).
[0016] In a third aspect, the present application provides a method for preparing a self-cascading reactive oxygen species capture hydrogel system based on hexagonal boron nitride, using the following technical solution:
[0017] The preparation method comprises the following steps:
[0018] (1) preparing methacrylated pectin;
[0019] (2) preparing glutathione-modified silk protein;
[0020] (3) preparing glutathione-modified silk protein into a 10-20% (w / v) solution, preferably, adding 10 mmol / L of tris(2-carboxyethyl)phosphate hydrochloride (TCEP·HCl), and stirring for 15 minutes;
[0021] (4) methacrylated pectin, the glutathione-modified silk protein optimized in step (3), and a photoinitiator, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), are uniformly mixed in a volume fraction ratio of 2:1:1 to obtain a hydrogel precursor solution;
[0022] (5) 10% by mass of hexagonal boron nitride (hBN) was added, mixed evenly, and irradiated with ultraviolet light (wavelength 405 nm) for 30 seconds to obtain a self-cascading active oxygen capture hydrogel system based on hexagonal boron nitride.
[0023] In a fourth aspect, the present application provides a self-cascading reactive oxygen species capture hydrogel system based on hexagonal boron nitride, which is prepared by the preparation method described in the third aspect.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] 1. The present invention provides a self-cascading reactive oxygen capture hydrogel system based on hexagonal boron nitride, which uses natural pectin and silk protein as raw materials for the hydrogel scaffold. The preparation method is simple, the operation is easy, and the biocompatibility is good.
[0026] 2. Different from the previous practice of simply adding antioxidants to the hydrogel system, the present invention provides a self-cascading reactive oxygen species capture hydrogel system based on hexagonal boron nitride, which places antioxidants at the cross-linking sites, acting as traps to capture excessive ROS and trigger subsequent self-cascade reactions to remove ROS.
[0027] 3. The addition of hBN not only effectively improves the mechanical properties of the hydrogel, but also, after the trap is "opened", hBN cascades the tube and removes the captured ROS. The oxidized hBN enters the cell as a self-cascade product of the hydrogel system, reverses the intracellular oxidative stress state, and regulates the intracellular redox balance.
[0028] 4. When the ROS scavenging reaction reaches equilibrium, it is necessary to further introduce reactants or consume products to maintain the forward progress of the reaction. Pectin oligosaccharides (POS) produced by pectin oxidative depolymerization have antioxidant effects, which help promote the self-cascade ROS capture reaction, improve the overall antioxidant properties of the hydrogel system, and synergize with oxidized hBN to reshape the oxidative stress state inside and outside the cell. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 :Hydrogel system (A) synthesis route and (BC) infrared spectrum.
[0030] Figure 2 : Scanning electron microscopy image of the hydrogel system (large image scale 100 μm, the upper right small image is an enlarged image of the red dotted box area in the figure, scale 50 μm).
[0031] Figure 3 : ROS responsiveness of the hydrogel system.
[0032] Figure 4 :Cell safety of the hydrogel system (A) CCK8 results; in vitro antioxidant capacity (B) GSSH / GSH ratio and (C) MitoSOX staining.
[0033] Figure 5 : In vitro osteogenic properties of the hydrogel system (A) ALP and ARS staining, (B) OCN immunofluorescence staining (blue: DAPI, green: Actin, red: OCN) and (C) expression of osteogenic genes Alp, Runx2, and Bmp-2.
[0034] Figure 6:The effect of the hydrogel system on promoting bone regeneration in vivo (A) Schematic diagram of the rat periodontitis model and (B) Micro-CT images and CEJ-ABC distance, BV / TV ratio, and BMD values 4 and 8 weeks after surgery. DETAILED DESCRIPTION
[0035] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It is to be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention. It should also be noted that the experimental methods used in the following embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial sources.
[0036] Materials and Reagents:
[0037] Pectin, P9135, Sigma Aldrich, molecular weight ∼485 kDa;
[0038] NaCl, Sinopharm Chemical Reagent, molecular weight 58.4428;
[0039] MES buffer, Aladdin Agent Co., Ltd., molecular weight 195.24;
[0040] NHS, Aladdin Agent Co., Ltd., molecular weight 115.09;
[0041] EDC, Aladdin Agent Co., Ltd., molecular weight 191.70;
[0042] AEMA, Aladdin Agent Co., Ltd, molecular weight 165.62;
[0043] GSH, Aladdin Agent Co., Ltd., molecular weight 307.32.
[0044] Example 1: Preparation of a self-cascading reactive oxygen species capture hydrogel system based on hexagonal boron nitride
[0045] The hydrogel system of this embodiment is prepared by the following steps:
[0046] (1) Preparation of methacrylated pectin:
[0047] 1 g of pectin was added to 100 mL of MES buffer solution containing 2.925 g of NaCl, and stirred at room temperature until completely dissolved; 1.035 g of NHS and 3.45 g of EDC were added, and stirred for 10 minutes; 1.491 g of AEMA was added to the solution, and reacted in the dark at room temperature for 24 hours;
[0048] An excess of acetone was added to the precipitation solution, and the obtained polymer was dried under a fume hood; the obtained polymer was dissolved in 100 ml of deionized water for purification, dialyzed with deionized water at room temperature for 72 hours, and freeze-dried to obtain a methacrylated pectin freeze-dried sample.
[0049] (2) Preparation of glutathione modified silk protein:
[0050] 0.8 g of natural silk protein was dissolved in 10 mL of MES, and 0.5 g of EDC and 0.7 g of NHS were added, and the mixture was reacted at room temperature for 15 minutes under magnetic stirring. 2 g of GSH was added to the above solution, and the mixture was reacted at room temperature for 15 minutes under magnetic stirring. The reaction solution was dialyzed with deionized water at room temperature for 24 hours, and the glutathione-modified silk protein freeze-dried sample was obtained after freeze-drying.
[0051] (3) 10 mmol / L TCEP·HCl was added to prepare a 10% (w / v) solution of glutathione-modified silk protein, and the solution was stirred for 15 minutes.
[0052] (4) The methacrylated pectin, the glutathione-modified silk protein solution in step (3), and the photoinitiator LAP are uniformly mixed in a volume fraction ratio of 2:1:1 to obtain a hydrogel precursor solution.
[0053] (5) 10% by mass of hBN was added, mixed evenly, and irradiated with ultraviolet light (wavelength 405 nm) for 30 seconds to obtain a hexagonal boron nitride-based self-cascading active oxygen capture hydrogel system.
[0054] The synthetic route and infrared spectrum of the hydrogel system in this embodiment are shown in Figure 1 As shown in the scanning electron microscope image Figure 2 shown.
[0055] It is worth noting that, in other implementations:
[0056] For the preparation of methacrylated pectin in step (1), the following raw material composition and ratio are also used:
[0057] ① Pectin 0.5g, NaCl 1.4625g, MES buffer 50mL, NHS 1g, EDC 3g and AEMA 1.43g;
[0058] ② Pectin 0.25g, NaCl 0.734g, MES buffer 25mL, NHS 0.0882g, EDC 0.2875g and AEMA 0.1243g.
[0059] For the preparation of glutathione-modified silk protein in step (2), the following raw material composition and ratio are also used:
[0060] ① Natural silk protein 0.6g, MES buffer 6mL, NHS 4.2mg, EDC 4mg and GSH 0.014g;
[0061] ② Natural silk protein 1g, MES buffer 12mL, NHS 8.4mg, EDC 6mg and GSH 0.026g.
[0062] For step (3), a 20% (w / v) glutathione-modified silk protein solution was also prepared.
[0063] In step (4), the methacrylated pectin, the glutathione-modified silk protein solution in step (3), and the photoinitiator LAP are also mixed in a volume fraction ratio of 2:2:2.
[0064] For step (5), 5% by mass of hBN was also added for preparation.
[0065] The synthetic route and infrared spectrum of the above-mentioned embodiment are uniform Figure 1 The results are the same or similar, indicating that the hydrogel system was successfully prepared.
[0066] Example 2: Effect of the hydrogel system on the antioxidant capacity of BMSCs
[0067] Rat BMSCs were isolated from the femur and tibia of 4-week-old male Sprague Dawley (SD) rats and cultured in high-glucose Dulbecco's modified Eagle (DMEM) medium containing 10% fetal bovine serum (37°C, 5% CO2). Cells from passages 2 to 5 were used for subsequent experimental tests.
[0068] The Ctrl (control) group was set up: cells were cultured only with high-glucose DMEM medium containing 10% fetal bovine serum.
[0069] G8-10 (experimental) group was set up: cells were cultured using conditioned medium. 1 mL of freshly prepared hydrogel system (prepared in Example 1) was sterilized under UV light for 24 hours, and the hydrogel sample was immersed in 20 mL of high-glucose DMEM medium containing 10% fetal bovine serum, and cultured continuously for 3 days on a shaker at 37°C with a shaking speed of 50 rpm. The supernatant was collected as the conditioned medium for cell culture.
[0070] The cell activity of BMSCs was detected by CCK-8 method. BMSCs were seeded in 12-well plates and cultured overnight with conditioned medium. 10% CCK-8 detection solution was added to the culture medium at 1, 4, and 7 days, and cultured at 37°C for 30 minutes. The absorbance at 450nm was detected using an ELISA reader. Figure 4 A The results showed that the hydrogel system significantly improved the cell activity of BMSCs.
[0071] BMSCs were seeded in 12-well plates and cultured in a conditioned medium containing 10 μM hydrogen peroxide (H2O2). Glutathione / oxidized glutathione (GSSG / GSH) was measured using a quantification kit. The absorbance at 415 nm was measured using an ELISA reader. Figure 4 B The results showed that the hydrogel system significantly improved the antioxidant capacity of BMSCs.
[0072] BMSCs were seeded in 12-well plates and cultured overnight with conditioned medium, and then cultured for 3 days with conditioned medium containing 10 μM H2O2. The cells were stained with 2,7-dichlorofluorescein diacetate (DCFH-DA) for 30 minutes (37°C), and images were taken using a fluorescence microscope. Figure 4 C The results showed that the hydrogel system significantly improved the antioxidant capacity of BMSCs.
[0073] Example 3: Effect of the hydrogel system on promoting osteogenic differentiation of BMSCs
[0074] The Ctrl (control) group was set up: cells were cultured only with high-glucose DMEM medium containing 10% fetal bovine serum.
[0075] G8-10 (experimental) group was set up: cells were cultured using the above-mentioned conditioned medium.
[0076] BMSCs were seeded in 12-well plates and cultured overnight with conditioned medium. Conditioned medium containing 10 μM H2O2 was added and cultured for 7 and 14 days. The cells were fixed and stained using an alkaline phosphatase (ALP) kit and an Alizarin Red (ARS) kit. Figure 5 A The results showed that the hydrogel system significantly enhanced the osteogenic differentiation of BMSCs.
[0077] BMSCs were seeded in 12-well plates and cultured with conditioned medium for 14 days. The cells were fixed, permeabilized and blocked. Osteocalcin (OCN) primary antibody was added and incubated at 4°C overnight. Fluorescent secondary antibody was added and incubated at room temperature in the dark for 1 hour. FITC-labeled phalloidin (1:200 dilution) was added and incubated in the dark for 30 minutes. After washing, the cells were stained with 1 μg / mL 4',6-diamidino-2-phenylindole (DAPI) stain for 3 minutes and observed using a laser confocal microscope after washing. Figure 5 B The results showed that the hydrogel system significantly enhanced the osteogenic differentiation of BMSCs.
[0078] BMSCs were seeded in 12-well plates and cultured in conditioned medium containing 10 μM H2O2 for 7 days. Total RNA was extracted using RNAisoPlus reagent, RNA concentration was adjusted, reaction system was configured, and cDNA was synthesized by reverse transcription using HifairⅢ1st Strand cDNASynthesis SuperMix for qPCR kit. After reverse transcription, qPCRSYBR Green Master Mix reagent was placed in a Roche high-throughput PCR instrument for detection reaction. Rat Gapdh was used as the housekeeping gene. -ΔΔCT The expression level of the target gene was calculated. Figure 5 C The results showed that the hydrogel system had a positive effect on promoting the osteogenic differentiation of BMSCs.
[0079] Example 4: Effect of the hydrogel system on the regeneration of alveolar bone in rats with periodontitis
[0080] Twenty-four male, 8-week-old SPF SD rats were selected and raised in an environment with balanced temperature and humidity, with 12 hours of light and 12 hours of darkness. All rats were adapted to the environment and diet in the first week and divided into three groups: BLANK (blank group), Ctrl (model group) and G8-10 (experimental group), with 8 rats in each group, and 4 rats at each time point of 4 and 8 weeks.
[0081] During the experiment, 4-0 silk sutures were used to ligature the maxillary second molars of the rats on both sides without damaging the nearby gingival tissue. 1 mg / mL lipopolysaccharide was applied to the ligature area.
[0082] BLANK group: healthy SD rats were set up. The rats were killed and samples were collected at 4 and 8 weeks.
[0083] Ctrl (model group): After 10 days of ligation, the silk thread was removed and PBS buffer was injected into the periodontal pockets of the maxillary second molars on both sides of the rats. The rats were killed and the samples were collected 4 and 8 weeks after the injection.
[0084] Setting G8-10 (experimental group): After 10 days of ligation, the silk thread was removed and the hydrogel system (prepared in Example 1) was injected into the periodontal pockets of the maxillary second molars on both sides of the rats. The rats were killed and samples were collected 4 and 8 weeks after the injection.
[0085] Figure 6 The results showed that (A) was a schematic diagram of the rat periodontitis model, (B) was a two-dimensional and three-dimensional Micro-CT reconstruction image after sampling of the rat, and the red rectangular box indicated the alveolar bone regeneration; the distance from the cementum-enamel junction to the alveolar bone ridge (CEJ-ABC), bone volume / tissue volume (BV / TV) and bone mineral density (BMD) results all showed that the injection of the hydrogel system could promote alveolar bone regeneration.
[0086] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.
Claims
1. A method for preparing a self-cascading reactive oxygen species capture hydrogel system based on hexagonal boron nitride, characterized in that: The following steps are involved: (1) preparing methacrylated pectin; (2) preparing glutathione-modified silk protein; (3) adding TCEP·HCl to the glutathione-modified silk protein to prepare a 10-20% (w / v) glutathione-modified silk protein solution; (4) mixing the methacrylated pectin, the glutathione-modified silk protein solution in step (3), and the photoinitiator LAP to obtain a hydrogel precursor solution; (5) adding hBN, mixing evenly, and irradiating with ultraviolet light to obtain a hexagonal boron nitride-based self-cascading reactive oxygen species capture hydrogel system.
2. The preparation method according to claim 1, characterized in that: The preparation process of step (1) is: Pectin was added to MES buffer containing NaCl, stirred at room temperature until completely dissolved, NHS and EDC were added, stirred, AEMA was added to the solution, and reacted in the dark at room temperature; An excess amount of acetone was added to the precipitation solution, and the obtained polymer was dried under a fume hood. The obtained polymer was dissolved in deionized water for purification, dialyzed with deionized water at room temperature, and freeze-dried to obtain a methacrylated pectin freeze-dried sample.
3. The preparation method according to claim 2, characterized in that: The NHS:EDC:AEMA ratio was 90 mM:180 mM:90 mM.
4. The preparation method according to claim 2, characterized in that: The raw material composition and proportion involved in the preparation process are: 1 g pectin, 2.925 g NaCl, 100 mL MES buffer, 1.035 g NHS, 3.45 g EDC and 1.491 g AEMA.
5. The preparation method according to claim 1, characterized in that: The preparation process of step (2) is: The natural silk protein was dissolved into a solution using MES buffer, EDC and NHS were added, and the reaction was carried out at room temperature under magnetic stirring. GSH was added to the above solution, and the reaction was carried out at room temperature under magnetic stirring. The reaction solution was dialyzed with deionized water at room temperature, and a glutathione-modified silk protein freeze-dried sample was obtained after freeze-drying.
6. The preparation method according to claim 5, characterized in that: The raw material composition and proportion involved in the preparation process are: 0.8 g of natural silk protein, 10 mL of MES buffer, 0.7 mg of NHS, 0.5 mg of EDC and 0.02 g of GSH.
7. The preparation method according to claim 1, characterized in that: The methacrylated pectin in step (4), the glutathione-modified silk protein solution in step (3), and the photoinitiator LAP are mixed in a volume fraction ratio of 2:1:
1.
8. A self-cascading reactive oxygen species capture hydrogel system based on hexagonal boron nitride obtained by the preparation method according to any one of claims 1 to 7.
9. Use of the hexagonal boron nitride-based self-cascading reactive oxygen species capture hydrogel system according to claim 8 in the preparation of a drug for treating periodontitis.
10. A drug, characterized in that The drug comprises the hexagonal boron nitride-based self-cascading reactive oxygen species capture hydrogel system according to claim 8 as an active ingredient.