Nucleoside supramolecular hydrogel and preparation method and use thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a nucleoside supramolecular hydrogel, its preparation method, and its uses. Background Technology

[0002] Due to the complex oral environment and anatomical factors in the alveolar bone defect area, continuous bone resorption is inevitable after tooth extraction, leading to a reduction in the size of the alveolar ridge. This dimensional change affects the patient's subsequent aesthetic and functional restoration. Therefore, promoting bone repair in the alveolar bone defect area is an urgent problem to be solved. Currently, commercially available and research-developed biomaterials for promoting alveolar bone defect repair (mainly divided into three categories: platelet concentrates, bone homologous and xenogeneic materials, and composite and hybrid materials) have shortcomings such as limited functional properties, complex preparation methods, non-injectability, poor mechanical properties, slow degradation hindering healing, and poor stability. Existing domestic and international products are limited to traditional gelatin sponges, fibrin-based mechanically active filling materials, polymeric double-layer patches, and collagen-guided bone regeneration membranes, which have potential risks of infection and allergies. Existing biomaterials often only have single functions such as hemostasis, antibacterial properties, and osteogenic properties; their preparation is complex and costly; they cannot be adapted to fit irregular alveolar bone; clinical operation is complex; and patient comfort is low. Therefore, the development of novel biomaterials to promote alveolar bone defect repair has significant clinical implications.

[0003] Although the literature (DOI:10.1002 / adma.202108300) reports the formation of a hydrogel using the self-assembly of 2-amino-2'-fluoro-2'-deoxyadenosine (2-FA), and further describes the preparation of a supramolecular hydrogel using 2-FA and cyanuric acid (CA), the 2-FA hydrogel can be used as a tooth extraction socket healing agent. However, as shown in Figure S8.a of that literature, the supramolecular hydrogel composed of 2-FA and CA (with a molar ratio of CA to 2-FA of 1:1) suffers from poor stability, disintegrating within 7 minutes, and is therefore unsuitable for in vivo application. Summary of the Invention

[0004] The purpose of this invention is to provide a nucleoside supramolecular hydrogel, its preparation method, and its uses.

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

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

[0007] Furthermore, 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.

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

[0009] Furthermore, the reaction conditions are as follows: heating at 80-100℃ until dissolved, and placing in a 4℃ 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 and ethyl trifluoromercaptoethyl ester were reacted to give intermediate product NU1;

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

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

[0015] In step (2), the silanizing agent is N,O-bis(trimethylsilylacetamide); the catalyst is trimethylsilyltrifluoromethanesulfonate; the molar ratio of the intermediate NU1, N,N'-(1H-purine-2,6-diyl)diacetamide, the silanizing agent and the catalyst is 1:1~2.5:5~10:12~14.5; the solvent for the reaction is an organic solvent; the reaction temperature is 70~90℃ and the time is 10~20min.

[0016] Further, in step (1), the molar ratio of 2'-amino-D-uridine to ethyl trifluoromercaptoethyl ester is 1:1.6; the solvent for the reaction is methanol; the reaction temperature 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)diacetamide, silanizing agent and catalyst is 1:1.85:7:13.8; the solvent for the reaction is acetonitrile; the reaction temperature is 80℃ and the reaction time is 2h15min.

[0018] Furthermore, after step (1) is completed, the following purification steps are also 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 steps are also included: pour the reaction solution into saturated methanol ammonia water, let it stand at room temperature for 24 hours, then evaporate the solution, extract, and separate by chromatographic column to obtain the final product.

[0020] Further, the extraction solvent is a 0.1M triethylamine aqueous solution and chloroform; the chromatographic column is a Dowex 1X4 (OH) column. - The chromatographic column was used, and the separation conditions were: first elution with 800 mL of 10% methanol-water, then elution with 20% methanol-water to obtain the product.

[0021] This invention also provides the use of the above-mentioned nucleoside supramolecular hydrogel in the preparation of bone defect repair materials, wherein the material is preferably an alveolar bone defect repair material or a periodontal bone defect material.

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

[0023] This invention prepares a nucleoside supramolecular hydrogel using 2,2'-diamino-2'-deoxyadenosine and cyanuric acid (CA) as raw materials. In the literature (DOI:10.1002 / adma.202108300), the supramolecular hydrogel composed of 2-FA and CA disintegrates within 7 minutes, exhibiting poor stability; while the nucleoside supramolecular hydrogel of this invention does not disintegrate even after 3 days, demonstrating significantly improved stability. This nucleoside supramolecular hydrogel can effectively promote the repair of alveolar bone defects and periodontal bone defects, showing broad application prospects in the preparation of bone defect repair materials.

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

[0025] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0026] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0027] Figure 1 High-resolution mass spectrometry for 2-NA.

[0028] Figure 2 Characterization results of 2-NC nucleoside supramolecular hydrogels: (A) Schematic diagram of 2-NA and CA aqueous solution self-assembling to form 2-NC hydrogel after mixing in a certain proportion; (B) Stable 2-NC hydrogel can be formed by heating and then cooling 2-NA and CA aqueous solution after mixing, and it does not collapse after 3 days; (C) 2-NC hydrogel is formed by mixing 2-NA and CA at different concentrations; (D) 2-NC hydrogel is formed by mixing 2-NA and CA at different volume ratios.

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

[0030] Figure 4 Experimental results on the in vivo alveolar bone repair performance of 2-NA: (A) Micro-CT images of the maxillary alveolar bone defect area at 7 and 21 days; (BC) Micro-CT images of the maxillary alveolar bone defect area at 7 days; (B) Bone volume fraction and (C) Bone mineral density statistics of the maxillary alveolar bone defect area at 7 days; (DE) Micro-CT images of the maxillary alveolar bone defect area at 21 days; (D) Bone volume fraction and (E) Bone mineral density statistics of the maxillary alveolar bone defect area at 21 days; (F) Mean buccal bone height of the maxillary alveolar bone defect area at 7 and 21 days; (G) Mean palatal bone height of the maxillary alveolar bone defect area at 7 and 21 days; (H) Mean maximum bone width of the maxillary alveolar bone defect area at 7 and 21 days; (I) H&E staining of the maxillary alveolar bone defect area at 7 days and (J) at 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 Experimental results on the in vivo bone repair performance of 2-NA for periodontal bone defects: (A) MICRO-CT images of the maxillary periodontal bone defect area after 10 days; (B) CEJ-ABJ statistical results of the maxillary periodontal bone defect area after 10 days. Detailed Implementation

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

[0033] In this invention, "room temperature" is 25±10℃ and "overnight" is 12±5 hours.

[0034] In the specific embodiments of the present invention, the 2,2'-diamino-2'-deoxyadenosine used for synthesizing nucleoside supramolecular hydrogels can be obtained by purchasing commercially available products (CAS No.: 215943-79-6), or it 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) in this invention are as follows:

[0037] The structural formula of 1,2'-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)diacetamide (CAS No.: 34097-37-5; MW: 234.21) is as follows:

[0042]

[0043] The following synthetic route was used to prepare 2,2'-diamino-2'-deoxyadenosine:

[0044]

[0045]

[0046] The specific steps are as follows:

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

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

[0049] (3) Evaporate the solvent, leaving 50 mL. Add chloroform to the residue to precipitate until no more precipitate forms. Filter out the precipitate and wash with chloroform.

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

[0051] (5) The intermediate NU1 (1.36 g; 4 mmol) and N,N'-(1H-purine-2,6-diyl)diacetamide (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 the silanization reaction. The mixture was treated at reflux temperature (80 °C) for 15 min.

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

[0053] (7) After standing at room temperature for 24 hours, evaporate the solution and add 300 mL of 0.1 M triethylamine aqueous solution and 300 mL of chloroform to the residue for extraction and phase separation.

[0054] (8) After concentrating the water layer to half its volume, use Dowex 1X4(OH) - Separation was performed using a chromatographic column (size: 3.1X30cm). The product was first eluted with 800mL of 10% methanol-water, and then eluted with 1500mL of 20% methanol-water to obtain 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 (151MHz, 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 values: C 10 H 15 N7O3[M+H + Actual value: 281.1236.

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

[0059] Example 2: Preparation of nucleoside supramolecular hydrogels

[0060] Weigh 2-NA and cyanuric acid (CA) and prepare 2-NA aqueous solutions (concentrations of 15 mM, 20 mM, 30 mM, 40 mM, and 50 mM) and CA aqueous solutions (concentrations of 15 mM, 20 mM, 30 mM, 40 mM, and 50 mM), respectively. Mix the equal concentrations of 2-NA and CA aqueous solutions at a 1:1 volume ratio, heat at 100°C until completely dissolved, and immediately place in a 4°C water bath for 60 min 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 hydrogels

[0062] Following the method of Example 2, except that the volume ratio of 2-NA to CA was replaced with 2:1, nucleoside supramolecular hydrogel 2-NC was prepared. In nucleoside supramolecular hydrogel 2-NC, the molar ratio of 2-NA to CA was 2:1.

[0063] Example 4: Preparation of nucleoside supramolecular hydrogels

[0064] Following the method of Example 2, except that the volume ratio of 2-NA to CA was replaced with 1:3, nucleoside supramolecular hydrogel 2-NC was prepared. In nucleoside supramolecular hydrogel 2-NC, the molar ratio of 2-NA to CA was 1:3.

[0065] The following experimental examples demonstrate the beneficial effects of the present invention.

[0066] Experimental Example 1: Screening of conditions for the synthesis of 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 durations, and the optimal conditions were screened based on the final product.

[0069] The specific steps are as follows:

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

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

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

[0073] Screening condition 2: The mixture is treated at reflux temperature (80℃) for 15 min.

[0074] Screening condition 3: The mixture is treated at reflux temperature (80℃) for 20 min.

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

[0076] 2. Experimental Results

[0077] Screening condition 1: The mixture was treated at reflux temperature (80℃) for 10 min, and the reaction was partially completed. Subsequent steps yielded a white powder of 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℃) for 15 min to obtain white powder 2,2'-diamino-2'-deoxyadenosine, 907 mg, yield 80.3%, purity 98.90%.

[0079] Screening condition 3: The mixture was treated at reflux temperature (80℃) for 20 min. If the reaction time was too long, by-products may be generated, and the yield of subsequent steps would decrease to 717.24 mg, yield 63.5%, purity 97.60%.

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

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

[0082] 1. Experimental Methods

[0083] The stability of the prepared 2-NC hydrogel was tested by inverting a vial.

[0084] 2. Experimental Results

[0085] Figure 2 A is a schematic diagram illustrating the self-assembly of 2-NA and CA aqueous solutions in Example 2, where a 2-NC hydrogel is formed by mixing them in a 1:1 ratio. This was demonstrated in an inverted vial experiment. Figure 2 In Example B), the mixture of 2-NA and CA aqueous solution, heated and then cooled, forms a stable 2-NC hydrogel that does not collapse after 3 days, demonstrating long-term stability.

[0086] Different concentrations (10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM) of 2-NA aqueous solution and different concentrations (10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM) of CA aqueous solution were prepared. The 2-NA and CA aqueous solutions of equal concentrations were mixed at a certain volume ratio (1:1). The 10 mM 2-NC hydrogel was unstable and collapsed after 24 hours, while the others all formed stable 2-NC hydrogels. Figure 2 C).

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

[0088] Experimental Example 3: Performance Test of 2-NA in Vitro Osteogenesis Promotion

[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 passages, when the cells reached 90% confluence, they were digested with trypsin, centrifuged, and resuspended to obtain a single-cell suspension. The cells were then seeded into well plates. When the cells reached 90% confluence, the in vitro osteogenic properties of the 2-NA component were tested using osteogenic-inducing medium (OM) as a control and OM with added adenosine (A) as a positive control.

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

[0092] (3) After culturing for 14 days, fix with 4% paraformaldehyde at room temperature for 15 min, wash three times with PBS, add alizarin red staining solution and stain at room temperature in the dark for 2 min, wash three times with PBS, air dry and take pictures, and use Image J software to perform quantitative analysis of positive staining area.

[0093] (4) After culturing for 2 and 7 days, the protein was extracted and Western blot was used to characterize the expression of classic osteogenic markers of 2-NA promoting rat bone marrow mesenchymal stem cells (rBMSCs) and mouse embryonic osteoblast precursor cells (MC3T3-E1).

[0094] 2. Experimental Results

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

[0096] Experiment Example 4: Performance Testing of Nucleoside Supramolecular Hydrogels in Promoting Alveolar Bone Defect Repair in Vitro

[0097] 1. Experimental Methods

[0098] (1) Establishment of a rat model of alveolar bone defect in the maxillary first molar: 4-5 week old female SD rats were randomly divided into three groups: blank control group (Ctrl group), gelatin sponge filling group (i.e., carrier group, GS group), and 2-NC group (15mM 2-NC hydrogel). After one week of acclimatization, the rats were anesthetized, and the gingiva around the maxillary first molar was separated using a disposable probe. The maxillary first molar was loosened completely using a 15mL syringe needle, and the alveolar socket was scraped with a probe to ensure no root fracture.

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

[0100] 1) Ctrl group: No intervention measures are taken after tooth extraction.

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

[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. Maxillae were collected, fixed with 4% paraformaldehyde for 48 hours, and then transferred to PBS for preservation. Maxillae samples were scanned using MICRO-CT (scanning parameters: voltage 40kV, current 250mA, scanning precision 10μm). After scanning, the samples were reconstructed using Scanco software and analyzed using SkyScan software. Subsequently, three measurement points were set at the midline 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. The vertical distance from the buccal alveolar ridge crest and the palatal alveolar ridge crest to the most prominent point of the alveolar bone bottom in the corresponding XZ plane was measured and recorded as the buccal / palatal bone height; the maximum bone width of the alveolar ridge was measured and recorded as the maximum bone width. Subsequently, the bone mineral density (BMD) and bone volume / total volume (BV / TV) of the defect area corresponding to the largest mesial root of the first molar were measured. The maxillary bone sample that had undergone the above steps was placed in 10% EDTA decalcification solution, and after decalcification, it was dehydrated, paraffin-embedded, sectioned, and stained with H&E.

[0104] 2. Experimental Results

[0105] Micro-CT scans of the maxilla were performed 7 and 21 days post-surgery. In SkyScan software, the minimum grayscale threshold was set to 48 to remove soft tissue. Pseudo-color was applied to the cross-sectional images based on the grayscale values. Figure 4 As shown in Figure A, the 2-NC group exhibited greater new bone formation and higher degree of new bone mineralization at postoperative times 7 and 21. Statistical results indicated that at postoperative time 7, the bone volume fraction in the 2-NC group was significantly higher than that in the Ctrl control group and the GS group, and a trend of increasing bone mineral density was observed, but without statistical significance. At postoperative time 21, the bone volume fraction and bone mineral density in the 2-NC group were significantly higher than those in the Ctrl group (…). Figure 4 BE). Furthermore, it was observed that the buccal and palatal bone heights in the 2-NC group were significantly higher than those in the Ctrl group, and the average maximum bone width showed a trend of increase. Figure 4FH). Further H&E results showed that 2-NA significantly promoted new bone formation within alveolar bone defects and improved the quality of new bone formation (FH). Figure 4 IJ).

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

[0107] Experimental Example 5: In vivo testing of the bone repair performance of nucleoside supramolecular hydrogels in promoting periodontal bone defects.

[0108] 1. Experimental Methods

[0109] (1) Establishment of a mouse model of periodontitis and bone defects: Six- to eight-week-old male C57BL / 6 mice were randomly divided into three groups: a periodontitis group (Ligation group), a group injected with 15 mM 2-NC hydrogel (15 mM 2-NC group), and a group injected with 30 mM 2-NC hydrogel (30 mM 2-NC group). After one week of acclimatization, the mice were anesthetized, and the neck of the maxillary second molar (M2) was ligated with 6-0 silk suture. On the second day after ligation, 20 μL of 2-NC hydrogel was injected around M2 using a microsyringe. On the 10th day after the establishment of the periodontitis model, the mice were euthanized, and the maxillary bone was fixed in 4% paraformaldehyde for 24 hours and then transferred to PBS for preservation.

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

[0111] 1) Ligation group: No intervention measures were taken after the periodontitis model was established according to the above method.

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

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

[0114] (2) Histological analysis: Mouse maxillary bone samples were fixed with 4% paraformaldehyde for 24 hours and then scanned using MICRO-CT (scanning parameters: voltage 40kV, current 250mA, scanning precision 10μm). After scanning, the samples were reconstructed using Scanco software and analyzed using SkyScan software. Vertical resorption of alveolar bone was measured, specifically the distance between the cementoenamel junction and the alveolar ridge crest (CEJ-ABJ).

[0115] 2. Experimental Results

[0116] Ten days after surgery, the maxilla was scanned using micro-CT and then three-dimensionally reconstructed to measure CEJ-ABJ. Figure 5 As shown in Figure A, significant alveolar bone resorption was observed in the Ligation group, and the alveolar bone defect model of periodontitis was successfully established. The CEJ-ABJ distance was significantly reduced using 15 mM 2-NC hydrogel and 30 mM 2-NC hydrogel. Figure 5 B) indicates that 2-NC hydrogel can treat bone defects caused by periodontitis in mice.

[0117] In summary, this invention provides a nucleoside supramolecular hydrogel, its preparation method, and its applications. This invention prepares a nucleoside supramolecular hydrogel using 2,2'-diamino-2'-deoxyadenosine and cyanuric acid as raw materials. This nucleoside supramolecular hydrogel exhibits good stability and can effectively promote the repair of alveolar bone defects and periodontal bone defects, showing 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 from 2,2'-diamino-2'-deoxyadenosine and cyanuric acid, with the molar ratio of 2,2'-diamino-2'-deoxyadenosine to cyanuric acid being 1~2:1~3.

2. A method for preparing the nucleoside supramolecular hydrogel of claim 1, characterized in that, The method includes the following steps: mixing an aqueous solution of 2,2'-diamino-2'-deoxyadenosine and an aqueous solution of cyanuric acid, and reacting 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 as follows: heating at 70-110℃ until dissolved, and placing in a water bath at 0-8℃ for 50-70 minutes.

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

6. A method for synthesizing 2,2'-diamino-2'-deoxyadenosine, characterized in that, The method includes the following steps: (1) 2'-amino-D-uridine and ethyl trifluoromercaptoethyl ester react to give intermediate product NU1; (2) The intermediate product NU1, N,N'-(1H-purine-2,6-diyl)diacetamide, silanizing agent and catalyst are reacted to give 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 for the reaction is an organic solvent; the reaction temperature is 10~40℃ and the time is 20~30h; In step (2), the silanizing agent is N,O-bis(trimethylsilylacetamide); the catalyst is trimethylsilyltrifluoromethanesulfonate; the molar ratio of the intermediate NU1, N,N'-(1H-purine-2,6-diyl)diacetamide, the silanizing agent and the catalyst is 1:1~2.5:5~10:12~14.5; the solvent for the reaction is an organic solvent; the reaction temperature is 70~90℃ and the time is 10~20min.

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 for the reaction is methanol; the reaction temperature is 15~35℃ and the reaction time is 24h. In step (2), the molar ratio of the intermediate product NU1, N,N'-(1H-purine-2,6-diyl)diacetamide, silanizing agent and catalyst is 1:1.85:7:13.8; the solvent for the reaction is acetonitrile; the reaction temperature is 80℃ and the time is 15min.

9. The method according to claim 6, characterized in that, After step (1) is completed, the following purification steps are also 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 steps are also included: pour the reaction solution into saturated methanol ammonia water, let it stand at room temperature for 24 h, evaporate the solution, extract, and separate by chromatographic column to obtain the final product.

10. The use of the nucleoside supramolecular hydrogel of claim 1 in the preparation of bone defect repair materials.

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