Nucleoside supramolecular hydrogel as well as preparation method and application thereof

Abstract

The invention provides nucleoside supramolecular hydrogel as well as a preparation method and application thereof, and belongs to the field of biological materials. The nucleoside supramolecular hydrogel is prepared by taking 2, 2 '-diamino-2'-deoxyadenosine and cyanuric acid as raw materials, is good in stability, can effectively promote alveolar bone defect bone repair and periodontitis bone defect repair, and has a wide application prospect in the aspect of preparation of bone defect repair materials.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a nucleoside supramolecular hydrogel and a preparation method and application thereof. Background Art

[0002] Due to the complex environment of the oral cavity and the anatomical factors of the alveolar bone defect area, the alveolar bone defect area will inevitably continue to absorb bone after tooth extraction, which will lead to the reduction of the alveolar ridge size. The change in size affects the aesthetics and functional repair of patients in the later stage. Therefore, promoting bone repair in the alveolar bone defect area is an urgent problem to be solved. At present, the biomaterials that promote the repair of alveolar bone defects in commercial and research and development (mainly divided into three categories: platelet concentrates, bone homologous and heterologous materials, composite and mixed materials) have the disadvantages of single functional properties, complex preparation methods, non-injectable, poor mechanical properties, slow degradation, hindering healing, and poor stability. Existing domestic and foreign products are limited to traditional gelatin sponges, fibrin-based mechanically active filling materials, polymer double-layer patch materials, collagen guided bone regeneration membranes, etc., with potential risks of infection and allergies. Existing biomaterials often have only single functions such as hemostasis, antibacterial, and osteogenesis, are complex to prepare and costly, cannot adapt to irregular alveolar bones, and have complex clinical operations and low patient comfort. Therefore, it is of great clinical significance to develop new biomaterials to promote the repair of alveolar bone defects.

[0003] Although the literature (DOI:10.1002 / adma.202108300) reported the hydrogel formed by self-assembly of 2-amino-2'-fluoro-2'-deoxyadenosine (2-FA), 2-FA and cyanuric acid (CA) were further used to prepare a supramolecular hydrogel. 2-FA hydrogel can be used as a healing agent for tooth extraction sockets. However, according to Figure S8.a of the literature, it can be seen that the supramolecular hydrogel composed of 2-FA and CA (the molar ratio of CA to 2-FA is 1:1) has poor stability and disintegrates within 7 minutes, and cannot be used as a hydrogel for in vivo application. Summary of the invention

[0004] The purpose of the present invention is to provide a nucleoside supramolecular hydrogel and a preparation method and use thereof.

[0005] The invention provides a nucleoside supramolecular hydrogel. The nucleoside supramolecular hydrogel is prepared from 2,2'-diamino-2'-deoxyadenosine and cyanuric acid as raw materials. The molar ratio of the 2,2'-diamino-2'-deoxyadenosine to cyanuric acid is 1-2:1-3.

[0006] The present invention also provides a method for preparing the nucleoside supramolecular hydrogel, which comprises the following steps: mixing an aqueous solution of 2,2'-diamino-2'-deoxyadenosine and an aqueous solution of cyanuric acid, and reacting them to obtain the nucleoside supramolecular hydrogel.

[0007] Furthermore, the concentration of the aqueous solution of 2,2'-diamino-2'-deoxyadenosine is 15 to 50 mM; the concentration of the aqueous solution of cyanuric acid is 15 to 50 mM.

[0008] Furthermore, the reaction conditions are: heating at 70-110° C. until dissolved, and placing in a 0-8° C. water bath for 50-70 min.

[0009] Furthermore, the reaction conditions are: heating at 80-100°C until dissolved, and placing in a 4°C water bath for 60 minutes.

[0010] The present invention also provides a method for synthesizing 2,2'-diamino-2'-deoxyadenosine, the method comprising the following steps:

[0011]

[0012] (1) 2'-amino-D-uridine reacts with ethyl trifluoromercaptoethyl ester to obtain the intermediate product NU1;

[0013] (2) The intermediate product NU1, N,N'-(1H-purine-2,6-diyl) diethylamide, a silanization agent and a catalyst are reacted to obtain 2,2'-diamino-2'-deoxyadenosine.

[0014] Furthermore, in step (1), the molar ratio of 2'-amino-D-uridine to ethyl trifluoromercaptoethyl ester is 1:1-2.5; the solvent of the reaction is an organic solvent; the temperature of the reaction is 10-40°C, and the time is 20-30h;

[0015] In step (2), the silanization agent is N, O-bis(trimethylsilyl)acetamide; the catalyst is trimethylsilyl trifluoromethanesulfonate; the molar ratio of the intermediate product NU1, N, N'-(1H-purine-2,6-diyl) diethylamide, the silanization agent and the catalyst is 1:1-2.5:5-10:12-14.5; the solvent of the reaction is an organic solvent; the reaction temperature is 70-90°C and the reaction time is 10-20 min.

[0016] Furthermore, in step (1), the molar ratio of 2'-amino-D-uridine to ethyl trifluoromercaptoethyl ester is 1:1.6; the solvent of the reaction is methanol; the temperature of the reaction is 15-35°C, and the reaction time is 24h;

[0017] In step (2), the molar ratio of the intermediate product NU1, N,N'-(1H-purine-2,6-diyl) diethylamide, silanization agent and catalyst is 1:1.85:7:13.8; the solvent of the reaction is acetonitrile; the reaction temperature is 80°C and the reaction time is 2h15min.

[0018] Furthermore, after step (1) is completed, the following purification step is further included: evaporating the solvent, treating the residue with chloroform, filtering, washing, and drying to obtain the intermediate product NU1;

[0019] After step (2) is completed, the following purification step is further included: pouring the reaction solution into saturated methanol and ammonia water, leaving it at room temperature for 24 hours, evaporating the solution, extracting, and separating by chromatographic column to obtain the product.

[0020] Furthermore, the extraction solvent is 0.1M triethylamine aqueous solution and chloroform; the chromatographic column is Dowex1X4 (OH - ) column, and the separation conditions were: first eluting with 800 mL of 10% methanol-water and then eluting with 20% methanol-water to obtain the product.

[0021] The present invention also provides the use of the nucleoside supramolecular hydrogel in preparing a bone defect repair material, wherein the material is preferably an alveolar bone defect repair material or a periodontitis bone defect material.

[0022] The present invention has achieved the following beneficial effects:

[0023] The present invention uses 2,2'-diamino-2'-deoxyadenosine and cyanuric acid (CA) as raw materials to prepare a nucleoside supramolecular hydrogel. The supramolecular hydrogel composed of 2-FA and CA in the document (DOI: 10.1002 / adma.202108300) disintegrated within 7 minutes, with poor stability; while the nucleoside supramolecular hydrogel of the present invention did not disintegrate after 3 days, and its stability was significantly improved. The nucleoside supramolecular hydrogel of the present invention can effectively promote the repair of alveolar bone defects and periodontitis bone defects, and has broad application prospects in the preparation of bone defect repair materials.

[0024] The present invention also optimizes the reaction process conditions of 2,2'-diamino-2'-deoxyadenosine (2-NA), and screens and obtains the preferred process conditions shown in Example 1.

[0025] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0026] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a high-resolution mass spectrum of 2-NA.

[0028] Figure 2 Characterization results of 2-NC nucleoside supramolecular hydrogel: (A) Schematic diagram of 2-NA and CA aqueous solutions that can be self-assembled to form 2-NC hydrogel after mixing in a certain proportion; (B) 2-NA and CA aqueous solutions can be mixed and heated and then cooled to form a stable 2-NC hydrogel, which does not collapse after 3 days; (C) 2-NA and CA at different concentrations are mixed to form 2-NC hydrogel; (D) 2-NA and CA at different volume ratios are mixed to form 2-NC hydrogel.

[0029] Figure 3 The experimental results of 2-NA promoting osteogenic performance in vitro: (A) Alizarin red staining at 14 days of osteogenic induction; (B) Quantitative analysis of the positive area of ​​Alizarin red; (C) Expression of osteogenic markers RUNX2 and OCN after 2 and 7 days of osteogenic induction, **P<0.01, *P<0.05.

[0030] Figure 4 The experimental results of 2-NA promoting the bone repair performance of alveolar bone defects in vivo: (A) MICRO-CT images of maxillary alveolar bone defect area at 7 and 21 days; (BC) MICRO-CT (B) bone volume fraction and (C) bone mineral density statistics of maxillary alveolar bone defect area at 7 days; (DE) MICRO-CT (D) bone volume fraction and (E) bone mineral density statistics of maxillary alveolar bone defect area at 21 days; (F) average buccal bone height results of maxillary alveolar bone defect area at 7 and 21 days; (G) average palatal bone height results of maxillary alveolar bone defect area at 7 and 21 days; (H) average maximum bone width results of maxillary alveolar bone defect area at 7 and 21 days; (I) H&E staining of maxillary alveolar bone defect area at 7 days and (J) 21 days, black star: newly formed woven bone; white arrow: temporary matrix. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05.

[0031] Figure 5 The experimental results of 2-NA promoting bone repair performance in periodontitis bone defects in vivo: (A) MICRO-CT image of maxillary periodontitis bone defect area after 10 days; (B) CEJ-ABJ statistical results of maxillary periodontitis bone defect area after 10 days. DETAILED DESCRIPTION

[0032] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0033] In the present invention, "room temperature / normal temperature" is 25±10°C, and "overnight" is 12±5 hours.

[0034] In the specific embodiment of the present invention, 2,2'-diamino-2'-deoxyadenosine used for synthesizing the nucleoside supramolecular hydrogel can be obtained by purchasing a commercial product (CAS No.: 215943-79-6), or can be prepared according to the method of Example 1.

[0035] Example 1. Preparation of 2,2'-diamino-2'-deoxyadenosine

[0036] The raw materials for synthesizing 2,2'-diamino-2'-deoxyadenosine (2-NA) of the present invention are as follows:

[0037] 1. The structural formula of 2'-amino-D-uridine (2'-NH2-dU) (CAS No.: 26889-39-4; MW: 243.22) is as follows:

[0038]

[0039] 2. The structural formula of S-ethyl trfluorothioacetate (CAS No.: 383-64-2; MW: 158.14) is as follows:

[0040]

[0041] 3. The structural formula of N,N'-(1H-purine-2,6-diyl)diethylamide (CAS No.: 34097-37-5; MW: 234.21) is as follows:

[0042]

[0043] The preparation of 2,2'-diamino-2'-deoxyadenosine was carried out according to the following synthetic route:

[0044]

[0045]

[0046] The specific operations are as follows:

[0047] (1) 2'-NH2-dU (6 g; 24.7 mmol) was suspended in 500 mL of methanol, stirred and ethyl trifluoromercaptoethyl (5 mL; 39 mmol) was added, and the suspension gradually turned into a colorless clear solution.

[0048] (2) The colorless clear solution was stirred at room temperature for 24 h.

[0049] (3) The solvent was evaporated to leave 50 mL. The residue was precipitated by adding chloroform until no more precipitate was produced. The precipitate was filtered out and washed with chloroform.

[0050] (4) Vacuum drying to obtain a white compound NU1, which was washed with CH2Cl2, ether, and n-hexane. (This step yielded 7.41 g of NU1, with a yield of 88.4%).

[0051] (5) The intermediate product NU1 (1.36 g; 4 mmol) and N,N'-(1H-purine-2,6-diyl)diethylamide (1.9 g; 7.4 mmol) were suspended in acetonitrile (24 mL), and N,O-bis(trimethylsilyl)acetamide (BSA) (7 mL; 28 mmol) was added for silylation reaction. The mixture was treated at reflux temperature (80°C) for 15 min.

[0052] (6) Trimethylsilyl trifluoromethanesulfonate (TMSTF) was added to the obtained clear solution as a catalyst for transglycosylation reaction (0.88 mL; 55.2 mmol), and the mixture was heated at reflux temperature (80° C.) for 2 h. The reaction mixture was poured into 120 mL of saturated methanolic ammonia solution.

[0053] (7) After standing at room temperature for 24 h, the solution was evaporated, and 300 mL of 0.1 M triethylamine aqueous solution and 300 mL of chloroform were added to the residue for phase separation.

[0054] (8) The aqueous layer was concentrated to half its volume and then washed with Dowex 1X4 (OH - ) column (specification: 3.1X30cm), first eluted with 800mL10% methanol-water, and then eluted with 1500mL20% methanol-water to obtain the product, a white powder, 2,2'-diamino-2'-deoxyadenosine (907mg, yield 80.3%, purity 98.90%).

[0055] Structural characterization data:

[0056] (1) 1 H NMR (600MHz, DMSO-d6) δ7.87(s,1H),6.78(s,2H),5.68(s,2H),5.57(s,1H),5.50(d,J=8.3Hz,1H),5.41(s,1H),3.98(d,J=5 .0Hz,1H),3.95-3.90(m,1H),3.84(dd,J=8.3,5.1Hz,1H),3.61(dd,J=11.7,3.5Hz,1H),3.52(d,J=11.6Hz,1H),1.78(s,2H).

[0057] (2) 13C NMR (151 MHz, DMSO-d6) δ 159.90, 156.27, 151.53, 136.58, 113.75, 88.24, 86.60, 71.90, 62.26, 57.03. ESI-TOF m / z calculated: C 10 H 15 N7O3[M+H + ]Actual value: 281.1236.

[0058] (3) High-resolution mass spectrometry: The molecular weight of the product was 282.13083, which was consistent with the target compound ( Figure 1 ).

[0059] Example 2: Preparation of Nucleoside Supramolecular Hydrogel

[0060] Weigh 2-NA and cyanuric acid (CA), respectively prepare 2-NA aqueous solution (concentration of 15mM, 20mM, 30mM, 40mM, 50mM) and CA aqueous solution (concentration of 15mM, 20mM, 30mM, 40mM, 50mM), respectively, mix the 2-NA aqueous solution and CA aqueous solution of equal concentration in a volume ratio of 1:1, heat to 100℃ until completely dissolved, and immediately place in a 4℃ water bath for 60min to obtain a stable nucleoside supramolecular hydrogel 2-NC. In the nucleoside supramolecular hydrogel 2-NC, the molar ratio of 2-NA to CA is 1:1.

[0061] Example 3: Preparation of Nucleoside Supramolecular Hydrogel

[0062] The method of reference example 2 is different only in that the volume ratio of 2-NA to CA is replaced with 2:1 to prepare the nucleoside supramolecular hydrogel 2-NC. In the nucleoside supramolecular hydrogel 2-NC, the molar ratio of 2-NA to CA is 2:1.

[0063] Example 4. Preparation of Nucleoside Supramolecular Hydrogel

[0064] The method of reference example 2 is different in that the volume ratio of 2-NA to CA is replaced with 1:3 to prepare the nucleoside supramolecular hydrogel 2-NC. In the nucleoside supramolecular hydrogel 2-NC, the molar ratio of 2-NA to CA is 1:3.

[0065] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0066] Experimental Example 1: Screening of conditions for synthesizing 2-NA

[0067] 1. Experimental methods

[0068] The conditions for synthesizing 2-NA were screened, including treating the mixture at reflux temperature (80°C) for different lengths of time, and the optimal conditions were screened through the final product.

[0069] The specific operations are as follows:

[0070] (1)-(4) as described in Example 1;

[0071] (5) The intermediate product NU1 (1.36 g; 4 mmol) and N,N'-(1H-purine-2,6-diyl)diethylamide (1.9 g; 7.4 mmol) were suspended in acetonitrile (24 mL), and N,O-bis(trimethylsilyl)acetamide (BSA) (7 mL; 28 mmol) was added to carry out silylation reaction.

[0072] Screening condition 1: The mixture is treated at reflux temperature (80°C) for 10 minutes.

[0073] Screening condition 2: The mixture was treated at reflux temperature (80°C) for 15 minutes.

[0074] Screening condition three: The mixture is treated at reflux temperature (80°C) for 20 minutes.

[0075] (6)-(8) As described in Example 1, 2,2'-diamino-2'-deoxyadenosine was obtained as a white powder.

[0076] 2. Experimental results

[0077] Screening condition 1: The mixture was treated at reflux temperature (80°C) for 10 min, the reaction was partially completed, and the subsequent step obtained white powder 2,2'-diamino-2'-deoxyadenosine, 550.07 mg, with a yield of 48.7% and a purity of 97.80%.

[0078] Screening condition 2: The mixture was treated at reflux temperature (80° C.) for 15 min to obtain 2,2'-diamino-2'-deoxyadenosine as a white powder, 907 mg, with a yield of 80.3% and a purity of 98.90%.

[0079] Screening condition three: The mixture was treated at reflux temperature (80°C) for 20 min. The reaction time was too long, which may lead to the production of by-products. The yield of the subsequent steps dropped to 717.24 mg, with a yield of 63.5% and a purity of 97.60%.

[0080] Therefore, in step (5) of preparing 2,2'-diamino-2'-deoxyadenosine, treating the mixture at reflux temperature (80° C.) for 15 min is the optimal condition, and the yield of 2,2'-diamino-2'-deoxyadenosine is higher.

[0081] Experimental Example 2: Structural Characterization of 2-NC Nucleoside Supramolecular Hydrogel

[0082] 1. Experimental methods

[0083] The prepared 2-NC hydrogel was subjected to a vial inversion experiment to test its stability.

[0084] 2. Experimental results

[0085] Figure 2 A is a schematic diagram of the self-assembly of 2-NA and CA aqueous solutions into 2-NC hydrogel after mixing in a certain ratio (1:1) in Example 2. Figure 2 In B), the 2-NA and CA aqueous solutions in Example 2 were mixed, heated, and then cooled to form a stable 2-NC hydrogel, which did not collapse after 3 days and had long-term stability.

[0086] Different concentrations (10mM, 15mM, 20mM, 30mM, 40mM, 50mM) of 2-NA aqueous solution and different concentrations (10mM, 15mM, 20mM, 30mM, 40mM, 50mM) of CA aqueous solution were prepared, and the 2-NA aqueous solution and CA aqueous solution of equal concentration were mixed at a certain volume ratio (1:1). Among them, the 10mM 2-NC hydrogel was unstable and collapsed for 24h, while the rest could form stable 2-NC hydrogels ( Figure 2 C).

[0087] Taking the lowest concentration of 15 mM as an example, 15 mM 2-NA and CA aqueous solutions of the same concentration were prepared and mixed in different volume ratios, including 2:1, 1:1 and 1:3, to form stable 2-NC hydrogels ( Figure 2 D).

[0088] Experimental Example 3: Test of the performance of 2-NA in promoting osteogenesis in vitro

[0089] 1. Experimental methods

[0090] (1) Rat bone marrow mesenchymal stem cells (rBMSCs) were extracted from the femur and tibia of 2-week-old SD rats. After three generations of culture, the cells were digested with trypsin and resuspended by centrifugation to obtain a single cell suspension after growing to a confluence of 90%. The cells were then inoculated into a well plate. When the confluence was 90%, osteogenic-inducing medium (OM) was used as a control and OM with adenosine (A) added was used as a positive control to detect the in vitro osteogenic properties of the 2-NA component.

[0091] (2) The specific components of each culture medium are as follows: 1) OM complete medium (OM group): α-MEM medium, 10% fetal bovine serum, 1% penicillin-streptomycin complete medium supplemented with 10mM β-glycerophosphate, 10mM ascorbic acid and 100nM dexamethasone; 2) Adenosine complete medium (Group A): The above OM complete medium was supplemented with 10μM adenosine; 3) 2-NA complete medium (2-NA group): The above OM complete medium was supplemented with 10μM 2-NA.

[0092] (3) After 14 days of culture, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, washed three times with PBS, and stained with Alizarin Red stain for 2 min at room temperature in the dark. After washing three times with PBS, the cells were dried and photographed, and Image J software was used to quantify the positive staining area.

[0093] (4) After 2 and 7 days of culture, protein was extracted and western blotting was used to characterize the expression of classic osteogenic markers in rat bone marrow mesenchymal stem cells (rBMSCs) and mouse embryonic osteoblast precursor cells (MC3T3-E1) promoted by 2-NA.

[0094] 2. Experimental results

[0095] Alizarin red staining and semi-quantitative analysis showed that after 14 days of osteogenic induction, the 2-NA group showed a stronger bone mineralization level ( Figure 3 AB). At the same time, after 2-7 days of osteogenic induction, compared with the OM group, protein immunoblotting showed that the expression of osteogenic markers RUNX2 and OCN in the 2-NA group was upregulated ( Figure 3 C) Therefore, 2-NA has the effect of promoting osteogenesis in vitro.

[0096] Experimental Example 4: Performance test of nucleoside supramolecular hydrogel in promoting alveolar bone defect repair in vitro

[0097] 1. Experimental methods

[0098] (1) Construction of alveolar bone defect model of maxillary first molar in rats: Female SD rats aged 4 to 5 weeks were randomly divided into three groups, namely, blank control group (Ctrl group), gelatin sponge filling group (i.e., vehicle group, GS group), and 2-NC group (2-NC hydrogel with a concentration of 15 mM). After one week of adaptive feeding, the rats were anesthetized, and the gingiva around the maxillary first molar was separated using a disposable probe. The maxillary first molar was loosened with a 15 mL syringe needle to completely dislocate it, and the alveolar socket was scraped with a probe to ensure that there was no broken root.

[0099] The specific operations of each group are as follows:

[0100] 1) Ctrl group: no intervention was performed after tooth extraction.

[0101] 2) GS group: After tooth extraction, gelatin sponge (1±0.1 g) was placed in the alveolar bone defect, and 20 μL of normal saline was injected.

[0102] 3) 2-NC group: After tooth extraction, 20 μL of 2-NC hydrogel was injected through a microsyringe and allowed to gel naturally in the alveolar bone defect.

[0103] (2) Histological analysis: Rats were euthanized 7 and 21 days after tooth extraction, and maxillae were obtained. They were fixed with 4% paraformaldehyde for 48 hours and then transferred to PBS for storage. Maxillary bone samples were scanned using MICRO-CT (scanning parameters: voltage 40kV, current 250mA, scanning accuracy 10μm). After scanning, the samples were reconstructed using Scanco software and analyzed using SkyScan software. Subsequently, three measurement points were set in the middle of the distal alveolar ridge corresponding to the three buccal roots of M1, corresponding to the distal 1 / 3, middle 1 / 3, and mesial 1 / 3 of the alveolar bone defect area of ​​the first molar, respectively. The vertical distances from the top of the buccal alveolar ridge and the top of the palatal alveolar ridge to the most protruding point of the alveolar bone bottom in the corresponding XZ plane were measured, respectively, and recorded as the buccal / palatal bone height; the maximum bone width of the alveolar ridge was measured, recorded as the maximum bone width (Max bone width). Subsequently, the bone mineral density (BMD) and bone volume fraction (BV / TV) at the defect area corresponding to the largest root of the first molar were measured. The maxillary bone samples that completed the above steps were placed in 10% EDTA decalcification solution, and after decalcification, they were dehydrated, paraffin-embedded, sectioned, and stained with H&E.

[0104] 2. Experimental results

[0105] The maxillary bones were scanned by MICRO-CT at 7 and 21 days after surgery. The minimum grayscale threshold was set to 48 in SkyScan software to remove soft tissue. The cross-sectional images were pseudo-colored according to the grayscale value. Figure 4 As shown in A, the 2-NC group had more new bone mass and higher new bone mineralization at 7 and 21 days after surgery. The statistical results showed that at 7 days after surgery, the bone volume fraction of the 2-NC group was significantly higher than that of the Ctrl control group and the GS group, and the bone mineral density showed an increasing trend, but there was no statistical difference. At 21 days after surgery, the bone volume fraction and bone mineral density of the 2-NC group were significantly higher than those of the Ctrl group ( Figure 4 BE). In addition, it can be seen that the buccal bone height and palatal bone height of the 2-NC group were significantly higher than those of the Ctrl group, and the average maximum bone width tended to increase ( Figure 4FH). H&E results further showed that 2-NA significantly promoted new bone formation in alveolar bone defects and improved the quality of new bone formation ( Figure 4 IJ).

[0106] The above experimental results show that the 2-NC nucleoside supramolecular hydrogel of the present invention can effectively promote bone repair of alveolar bone defects.

[0107] Experimental Example 5: Testing of the bone repair performance of nucleoside supramolecular hydrogel in periodontitis bone defects in vivo

[0108] 1. Experimental methods

[0109] (1) Construction of mouse periodontitis bone defect model: 6-8 week old male C57BL / 6 mice were randomly divided into three groups, namely, periodontitis group (Ligation group), group injected with 15mM 2-NC hydrogel (15mM 2-NC group), and group injected with 30mM 2-NC hydrogel (30mM2-NC group). After one week of adaptive feeding, the mice were anesthetized and ligated with 6-0 silk thread at the neck of the maxillary second molar (M2). On the second day after the silk thread ligation, 20μL of 2-NC hydrogel was injected around M2 by a microsyringe. On the 10th day after the establishment of the periodontitis model, the mice were euthanized, and the maxillary bones were fixed with 4% paraformaldehyde for 24 hours and then transferred to PBS for storage.

[0110] The specific operations of each group are as follows:

[0111] 1) Ligation group: The periodontitis model was established according to the above method without any intervention measures.

[0112] 2) 15 mM 2-NC group: After the periodontitis model was established as described above, 20 μL of 15 mM 2-NC hydrogel was injected through a microsyringe and allowed to gel naturally in the periodontitis bone defect area.

[0113] 3) 30 mM 2-NC group: After the periodontitis model was established according to the above method, 20 μL of 30 mM 2-NC hydrogel was injected through a microsyringe and allowed to gel naturally in the periodontitis bone defect area.

[0114] (2) Histological analysis: The mouse maxillary bone samples were fixed with 4% paraformaldehyde for 24 hours and then scanned using MICRO-CT (scanning parameters: voltage 40 kV, current 250 mA, scanning accuracy 10 μm). After scanning, the samples were reconstructed using Scanco software and analyzed using SkyScan software to measure the vertical resorption of alveolar bone, i.e., the distance between cemento-enamel junction and alveolar bone crest (CEJ-ABJ).

[0115] 2. Experimental results

[0116] The maxilla was scanned with micro-CT 10 days after surgery and three-dimensional reconstruction was performed to measure the CEJ-ABJ. Figure 5 As shown in A, the alveolar bone in the Ligation group was significantly absorbed, and the alveolar bone defect model of periodontitis was successfully established. The CEJ-ABJ distance was significantly reduced by using 15mM 2-NC hydrogel and 30mM 2-NC hydrogel ( Figure 5 B), indicating that 2-NC hydrogel can treat periodontitis bone defects in mice.

[0117] In summary, the present invention provides a nucleoside supramolecular hydrogel and a preparation method and use thereof. The present invention uses 2,2'-diamino-2'-deoxyadenosine and cyanuric acid as raw materials to prepare a nucleoside supramolecular hydrogel. The nucleoside supramolecular hydrogel has good stability, can effectively promote the repair of alveolar bone defects and periodontitis bone defects, and has broad application prospects in the preparation of bone defect repair materials.

Claims

1. A nucleoside supramolecular hydrogel, characterized in that: The nucleoside supramolecular hydrogel is prepared by using 2,2'-diamino-2'-deoxyadenosine and cyanuric acid as raw materials, and the molar ratio of the 2,2'-diamino-2'-deoxyadenosine to cyanuric acid is 1-2:1-3.

2. A method for preparing the nucleoside supramolecular hydrogel according to claim 1, characterized in that: The method comprises the following steps: mixing an aqueous solution of 2,2'-diamino-2'-deoxyadenosine and an aqueous solution of cyanuric acid, and reacting them to obtain the product.

3. The method according to claim 2, characterized in that The concentration of the aqueous solution of 2,2'-diamino-2'-deoxyadenosine is 15-50 mM; the concentration of the aqueous solution of cyanuric acid is 15-50 mM.

4. The method according to claim 2, characterized in that: The reaction conditions are: heating at 70-110° C. until dissolved, and placing in a 0-8° C. water bath for 50-70 min.

5. The method according to claim 4, characterized in that The reaction conditions are: heating at 80-100°C until dissolved, and placing in a 4°C water bath for 60 minutes.

6. A method for synthesizing 2,2'-diamino-2'-deoxyadenosine, characterized in that: The method comprises the following steps: (1) 2'-amino-D-uridine reacts with ethyl trifluoromercaptoethyl to obtain the intermediate product NU1; (2) The intermediate product NU1, N,N'-(1H-purine-2,6-diyl) diethylamide, a silanization agent and a catalyst are reacted to obtain 2,2'-diamino-2'-deoxyadenosine.

7. The method according to claim 6, characterized in that In step (1), the molar ratio of 2'-amino-D-uridine to ethyl trifluoromercaptoethyl ester is 1:1-2.5; the solvent of the reaction is an organic solvent; the temperature of the reaction is 10-40°C and the time is 20-30h; In step (2), the silanization agent is N, O-bis(trimethylsilyl)acetamide; the catalyst is trimethylsilyl trifluoromethanesulfonate; the molar ratio of the intermediate product NU1, N, N'-(1H-purine-2,6-diyl) diethylamide, the silanization agent and the catalyst is 1:1-2.5:5-10:12-14.5; the solvent of the reaction is an organic solvent; the reaction temperature is 70-90°C and the reaction time is 10-20 min.

8. The method according to claim 7, characterized in that In step (1), the molar ratio of 2'-amino-D-uridine to ethyl trifluoromercaptoethyl ester is 1:1.6; the solvent of the reaction is methanol; the temperature of the reaction is 15-35°C and the reaction time is 24h; In step (2), the molar ratio of the intermediate product NU1, N,N'-(1H-purine-2,6-diyl) diethylamide, silanization agent and catalyst is 1:1.85:7:13.8; the solvent of the reaction is acetonitrile; the reaction temperature is 80°C and the reaction time is 2h15min.

9. The method according to claim 6, characterized in that After step (1) is completed, the following purification step is further included: evaporating the solvent, treating the residue with chloroform, filtering, washing, and drying to obtain the intermediate product NU1; After step (2) is completed, the following purification step is further included: pouring the reaction solution into saturated methanol and ammonia water, leaving it at room temperature for 24 hours, evaporating the solution, extracting, and separating by chromatographic column to obtain the product.

10. Use of the nucleoside supramolecular hydrogel according to claim 1 in preparing a bone defect repair material, wherein the material is preferably an alveolar bone defect repair material or a periodontitis bone defect material.

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