Preparation method of bioactive glass for preventing recurrence and promoting tissue repair after melanoma operation

Abstract

The invention discloses a preparation method of bioactive glass for preventing recurrence and promoting tissue repair after a melanoma operation, which comprises the following steps: firstly, preparing mesoporous bioactive glass rich in cerium and neodymium by an organic template method, and then modifying the surface of the mesoporous bioactive glass to enable the surface of the mesoporous bioactive glass to be rich in amino groups; finally, a chemotherapeutic drug methotrexate is grafted on the active glass, carboxyl of methotrexate and the ammoniated active glass form an acid pH responsive broken amido bond, and methotrexate grafting modified mesoporous bioactive glass is obtained. The bioactive glass obtained by the invention has good biocompatibility and photo-thermal performance, and can eliminate postoperative residual tumors and promote skin tissue repair through photo-thermal combined chemotherapy.

Description

Technical Field

[0001] The invention relates to a method for preparing bioactive glass for preventing melanoma recurrence and promoting tissue repair after surgery, and belongs to the technical field of biomaterial preparation. Background Art

[0002] Melanoma is the most malignant tumor among skin cancers, with high invasiveness and mortality. When diagnosed with a thick tumor (late stage), the probability of death will be as high as 60%. Compared with Western countries, most of the patients in my country are in the middle and late stages (stages II, III, and IV) when they are first diagnosed, and the tumor thickness is thicker. Patients in the late stage / recurrence often face the risk of amputation, and have a shorter survival period and poor prognosis. The current clinical strategy is mainly surgical resection and chemotherapy, but surgical resection cannot guarantee whether the tumor tissue is cleaned up, and the risk of recurrence is relatively high; chemotherapy can cause irreversible damage to healthy cells, and patients often cannot bear this huge side effect; tissue healing is slow after surgical resection, which may eventually lead to tumor recurrence.

[0003] An ideal melanoma treatment method needs to be able to both eliminate residual tumor cells and promote tissue repair. Biomaterials with intelligent management that combine anti-cancer and tissue repair functions are undoubtedly ideal materials for treating melanoma. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing bioactive glass for preventing recurrence and promoting tissue repair of melanoma after surgery in view of the deficiencies of the above-mentioned prior art, which can effectively prevent recurrence and promote tissue repair of melanoma after surgery.

[0005] The implementation process of the present invention is as follows: A method for preparing bioactive glass for preventing melanoma recurrence and promoting tissue repair after surgery comprises the following steps: (1) preparing cerium-neodymium dual-doped mesoporous bioactive glass nanospheres, specifically comprising: adding calcium nitrate tetrahydrate, cerium nitrate hexahydrate and neodymium nitrate hexahydrate into deionized water containing hexadecyltrimethylammonium bromide, anhydrous ethanol and ammonia water, then adding triethyl phosphate and tetraethyl orthosilicate, stirring the reaction thoroughly, collecting the precipitate, and calcining to obtain cerium-neodymium dual-doped mesoporous bioactive glass nanospheres; (2) preparing surface-aminated mesoporous bioactive glass nanospheres, specifically comprising: dispersing the cerium-neodymium dual-doped mesoporous bioactive glass nanospheres synthesized in step (1) and 3-aminopropyltriethoxysilane in anhydrous ethanol, stirring at room temperature for reaction, and collecting the precipitate to obtain surface-aminated mesoporous bioactive glass nanospheres; (3) preparing methotrexate grafted modified mesoporous bioactive glass nanospheres, specifically comprising: dissolving methotrexate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in deionized water, adding the surface aminated mesoporous bioactive glass nanospheres synthesized in step (2), stirring at room temperature, dialyzing, lyophilizing and collecting the product to obtain methotrexate grafted modified mesoporous bioactive glass nanospheres.

[0006] In the above step (1), the molar ratio of the calcium nitrate tetrahydrate, the cerium nitrate hexahydrate and the neodymium nitrate hexahydrate is (2-3): (10-50): (10-50); the mass of the calcium nitrate tetrahydrate is 2-6 times the mass of the hexadecyltrimethylammonium bromide; the volume of triethyl phosphate is 0.1-0.2 times the mass of the calcium nitrate tetrahydrate; the volume of tetraethyl orthosilicate is 6-9 times the volume of triethyl phosphate; the volume ratio of tetraethyl orthosilicate to deionized water, ammonia water and ethanol is 1: (3-20): (2-5): (1-7), the volume unit of triethyl phosphate is mL, the mass unit of calcium nitrate tetrahydrate is g, and the concentration of ammonia water is 20-25%; the calcination temperature is 500-800°C, and the calcination time is 1-5 h.

[0007] In the above step (2), the dosage ratio of the cerium-neodymium dual-doped mesoporous bioactive glass nanospheres to 3-aminopropyltriethoxysilane is 1: (1-5) g / ml; the volume of the anhydrous ethanol is 100-150 times the mass of the dual-doped mesoporous bioactive glass nanosphere powder, the mass unit of the dual-doped mesoporous bioactive glass nanosphere powder is g, and the volume unit of the 3-aminopropyltriethoxysilane is mL.

[0008] In the above step (3), the volume of the deionized water is 150-220 times the mass of the aminated mesoporous bioactive glass nanosphere powder; the mass ratio of the methotrexate to the aminated mesoporous bioactive glass nanosphere powder is 1:(1.5-2.2); the molar ratio of methotrexate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1-1.7):(1-2); the mass unit of the aminated mesoporous bioactive glass nanosphere powder is g, and the volume unit of the deionized water is mL.

[0009] The cerium-neodymium dual-doped bioactive glass prepared by the above method is used in the preparation of tissue defect repair for the treatment of melanoma surgery and the inhibition of cancer recurrence.

[0010] Compared with the prior art, the present invention has the following advantages: (1) In the cerium-neodymium dual-doped bioactive glass nanospheres prepared by the present invention, cerium has high biocompatibility and the ability to promote cell proliferation; neodymium has a photothermal effect, so they can kill cancer cells and promote tissue repair at the same time; (2) The bioactive glass of the present invention is grafted with methotrexate through an amide bond after ammoniation. The amide bond is a chemical bond that breaks at acidic pH. When it infiltrates into the acidic microenvironment of the tumor, it breaks, thereby responsively releasing the chemotherapy drug methotrexate. (3) The methotrexate-grafted mesoporous bioactive glass nanospheres prepared by the present invention can be used as a targeting ligand for folate receptors by the methotrexate grafting, which can effectively achieve targeted treatment of melanoma by nanospheres and reduce treatment side effects; (4) The mesoporous bioactive glass nanospheres obtained by the preparation method of the present invention have stable performance and significant biological properties, meeting the needs of postoperative treatment of melanoma; (5) The preparation method of the present invention is simple, the raw materials are cheap and readily available, the process is environmentally friendly, and it is easy to scale up, and has good market application value. Instruction Manual

[0011] Figure 1 This is a SEM image of the mesoporous bioactive glass nanospheres modified by methotrexate grafting in Example 1; Figure 2 TEM image of the mesoporous bioactive glass nanospheres modified by methotrexate grafting in Example 1; Figure 3 The XRD pattern of the mesoporous bioactive glass nanospheres modified by methotrexate grafting in Example 1; Figure 4 This is a particle size analysis diagram of the mesoporous bioactive glass nanospheres modified by methotrexate grafting in Example 1; Figure 5 is the drug release curve of methotrexate in Example 1; Figure 6 Schematic diagram of the cytotoxicity of the methotrexate-grafted mesoporous bioactive glass nanospheres of Example 1 to B16F10. DETAILED DESCRIPTION

[0012] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used, unless otherwise specified, can be obtained from commercial sources. The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Example 1

[0013] A method for preparing a bioactive glass grafted with methotrexate that can be used to prevent recurrence of melanoma after surgery and promote tissue repair comprises the following steps: (1) Preparation of mesoporous bioactive glass nanospheres doped with cerium and neodymium, specifically comprising: Under the condition of magnetic stirring at 600 rpm, 2.36 g hexadecyltrimethylammonium bromide, 19.84 mL anhydrous ethanol and 14.17 mL ammonia water were dissolved in 56.68 mL deionized water. After being mixed evenly, 4.72 g calcium nitrate tetrahydrate, 2.17 g cerium nitrate hexahydrate and 0.44 g neodymium nitrate hexahydrate were added and fully dissolved, and then 0.47 mL triethyl phosphate and 2.84 mL tetraethyl orthosilicate were added. After stirring for 4 h, centrifugation was continued, and the mixture was washed with anhydrous ethanol for 3 times, dried and calcined to obtain mesoporous bioactive glass nanospheres doped with cerium and neodymium elements. (2) Preparing surface-aminated mesoporous bioactive glass nanospheres, specifically comprising: 0.1 g of the dual-doped mesoporous bioactive glass nanospheres synthesized in step 1 and 0.5 mL of 3-aminopropyltriethoxysilane were dispersed in 10 mL of anhydrous ethanol, stirred at room temperature for 12 h, centrifuged, washed with anhydrous ethanol three times, and dried to obtain surface-ammonified mesoporous bioactive glass nanospheres; (3) Preparing methotrexate-grafted mesoporous bioactive glass nanospheres, specifically comprising: 0.16 g of methotrexate, 0.068 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.041 g of N-hydroxysuccinimide were dissolved in 36 mL of deionized water. After stirring for 5 h, 0.24 g of the aminated mesoporous bioactive glass nanospheres synthesized in step 2 were added. After being fully stirred at room temperature for 24 h, the methotrexate-grafted mesoporous bioactive glass nanospheres were collected by dialysis (MWXO = 3500Da) and freeze-drying.

[0014] Material testing and performance evaluation:

[0015] The SEM image of the mesoporous bioactive glass nanospheres modified by methotrexate grafting in Example 1 is as follows: Figure 1 As shown. Figure 1 It can be seen that the nanospheres are about 125 nm, and the surface of the spheres is rough due to the grafting of drugs.

[0016] The TEM image of the mesoporous bioactive glass nanospheres modified by methotrexate grafting in Example 1 is as follows: Figure 2 As shown in Figure 2, corresponding to the SEM image, the coated drug can be clearly observed on the surface of the sphere.

[0017] The XRD pattern of the mesoporous bioactive glass nanospheres modified by methotrexate grafting in Example 1 is as follows: Figure 3 As shown, according to Figure 3It can be seen that the prepared nanoparticles have an amorphous structure and the broad peak range of amorphous silicate is 2θ = 20°-34°, which is a characteristic of amorphous silicate bioactive glass.

[0018] The particle size analysis diagram of Example 1 is as follows Figure 4 As shown, according to Figure 4 It can be seen that the size distribution of the mesoporous bioactive glass nanospheres modified by methotrexate grafting is about 100-150 nm, which corresponds to the size of the microspheres shown by SEM and TEM.

[0019] Cytological experiments on mesoporous bioactive glass nanospheres grafted with methotrexate Figure 5 and Figure 6 As shown, it is based on the mesoporous bioactive glass nanospheres modified by methotrexate grafting as the research object to investigate its efficacy in inhibiting or killing cancer. The test methods include: (1) Preparation of 1640 extract of mesoporous bioactive glass modified by methotrexate grafting ①1640 cell complete culture medium: add 10% fetal bovine serum, 100 μg / ml streptomycin, and 100 U / ml penicillin to 500 mL of 1640 basal culture medium, mix well, and place in a 4°C refrigerator for later use; ② Preparation of PBS solution: Weigh 0.01 g potassium chloride, 0.40 g sodium chloride, 0.01 g dipotassium hydrogen phosphate, and 0.17 g disodium hydrogen phosphate, respectively, add 1000 ml double distilled water, dissolve by ultrasonic, adjust the pH to 7.40, sterilize at 121 °C for 30 min, and store in a refrigerator at 4 °C for later use; ③ Preparation of 0.25% pancreatic enzyme: weigh 100 mg of pancreatic enzyme, add 40 ml PBS, filter with a 0.22 μm filter membrane, and store in a 4°C refrigerator for later use; ④ Preparation of 1640 extract of mesoporous bioactive glass grafted with methotrexate: Weigh the grafted modified mesoporous bioactive glass in Example 1 and soak it in 1640 complete cell culture medium. Place it in a 37°C incubator for three days, then filter it with a 0.22 μm filter membrane to obtain the extract of the grafted modified mesoporous bioactive glass. Seal it and place it in a 4°C refrigerator for use.

[0020] (2) Cell culture B16F10 cells were selected as model cells of cancer cells and cultured with the extracts of grafted modified mesoporous bioactive glass to investigate the effects of the extracts on the growth of model cells.

[0021] ①Cell passaging Transfer B16F10 cells into a culture dish containing 1640 culture medium and culture in a 37°C, 5% CO2 incubator for 12 h. Discard the culture medium, add 3 ml PBS to wash the cells, add 2 ml trypsin, and place in an incubator for digestion for 1.5 min. Take out the culture dish and add 3 ml culture medium to terminate digestion. Use a pipette to blow the cell culture medium several times, then transfer the culture medium containing B16F10 cells into 15 ml centrifuge tubes, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 ml culture medium to each centrifuge tube, blow evenly, divide into two portions, add to new culture dishes containing 9 ml 1640 culture medium, shake crosswise to mix well, and place flat in a 37°C, 5% CO2 incubator for 24 h.

[0022] ②Cell layout Take the cells cultured in step ① out of the incubator, discard the culture medium, add 3 ml PBS to wash the cells, add 2 ml trypsin, and put them into the incubator for digestion for 1.5 min. After digestion is completed, blow the cell culture medium several times with a pipette, add 5-8 ml culture medium and blow the cells several times, then pipette 50 μl of each cell sample into the cell counting plate, count, dilute the cells according to a certain ratio, and then plate the cells in a 96-well plate. The cell density is about 1×10 4 The cells were placed in a 37°C, 5% CO2 incubator and cultured for another 24 h.

[0023] ③Cell culture in the extract of grafted modified mesoporous bioactive glass The 96-well plate containing cells cultured in ② was taken out, the culture medium was discarded, and the grafted modified mesoporous bioactive glass extract prepared in (1) of this embodiment (experimental group) and an equal volume of 1640 culture medium (control group) were added thereto, respectively, and placed in an incubator for culture, with 5 replicate wells set for each concentration. After 24 hours of culture, 50 μL of MTT was added to each well, and after further culture for 2-4 hours, 150 μL of DMSO was added to each well, and the absorbance (OD) value corresponding to each well was detected at 490 nm using an ELISA instrument, and the cell survival rate was calculated based on the OD value.

[0024] The experimental results show that the prepared methotrexate grafted modified mesoporous bioactive glass nanosphere extract has strong toxicity to B16F10 cells, and the cell viability of B16F10 decreases with the increase of nanoparticle concentration. When the concentration of nanospheres is 32 μg / mL, the cell viability is less than 50% after 48 hours of cell culture. This shows that the 1640 extract of nanospheres has a strong inhibitory and killing effect on B16F10 cells, that is, nanospheres have the ability to effectively inhibit and kill tumor cells.

[0025] Cytological experiments show that the present invention provides mesoporous bioactive glass nanospheres doped with cerium and neodymium elements, and the surface of the nanospheres is aminated to successfully graft methotrexate. A new type of mesoporous bioactive glass nanospheres that promote cell growth and inhibit cancer cell growth are prepared, which can effectively repair tissue defects after melanoma surgery and inhibit cancer recurrence. Example 2

[0026] A method for preparing a bioactive glass grafted with methotrexate that can be used to prevent recurrence of melanoma after surgery and promote tissue repair comprises the following steps: (1) Providing mesoporous bioactive glass nanospheres doped with cerium and neodymium, specifically including: Under the condition of magnetic stirring at 600 rpm, 2.01 g hexadecyltrimethylammonium bromide, 24.31 mL anhydrous ethanol and 16.21 mL ammonia water were dissolved in 60.78 mL deionized water. After being mixed evenly, 5.19 g calcium nitrate tetrahydrate, 1.95 g cerium nitrate hexahydrate and 0.87 g neodymium nitrate hexahydrate were added and fully dissolved, and then 0.62 mL triethyl phosphate and 4.05 mL tetraethyl orthosilicate were added. After stirring for 4 h, centrifugation was continued, and the mixture was washed with anhydrous ethanol for 3 times, dried and calcined to obtain mesoporous bioactive glass nanospheres doped with cerium and neodymium elements. (2) Providing surface-ammoniated mesoporous bioactive glass nanospheres, specifically comprising: Weigh 0.2 g of the dual-doped mesoporous bioactive glass nanospheres synthesized in step 1 and 0.8 mL of 3-aminopropyltriethoxysilane and disperse them in 21 mL of anhydrous ethanol. Stir at room temperature for 12 h, centrifuge, wash with anhydrous ethanol three times, and dry to obtain surface-ammoniated mesoporous bioactive glass nanospheres. (3) Providing mesoporous bioactive glass nanospheres grafted with methotrexate, specifically comprising: 0.16 g of methotrexate, 0.074 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.045 g of N-hydroxysuccinimide were dissolved in 41 mL of deionized water. After stirring for 5 h, 0.25 g of the aminated mesoporous bioactive glass nanospheres synthesized in step 2 were added. After being fully stirred at room temperature for 24 h, the methotrexate-grafted mesoporous bioactive glass nanospheres were collected by dialysis (MWXO = 3500Da) and freeze-drying.

[0027] The physical and chemical properties of the modified bioactive glass nanospheres in this example are similar to those in Example 1. Example 3

[0028] A method for preparing a bioactive glass grafted with methotrexate that can be used to prevent recurrence of melanoma after surgery and promote tissue repair comprises the following steps: (1) Providing mesoporous bioactive glass nanospheres doped with cerium and neodymium, specifically including: Under the condition of magnetic stirring at 600 rpm, 1.81 g hexadecyltrimethylammonium bromide, 24.71 mL anhydrous ethanol and 17.79 mL ammonia water were dissolved in 49.42 mL deionized water. After being mixed evenly, 5.43 g calcium nitrate tetrahydrate, 1.74 g cerium nitrate hexahydrate and 1.09 g neodymium nitrate hexahydrate were added and fully dissolved, and then 0.71 mL triethyl phosphate and 4.94 mL tetraethyl orthosilicate were added. After stirring for 4 h, centrifugation was continued, and the mixture was washed with anhydrous ethanol for 3 times, dried and calcined to obtain mesoporous bioactive glass nanospheres doped with cerium and neodymium elements. (2) Providing surface-ammoniated mesoporous bioactive glass nanospheres, specifically comprising: Weigh 0.3 g of the dual-doped mesoporous bioactive glass nanospheres synthesized in step 1 and 1.05 mL of 3-aminopropyltriethoxysilane and disperse them in 33 mL of anhydrous ethanol. Stir at room temperature for 12 h, centrifuge, wash with anhydrous ethanol three times, and dry to obtain surface-ammonified mesoporous bioactive glass nanospheres. (3) Providing mesoporous bioactive glass nanospheres grafted with methotrexate, specifically comprising: 0.16 g of methotrexate, 0.081 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.049 g of N-hydroxysuccinimide were dissolved in 46 mL of deionized water. After stirring for 5 h, 0.27 g of the aminated mesoporous bioactive glass nanospheres synthesized in step 2 were added. After being fully stirred at room temperature for 24 h, the methotrexate-grafted mesoporous bioactive glass nanospheres were collected by dialysis (MWXO = 3500Da) and freeze-drying.

[0029] The physical and chemical properties of the modified bioactive glass nanospheres in this example are similar to those in Example 1. Example 4

[0030] A method for preparing a novel bioactive glass grafted with methotrexate that can be used to prevent recurrence of melanoma after surgery and promote tissue repair comprises the following steps: (1) Providing mesoporous bioactive glass nanospheres doped with cerium and neodymium, specifically including: Under the condition of magnetic stirring at 600 rpm, 1.69 g hexadecyltrimethylammonium bromide, 25.86 mL anhydrous ethanol and 19.39 mL ammonia water were dissolved in 51.72 mL deionized water. After being mixed evenly, 5.90 g calcium nitrate tetrahydrate, 1.30 g cerium nitrate hexahydrate and 1.32 g neodymium nitrate hexahydrate were added and fully dissolved, and then 0.88 mL triethyl phosphate and 6.46 mL tetraethyl orthosilicate were added. After stirring for 4 h, centrifugation was continued, and the mixture was washed with anhydrous ethanol for 3 times, dried and calcined to obtain mesoporous bioactive glass nanospheres doped with cerium and neodymium elements. (2) Providing surface-ammoniated mesoporous bioactive glass nanospheres, specifically comprising: Weigh 0.4 g of the dual-doped mesoporous bioactive glass nanospheres synthesized in step 1 and 1.2 mL of 3-aminopropyltriethoxysilane and disperse them in 48 mL of anhydrous ethanol. Stir at room temperature for 12 h, centrifuge, wash with anhydrous ethanol three times, and dry to obtain surface-ammoniated mesoporous bioactive glass nanospheres. (3) Providing mesoporous bioactive glass nanospheres grafted with methotrexate, specifically comprising: 0.16 g of methotrexate, 0.087 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.058 g of N-hydroxysuccinimide were dissolved in 52 mL of deionized water. After stirring for 5 h, 0.29 g of the aminated mesoporous bioactive glass nanospheres synthesized in step 2 were added. After being fully stirred at room temperature for 24 h, the methotrexate-grafted mesoporous bioactive glass nanospheres were collected by dialysis (MWXO = 3500Da) and freeze-drying.

[0031] The physical and chemical properties of the modified bioactive glass nanospheres in this example are similar to those in Example 1. Example 5

[0032] A method for preparing a novel bioactive glass grafted with methotrexate that can be used to prevent recurrence of melanoma after surgery and promote tissue repair comprises the following steps: (1) Providing mesoporous bioactive glass nanospheres doped with cerium and neodymium, specifically including: Under the condition of magnetic stirring at 600 rpm, 1.53 g hexadecyltrimethylammonium bromide, 23.79 mL anhydrous ethanol and 22.21 mL ammonia water were dissolved in 47.60 mL deionized water. After being mixed evenly, 6.14 g calcium nitrate tetrahydrate, 1.09 g cerium nitrate hexahydrate and 1.75 g neodymium nitrate hexahydrate were added and fully dissolved, and then 1.04 mL triethyl phosphate and 7.93 mL tetraethyl orthosilicate were added. After stirring for 4 h, centrifugation was continued, and the mixture was washed with anhydrous ethanol for 3 times, dried and calcined to obtain mesoporous bioactive glass nanospheres doped with cerium and neodymium elements. (2) Providing surface-ammoniated mesoporous bioactive glass nanospheres, specifically comprising: Weigh 0.5 g of the dual-doped mesoporous bioactive glass nanospheres synthesized in step 1 and 1.25 mL of 3-aminopropyltriethoxysilane and disperse them in 65 mL of anhydrous ethanol. Stir at room temperature for 12 h, centrifuge, wash with anhydrous ethanol three times, and dry to obtain surface-ammoniated mesoporous bioactive glass nanospheres. (3) Providing mesoporous bioactive glass nanospheres grafted with methotrexate, specifically comprising: 0.16 g of methotrexate, 0.095 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.065 g of N-hydroxysuccinimide were dissolved in 58 mL of deionized water. After stirring for 5 h, 0.304 g of the aminated mesoporous bioactive glass nanospheres synthesized in step 2 were added. After being fully stirred at room temperature for 24 h, the methotrexate-grafted mesoporous bioactive glass nanospheres were collected by dialysis (MWXO = 3500Da) and freeze-drying.

[0033] The physical and chemical properties of the mesoporous bioactive glass nanospheres in this example are similar to those in Example 1. Example 6

[0034] A method for preparing a novel bioactive glass grafted with methotrexate that can be used to prevent recurrence of melanoma after surgery and promote tissue repair comprises the following steps: (1) Providing mesoporous bioactive glass nanospheres doped with cerium and neodymium, specifically including: Under the condition of magnetic stirring at 600 rpm, 1.26 g hexadecyltrimethylammonium bromide, 18.36 mL anhydrous ethanol and 21.12 mL ammonia water were dissolved in 36.73 mL deionized water. After being mixed evenly, 6.38 g calcium nitrate tetrahydrate, 0.87 g cerium nitrate hexahydrate and 1.97 g neodymium nitrate hexahydrate were added and fully dissolved, and then 1.15 mL triethyl phosphate and 9.18 mL tetraethyl orthosilicate were added. After stirring for 4 h, centrifugation was continued, and the mixture was washed with anhydrous ethanol for 3 times, dried and calcined to obtain mesoporous bioactive glass nanospheres doped with cerium and neodymium elements. (2) Providing surface-ammoniated mesoporous bioactive glass nanospheres, specifically comprising: Weigh 0.6 g of the dual-doped mesoporous bioactive glass nanospheres synthesized in step 1 and 1.2 mL of 3-aminopropyltriethoxysilane and disperse them in 84 mL of anhydrous ethanol. After stirring at room temperature for 12 h, centrifuge, wash with anhydrous ethanol three times, and dry to obtain surface-ammoniated mesoporous bioactive glass nanospheres. (3) Providing mesoporous bioactive glass nanospheres grafted with methotrexate, specifically comprising: 0.16 g of methotrexate, 0.10 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.073 g of N-hydroxysuccinimide were dissolved in 64 mL of deionized water. After stirring for 5 h, 0.32 g of the aminated mesoporous bioactive glass nanospheres synthesized in step 2 were added. After being fully stirred at room temperature for 24 h, the methotrexate-grafted mesoporous bioactive glass nanospheres were collected by dialysis (MWXO = 3500Da) and freeze-drying.

[0035] The physical and chemical properties of the modified bioactive glass nanospheres in this example are similar to those in Example 1. Example 7

[0036] A method for preparing a novel bioactive glass grafted with methotrexate that can be used to prevent recurrence of melanoma after surgery and promote tissue repair, comprising the following steps: (1) Providing mesoporous bioactive glass nanospheres doped with cerium and neodymium, specifically including: Under the condition of magnetic stirring at 600 rpm, 1.18 g hexadecyltrimethylammonium bromide, 12.75 mL anhydrous ethanol and 25.5 mL ammonia water were dissolved in 38.26 mL deionized water. After being mixed evenly, 7.08 g calcium nitrate tetrahydrate, 0.43 g cerium nitrate hexahydrate and 2.19 g neodymium nitrate hexahydrate were added and fully dissolved, and then 1.42 mL triethyl phosphate and 12.75 mL tetraethyl orthosilicate were added. After stirring for 4 h, centrifugation was continued, and the mixture was washed with anhydrous ethanol for 3 times, dried and calcined to obtain mesoporous bioactive glass nanospheres doped with cerium and neodymium elements. (2) Providing surface-ammoniated mesoporous bioactive glass nanospheres, specifically comprising: Weigh 0.7 g of the dual-doped mesoporous bioactive glass nanospheres synthesized in step 1 and 0.7 mL of 3-aminopropyltriethoxysilane and disperse them in 105 mL of anhydrous ethanol. After stirring at room temperature for 12 h, centrifuge, wash with anhydrous ethanol three times, and dry to obtain surface-ammoniated mesoporous bioactive glass nanospheres. (3) Providing mesoporous bioactive glass nanospheres grafted with methotrexate, specifically comprising: 0.16 g of methotrexate, 0.11 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.076 g of N-hydroxysuccinimide were dissolved in 74 mL of deionized water. After stirring for 5 h, 0.34 g of the aminated mesoporous bioactive glass nanospheres synthesized in step 2 were added. After being fully stirred at room temperature for 24 h, the methotrexate-grafted mesoporous bioactive glass nanospheres were collected by dialysis (MWXO = 3500Da) and freeze-drying.

[0037] The physical and chemical properties of the modified bioactive glass nanospheres in this example are similar to those in Example 1.

[0038] Finally, it should be noted that although the present invention has been described above in conjunction with the embodiments, the present invention is not limited to the above embodiments and application fields, and the above schemes are only for illustration and guidance purposes, rather than restrictive. Any simple modification, change and equivalent structural change made to the above embodiments by ordinary technicians in this field under the inspiration of this specification and without departing from the scope of protection of the claims of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing bioactive glass for preventing recurrence and promoting tissue repair after melanoma surgery, characterized in that The following steps are involved: (1) preparing cerium-neodymium dual-doped mesoporous bioactive glass nanospheres, specifically comprising: adding calcium nitrate tetrahydrate, cerium nitrate hexahydrate and neodymium nitrate hexahydrate into deionized water containing hexadecyltrimethylammonium bromide, anhydrous ethanol and ammonia water, then adding triethyl phosphate and tetraethyl orthosilicate, stirring the reaction thoroughly, collecting the precipitate, and calcining to obtain cerium-neodymium dual-doped mesoporous bioactive glass nanospheres; (2) preparing surface-aminated mesoporous bioactive glass nanospheres, specifically comprising: dispersing the cerium-neodymium dual-doped mesoporous bioactive glass nanospheres synthesized in step (1) and 3-aminopropyltriethoxysilane in anhydrous ethanol, stirring at room temperature for reaction, and collecting the precipitate to obtain surface-aminated mesoporous bioactive glass nanospheres; (3) preparing methotrexate grafted modified mesoporous bioactive glass nanospheres, specifically comprising: dissolving methotrexate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in deionized water, adding the surface aminated mesoporous bioactive glass nanospheres synthesized in step (2), stirring at room temperature, dialyzing, lyophilizing and collecting the product to obtain methotrexate grafted modified mesoporous bioactive glass nanospheres.

2. The method for preparing the bioactive glass according to claim 1, characterized in that: In step (1), the molar ratio of the calcium nitrate tetrahydrate, the cerium nitrate hexahydrate and the neodymium nitrate hexahydrate is (2-3): (10-50): (10-50).

3. The method for preparing the bioactive glass according to claim 1, characterized in that: In step (1), the mass of the calcium nitrate tetrahydrate is 2 to 6 times the mass of hexadecyltrimethylammonium bromide, the volume of triethyl phosphate is 0.1 to 0.2 times the mass of the calcium nitrate tetrahydrate, and the volume of tetraethyl orthosilicate is 6 to 9 times the volume of triethyl phosphate.

4. The method for preparing the bioactive glass according to claim 1, characterized in that: In step (1), the volume ratio of tetraethyl orthosilicate to deionized water, ammonia water, and ethanol is 1:(3-20):(2-5):(1-7), and the concentration of ammonia water is 20-25%.

5. The method for preparing the bioactive glass according to claim 1, characterized in that: In step (1), the calcination temperature is 500-800°C, and the calcination time is 1-5 h.

6. The method for preparing the bioactive glass according to claim 1, characterized in that: In step (2), the dosage ratio of the cerium-neodymium dual-doped mesoporous bioactive glass nanospheres to 3-aminopropyltriethoxysilane is 1: (1-5) g / ml; the volume of the anhydrous ethanol is 100-150 times the mass of the dual-doped mesoporous bioactive glass nanosphere powder.

7. The method for preparing the bioactive glass according to claim 1, characterized in that: In step (3), the volume of the deionized water is 150 to 220 times the mass of the ammoniation-modified mesoporous bioactive glass nanosphere powder.

8. The method for preparing the bioactive glass according to claim 1, characterized in that: In step (3), the mass ratio of methotrexate to aminated mesoporous bioactive glass nanosphere powder is 1:(1.5-2.2); the molar ratio of methotrexate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1-1.7):(1-2).

9. Cerium-neodymium dual-doped bioactive glass prepared by the method according to any one of claims 1 to 8.

10. Use of the cerium-neodymium dual-doped bioactive glass according to claim 9 in the preparation of drugs for repairing tissue defects after melanoma surgery and inhibiting cancer recurrence.

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