A dual-responsive copper death nanoparticle material, a preparation method and application thereof

Abstract

The application discloses a preparation method of a dual-response copper death nanoparticle material, and belongs to the technical field of nanometer materials.The method comprises the following steps: dissolving BSA to obtain solution A, mixing a sodium citrate solution and CuCl2 to obtain solution B, dropping the solution B into the solution A, dropping Na2S*9H2O, stirring and reacting, dialyzing and freeze-drying to obtain Cu2S@BSA; dissolving TEA, CTAB and NaSal to obtain solution C, then dropping a mixture of TEOS and BTESPTS into the solution C, removing a template after reaction to obtain MSN; mixing and stirring the MSN and the Cu2S@BSA to load, to obtain MSN@Cu2S; dissolving dextran into deionized water, adding sodium periodate, stirring and reacting in the dark, then adding glycerol to stop oxidation to obtain oDEX; and (5) dissolving the MSN@Cu2S, adding the oDEX to carry out capping treatment to obtain MD@Cu2S. The application also discloses the MD@Cu2S obtained by the preparation method and application of the MD@Cu2S in preparation of a drug for bone tumor or bone repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a dual-responsive copper death nanoparticle material, its preparation method, and its application. Background Technology

[0002] Osteosarcoma (OS) is a common type of bone tumor (accounting for 44.6% of all bone tumors), most commonly occurring between the ages of 10 and 25, and generally has a poor prognosis. Due to its malignant invasiveness and metastatic potential, OS is characterized by both local invasion and distant metastasis, leading to lung metastasis being the most common fatal complication in OS patients.

[0003] Osteosarcoma secretes large amounts of RANK ligands (RANKLS), causing pre-cells to form osteoclasts, which then perform osteoclast functions, leading to pathological fractures. Current treatments for overall survival (OS) include surgery, radiotherapy, and chemotherapy, but these still have many limitations, such as chemotherapy and radiotherapy resistance and low survival rates. Regarding drug resistance, Chinese patent CN107970241A discloses the application of a novel tyrosine kinase inhibitor—anlotinib—in osteosarcoma. Anlotinib has been found to inhibit osteosarcoma growth and metastasis and to enhance the killing effect of the chemotherapeutic drug cisplatin on osteosarcoma cells.

[0004] A promising drug for treating osteosarcoma (OS) needs to possess the following characteristics: (1) the ability to effectively kill tumor cells; (2) the ability to promote osteogenic repair and inhibit osteoclast formation in OS-induced pathological fracture repair. For example, Chinese Patent CN113908168A discloses the application of sennaroside in the preparation of anti-osteosarcoma drugs and anti-osteosarcoma drug formulations. Sennaroside can inhibit the growth and proliferation of various osteosarcoma cells, promote osteosarcoma cell apoptosis, interfere with the cell cycle, arrest cells in the G1 phase, and reduce the number of cells in the G2 phase, thus inhibiting cell division ability; it can also inhibit the invasion and migration of HOS and 143B osteosarcoma cells; it has been confirmed that sennaroside can significantly inhibit the volume of subcutaneous 143B cell tumors in nude mice and can effectively inhibit the growth of osteosarcoma; sennaroside is expected to become a drug for treating osteosarcoma and has broad prospects in the development of drugs for treating osteosarcoma.

[0005] Currently, materials that kill tumor cells, inhibit osteoclast differentiation, and promote pathological bone destruction are research hotspots and urgent technical problems to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing dual-responsive copper death nanoparticles. The preparation method is simple, and the prepared dual-responsive copper death nanoparticles have good anti-tumor, osteoblast differentiation and mineralization, and osteoclast formation inhibition properties. They can be used in the preparation of drugs for bone tumors or bone repair.

[0007] This invention provides the following technical solution:

[0008] A method for preparing dual-responsive copper death nanoparticles, the method comprising the following steps:

[0009] (1) Dissolve bovine serum albumin to obtain solution A, mix sodium citrate solution and CuCl2 to obtain solution B, add solution B dropwise to solution A, then add Na2S•9H2O (sodium sulfide nonahydrate), stir the reaction, dialyze and freeze dry to obtain Cu2S@BSA (fetal bovine serum ferrous sulfide).

[0010] (2) Dissolve TEA (triethanolamine), CTAB (hexadecyltrimethylammonium bromide) and NaSal (sodium salicylate) to obtain solution C. Then add a mixture of TEOS (tetraethyl silicate) and BTESPTS (bis-[γ-(triethoxysilyl)propyl]-tetrasulfide) dropwise to solution C. After the reaction, remove the template to obtain MSN (mesoporous silica).

[0011] (3) MSN and Cu2S@BSA were mixed and stirred before loading to obtain MSN@Cu2S;

[0012] (4) Dissolve dextran in deionized water, add NaIO4 (sodium periodate), stir the reaction in the dark, and then add glycerol to stop the oxidation to obtain oDEX (oxidized dextran).

[0013] (5) After dissolving MSN@Cu2S, add oDEX for capping treatment to obtain MD@Cu2S.

[0014] The dual-responsive copper death nanoparticle material provided by this invention is a pH and GSH (reduced glutathione) dual-responsive dendritic silica loaded with Cu2S, which can also be called responsive MSN loaded with Cu2S nanoparticles.

[0015] The technical principle of this invention lies in the following: Based on the slightly acidic and reducing characteristics of the tumor microenvironment, the dendritic MSN designed in this invention exhibits acidic and GSH-responsive properties, enabling the large-scale release of Cu2S at the tumor site, directly killing tumor cells. Furthermore, Cu2S has an absorption peak near 1064 nm in the ultraviolet absorbable spectrum, and the 1064 nm absorption peak is ideal for NIRII photothermal therapy; therefore, NIRII photothermal therapy can further kill tumor cells. Simultaneously, in osteoclasts, the release of a small amount of Cu2S can inhibit osteoclast energy metabolism, suppressing osteoclast formation and differentiation. Copper ions are key ions for osteoblast differentiation and mineralization. Therefore, the copper ions released by MD@Cu2S in osteoblasts can promote osteoblast mineralization. Thus, the responsive MSN carrying Cu2S nanoparticles prepared in this invention has broad application prospects in the treatment and repair of bone tumors.

[0016] In step (1), the concentration of BSA (fetal bovine serum) in solution A is 1-5 mg / ml; 4-6 ml of sodium citrate solution with a concentration of 10-16 mM and 4-6 ml of CuCl2 with a concentration of 10-16 mM are mixed to obtain solution B; 4-6 ml of Na2S•9H2O with a concentration of 10-16 mM is added dropwise.

[0017] In step (1), if the BSA concentration in solution A is too low, the Cu2S particles will be too large and difficult to load into MSN. Therefore, the BSA concentration should not be lower than 5 mg / ml. In addition, the particle size of Cu2S can be controlled by adjusting the amount of sodium citrate solution, CuCl2 (copper chloride), and Na2S•9H2O added.

[0018] In step (1), the dropping rate of solution B into solution A must not exceed 1 ml / min. If the dropping rate is not faster than 1 ml / min, the solution temperature will be lower than 37°C, and the low temperature will result in larger Cu2S particles.

[0019] In step (1), the dropping rate of Na2S•9H2O should not exceed 1 ml / min. The dropping rate of the Na2S•9H2O solution should not be too fast, not exceeding 1 ml per minute, otherwise the temperature will be too low, resulting in larger Cu2S particles. At the same time, too fast a rate will lead to incomplete reaction, which will also result in larger Cu2S particles.

[0020] In step (1), the Cu2S@BSA dialysis fluid is changed at least once every 12 hours, and dialysis is performed for at least 5 days. The dialysis fluid must be changed at least once every 8 hours to ensure that the BSA can be completely dialyzed out.

[0021] In step (1), solutions A and B must be dissolved at 37°C, with a stirring rate of 300 rpm, and stirred for at least 15 minutes.

[0022] In step (2), 40-60 μl of TEA, 0.2-0.5 g of CTAB, and 50-63 mg of NaSal are dissolved to obtain solution C; a mixture of 1-2 ml of TEOS and 1-2 ml of BTESPTS is added dropwise to solution C. The amount of TEA, CTAB, NaSal, and the mixture of TEOS and BTESPTS can be adjusted to form dendritic MSNs.

[0023] In step (2), the mixture of TEOS and BTESPTS should not be added at a rate faster than 1 ml / min. Excessive dropping speed will disrupt the formation of dendritic MSNs.

[0024] In step (2), TEA, CTAB, and NaSal are stirred in 50 ml ddH2O at a speed of at least 500 rpm, because if the speed is too low, CTAB will not dissolve evenly. Before adding the BTESPTS and TEOS mixture dropwise, it needs to be sonicated for at least 5 minutes to promote complete mixing.

[0025] In step (2), the reaction is followed by centrifugation and washing. The washing process is as follows: first, wash with anhydrous ethanol three times. After each centrifugation, the MSN is completely dispersed in anhydrous ethanol by spiral oscillation and ultrasonic oscillation. After washing with anhydrous ethanol three times, the same operation is performed with ddH2O three times to ensure that the raw materials are completely removed.

[0026] In step (2), the template is removed by washing it with a hot ethanol-hydrochloric acid mixture. Specifically, the ratio of anhydrous ethanol to concentrated hydrochloric acid is 5:1, and the mixture is stirred at 70 °C. After 12 hours, the mixture is collected by centrifugation. The above steps are repeated at least 5 times to completely remove the template.

[0027] In step (2), after the template is washed away by the hot ethanol-hydrochloric acid mixture, it needs to be washed again. The washing steps include at least 3 washes with anhydrous ethanol and at least 3 washes with ddH2O to completely remove concentrated hydrochloric acid.

[0028] In step (3), during the Cu2S loading test, the stirring time is greater than 12 hours and the stirring speed is not less than 300 rpm to promote complete contact between MSN and Cu2S during loading. After the Cu2S loading test is completed, the product is washed at least 3 times with ddH2O (or at least 3 times with ddH2O and 3 times with anhydrous ethanol if necessary) to completely remove excess Cu2S.

[0029] In step (4), the concentration of the dextran is 25-100 mg / ml, the concentration of the sodium periodate is 25-100 mg / ml, and the concentration of the glycerol is 25-100 mg / ml.

[0030] Preferably, in step (4), the oxidized dextran is dialyzed for at least 5 days to ensure that the raw materials are completely removed.

[0031] More preferably, step (4) specifically involves: (4) diluting the dextran in deionized water and stirring at 50°C. Then, dissolving NaIO4 in the mixture and stirring in the dark for 12 h. Adding glycerol to terminate the oxidation reaction and continuing to stir until the mixture is homogeneous. Dialyzing the solution with a dialysis membrane to remove unreacted substances, and then lyophilizing after 5 days of dialysis to obtain oxidized dextran.

[0032] Preferably, in step (5), the mass of the added oxidized dextran is greater than that of MSN@Cu2S.

[0033] In step (5), when the oxidized dextran caps to form Schiff base bonds, the mass of the oxidized dextran added must be at least greater than the mass of M@Cu2S to ensure that M@Cu2S forms MD@Cu2S. In step (4), when the oxidized dextran caps to form Schiff base bonds, it must be carried out in the dark to avoid the oxidized dextran being destroyed.

[0034] In step (5), after capping, wash at least 3 times with ddH2O (or at least 3 times with ddH2O or 3 times with anhydrous ethanol if necessary) to completely remove excess oDEX.

[0035] The present invention also provides a dual-response copper death nanoparticle material obtained by the above preparation method.

[0036] The present invention also provides an application of the above-mentioned dual-responsive copper death nanoparticle material in the preparation of drugs for bone tumors or bone repair.

[0037] The main advantages of this invention compared to existing technologies include:

[0038] (1) The preparation method provided by the present invention is simple to operate, the raw materials are readily available, the cost is low, there is no pollution, and it is easy to repeat and industrialize.

[0039] (2) The responsive MSN prepared by the present invention carries Cu2S nanoparticles and has good acidic and reducing release, while the release amount is very small in neutral and non-reducing environments.

[0040] (3) The responsive MSN prepared by this invention carries Cu2S nanoparticles, which have good tumor killing, promote osteoblast differentiation and mineralization, and inhibit osteoclast formation. It can be used as a highly efficient repair drug for bone destruction caused by bone tumors. Attached Figure Description

[0041] Figure 1 In the figures, A, G, and D represent the macroscopic photograph, UV-Vis absorbance spectrum, XPS image, MSN transmission electron microscope image, MSN porosity, MD transmission electron microscope image, and elemental distribution image of the small-sized Cu2S obtained in Example 1, respectively; the scale bars in the figures are: A = 50 nm, D = 100 nm, and F = 100 nm.

[0042] Figure 2 Disintegration and release tests (A, B, C, and D) for acidic and reducing properties of MD, scale bar in figure: 50 nm;

[0043] Figure 3 MD@Cu2S kills osteosarcoma cells 143B and U2OS: (A) CCK-8 assay, live / dead staining and statistical graphs (B and C).

[0044] Figure 4 For osteoclast toxicity (A, B and C) experiments, osteoclast marker gene inhibition (D), osteoclast differentiation assay (E and F), scale bar in figure 100 μm;

[0045] Figure 5 The toxicity assays of MD@Cu2S to osteoblasts (A, B, and C) and its effects on osteogenic marker genes (D and E).

[0046] Figure 6 A schematic diagram of osteosarcoma in situ (A), in vivo photothermal images (B, C, E and F), and a gross image of the experimental animal (D).

[0047] Figure 7 This refers to bone destruction caused by osteosarcoma in situ. Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0049] Example 1

[0050] Bovine serum albumin was dissolved in 1 ml of ddH2O solution and vigorously shaken to promote dissolution, resulting in solution A with a concentration of 5 mg / ml.

[0051] Solution B is obtained by mixing sodium citrate solution (5 ml, 16 mM) and CuCl2 (5 ml, 16 mM).

[0052] Liquid B was added dropwise to liquid A, and the mixture was stirred at 37°C for 15 minutes. Then, Na₂S•9H₂O (5 ml, 16 mM) was added dropwise, and stirring continued for 8 hours. The mixture was dialyzed for 5 days using a 10 kDa dialysis bag, and then freeze-dried to obtain Cu₂S@BSA.

[0053] 40 μl TEA, 0.2 g CTAB, 63 mg NaSal, and 50 ml ddH2O were mixed and stirred vigorously for 3 h to obtain solution C. Then, the mixture (2 ml TEOS and 1 ml BTESPTS) was added dropwise to solution C and the reaction continued for 16 h. The mixture was then collected by centrifugation (10000 rpm, 20 min) and washed. After centrifugation, the template was washed away with a hot ethanol-hydrochloric acid mixture and dried at 50 °C to obtain MSN.

[0054] 10 mg MSN and 5 mg Cu2S@BSA were mixed and loaded in ddH2O. Excess Cu2S@BSA was washed away after centrifugation.

[0055] Dextran at a concentration of 25 mg / ml was dissolved in deionized water, and sodium periodate at a concentration of 100 mg / ml was added to the dextran solution. The mixture was stirred in the dark for 12 hours, and 100 mg / ml glycerol was added to terminate the oxidation. The mixture was then dialyzed in the dark for 5 days, and lyophilized to obtain oxidized dextran powder oDEX.

[0056] MSN@Cu2S was dissolved in ddH2O at pH = 8, and oxidized dextran (oDEX) was added for capping treatment to obtain MD@Cu2S.

[0057] like Figure 1 As shown in Figure A, this represents the small-sized Cu₂S prepared in this embodiment. Figure 1 As shown in B, the ultraviolet absorption spectrum has an absorption peak at 1064, making it an ideal material for photothermal applications in the second region. Figure 1 C in the figure represents XPS data for small-sized Cu2S. Figure 1 In this context, D stands for MSN (Transmission Electron Microscope). Figure 1 The E in the figure represents the pore size of the MSN, which is approximately 11 nm. Figure 1 F in the figure represents the transmission electron microscope (TEM) of the MD. Figure 1 G in the figure represents the elemental analysis of MD@Cu2S, proving that Cu2S was successfully loaded onto MSN.

[0058] Example 2

[0059] Figure 2Figure A shows the electron microscopy results of MD disintegration under different concentrations of GSH and different pH values. We found that at pH 7.4, MD hardly disintegrated within 48 hours. However, under weakly acidic conditions, MD disintegration accelerated. Under the combined presence of GSH and weak acidity, disintegration accelerated further.

[0060] Figure 2 Figures B, C, and D show the release of MD@Cu2S under different GSH concentrations and pH values. Quantitative results show that under normal physiological conditions (pH = 7.4, GSH = 0 mM), the release rate of Cu2S within 60 h is approximately 8%, indicating that the material has good blocking ability. However, the release rate of Cu2S nanoparticles increases significantly with decreasing pH and increasing GSH concentration. For example, after 60 h of cultivation, the cumulative release rate of Cu2S nanoparticles reached 16% (pH = 6.5) and 23% (pH = 6.0), respectively. This is due to the breaking of Schiff base bonds under acidic conditions, indicating that the release of Cu2S increases with decreasing pH. When the pH is 6.5, the cumulative release rates of Cu2S nanoparticles are 18%, 45%, and 55% when 0 mM GSH, 5 mM GSH, and 10 mM GSH are applied, respectively.

[0061] Application Example 1: In vitro antitumor effect of MD@Cu2S

[0062] Figure 3 A in the experiment demonstrates, through a CCK-8 assay, that MD@Cu2S has a cytotoxic effect on two osteosarcoma cell lines, 143B and U2OS. The specific experimental method involved adding 3,000 143B and U2OS osteosarcoma cells and 100 μl of 10% fetal bovine serum-containing high-glucose medium to a 96-well plate and incubating overnight at 37°C with 5% CO2 to allow cell adhesion. Different concentrations of MD@Cu2S were then added to the cells, and the plates were incubated for 6 hours to allow for cell lysis and phagocytosis. Excess material was washed with serum-free high-glucose medium, and NIR II laser irradiation was performed for 5 minutes. At different time points, CCK-8 reagent was added, and the plates were incubated for 2 hours for analysis. The experiment demonstrated that MD@Cu2S has a cytotoxic effect on osteosarcoma cells, and the laser further killed both osteosarcoma cell lines. The effect of the material on 143B osteosarcoma cells was detected using a live-death staining method. The specific steps involved adding live-death staining solution to the treated cells and observing the results under a fluorescence microscope. The live / dead staining method allows for a more direct observation of the material's destructive effects and further validates the results of the CCK-8 test. Figure 3 (B and C in the text).

[0063] Application Example 2: In vitro inhibitory effect of MD@Cu2S on osteoclast differentiation and osteoclast function.

[0064] 3,000 BMMs (bone marrow mononuclear cells) and 100 μl of 10% fetal bovine serum alpha medium (with 50 ng / ml M-CSF) were added to 96-well plates and incubated overnight at 37°C with 5% CO2 to allow cell adhesion. Different concentrations of MD@Cu2S were added to the cells, and the plates were incubated for 6 hours to allow cell lysis and phagocytosis. Excess cells were washed with serum-free alpha medium, and NIR II laser irradiation was performed for 5 minutes. At different time points, CCK-8 solution was added, and the plates were incubated for 2 hours before detection. Figure 4 The A, CCK-8 results showed that after 24 and 72 hours of treatment, MD@Cu2S nanoparticles exhibited no significant difference in proliferation and toxicity compared to the control and MSN groups. Live / dead images showed that BMM cells in each group exhibited a spindle-shaped morphology. Figure 4 (B in the text). Quantitative results also showed that the cell viability of each group was close to 100%, indicating that the MD@Cu2S biomaterial has good cell compatibility. Figure 4 (C in the middle).

[0065] 3,000 BMMs (bone marrow mononuclear cells) and 100 μl of 10% fetal bovine serum α-medium were added to a 24-well plate and incubated overnight in a cell culture incubator (37°C, 5% CO2) to allow the cells to adhere. Different concentrations of MD@Cu2S were added to the cells, and the plates were allowed to disintegrate and phagocytose for 6 hours. Excess material was washed with serum-free α-medium, and the plates were simultaneously irradiated with NIR II laser for 5 minutes. The plates were then cultured in osteoclast conditioned medium for 5 days until mature osteoclasts were formed in the control group. Figure 4 The results of quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that the expression of c-Fos, DC-STAMP, NFATc1 and CTSK in BMMs co-cultured with the MD@Cu2S@NIRII group was relatively lower than that in the MD@Cu2S group, MSN group and control group, indicating that MD@Cu2S nanoparticles have a good inhibitory effect on BMMs.

[0066] Add 3,000 BMMs (bone marrow mononuclear cells) and 100 μl of 10% fetal bovine serum α medium to a 24-well plate, and incubate overnight in a cell culture incubator (37°C, 5% CO2) to allow the cells to adhere. Add different concentrations of MD@Cu2S to the cells, let them stand for 6 hours to allow the material to disintegrate and phagocytose, and wash off excess material with serum-free α medium. At the same time, irradiate with NIR II laser for 5 minutes, and then continue to culture in osteoclast conditioned medium for 5 days until mature osteoclasts are formed in the control group. Wash three times with PBS (phosphate-buffered saline), fix with 4% paraformaldehyde for 15 minutes, and wash three more times with PBS (phosphate-buffered saline). Add TRAP cell staining solution. Figure 4E and F TRAP staining results showed that the MD@Cu2S@NIRII group had a smaller number of osteoclasts formed by RANKL stimulation of BMMs than the other three groups.

[0067] Application Example 3: In vitro osteogenic differentiation and mineralization assay using MD@Cu2S.

[0068] 3,000 BMSCs (bone marrow mesenchymal stem cells) and 100 μl of 10% fetal bovine serum alpha medium were added to 96-well plates and incubated overnight at 37°C with 5% CO2 to allow cell adhesion. Different concentrations of MD@Cu2S were added to the cells, and the plates were incubated for 6 hours to allow for cell lysis and phagocytosis. Excess material was washed with serum-free alpha medium, and the plates were simultaneously irradiated with NIR II laser for 5 minutes. At different time points, CCK-8 solution was added, and the plates were incubated for 2 hours before analysis. Figure 5 The A CCK-8 assay showed that MSN, MD@Cu2S biomaterials, and thermotherapy were non-toxic to bone marrow mesenchymal stem cells. Live / death results showed that bone marrow mesenchymal stem cells containing the material did not show significant induced cell death. Figure 5 (BC in the text). Considering that osteogenic differentiation and mineralization of bone marrow mesenchymal stem cells are important advances in bone regeneration, their osteogenic and mineralization activities were evaluated using alkaline phosphatase (ALP), Alizarin Red S (ARS), and qRT-PCR. 3,000 BMSCs (bone marrow mesenchymal stem cells) and 100 μl of 10% fetal bovine serum α-medium were added to 24-well plates and incubated overnight (37°C, 5% CO2) to allow cell adhesion. Different concentrations of MD@Cu2S were added to the cells, and the plates were incubated for 6 hours to allow disintegration and phagocytosis. Excess material was washed with serum-free α-medium, and NIR II laser irradiation was performed for 5 minutes. Cells were then cultured in osteogenic induction medium for 7 and 14 days. Compared to the other three groups, the MD@Cu2S@NIRII group showed the highest qRT-PCR results for ALP, OPN, RUNX2, OCN, and BMP2. Figure 5 DE in the middle.

[0069] Application Example 4: In vivo antitumor assay of MD@Cu2S

[0070] To test the function of the biomaterial and in vivo irradiation, luciferase-labeled 143B was implanted to establish an in situ spontaneous metastasis OS model until the tumor tissue reached approximately 50 mm. 3 Nude mice were randomly divided into 6 groups (n=5): (1) PBS, (2) MSN, (3) Cu2S, (4) Cu2S@NIRII, (5) MD@Cu2S, (6) MD@Cu2S@NIRII.

[0071] The experimental procedure is as follows Figure 6As shown in Figure A, mice were irradiated with a 1064 nm laser for 5 min, 10 cycles, and 20 days after tumor cell injection. Photothermal results showed that the MD@Cu2S@NIRII group exhibited a higher temperature rise compared to the other two groups. Figure 6 (B) This is because Cu2S nanoparticles are relatively small in size and can be metabolized in vivo. Specifically, the temperature of the MD@Cu2S@NIRII group after 5 minutes of irradiation was 51.3℃, while the temperatures of the Cu2S@NIRII group and the Control@NIRII group increased to 39.5℃ and 34.8℃, respectively. Figure 6 (C) In addition, compared with the other 5 groups, the MD@Cu2S@NIRII group had the smallest tumor volume and tumor weight (C). Figure 6 The presence of DF in the data indicates that MMD@Cu2S@NIRII can effectively inhibit tumor development.

[0072] Application Example 5: In vivo anti-bone destruction test of MD@Cu2S against bone tumors

[0073] Previous experiments revealed that the MD@Cu2S@NIRII group inhibited osteoclast formation and promoted osteoblast function, suggesting that osteolysis-induced bone damage in vivo could be repaired through nanoparticle treatment. To verify this hypothesis, μCT and histological analysis were used to study bone tissue damage and degeneration in Application Example 6. Clearly, μCT scans showed different degrees of bone damage in the control group, MSN group, Cu2S group, Cu2S@NIRII group, MD@Cu2S@NIRII group, and MD@Cu2S@NIRIII group. Figure 7 The results showed that the tibia of the control group and MSN group had severe bone defects, while the tibia of the same area of ​​the MD@Cu2S group and MD@Cu2S@NIRII group was relatively intact.

[0074] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a dual-responsive copper death nanoparticle material in the preparation of a drug for treating bone tumors or bone repair, characterized in that, The preparation method of the dual-responsive copper death nanoparticle material comprises the following steps: (1) dissolving bovine serum albumin BSA to obtain solution A, mixing sodium citrate solution and CuCl2 to obtain solution B, dropping solution B into solution A, then dropping Na2S•9H2O, stirring and reacting, dialysis and freeze-drying to obtain Cu2S@BSA; (2) dissolving triethanolamine TEA, cetyltrimethylammonium bromide CTAB and sodium salicylate NaSal to obtain solution C, then dropping the mixture of tetraethyl silicate TEOS and bis-[γ-(triethoxysil) propyl]-tetrasulfide BTESPTS into solution C, removing the template after reaction to obtain mesoporous silica MSN; (3) mixing and stirring MSN and Cu2S@BSA to load, to obtain MSN@Cu2S; (4) dissolving dextran in deionized water, adding sodium periodate, stirring and reacting in the dark, then adding glycerol to stop oxidation, to obtain oxidized dextran; (5) dissolving MSN@Cu2S and adding oxidized dextran to cap to form Schiff base bond, to obtain MD@Cu2S; In step (1), the BSA concentration in solution A is 1-5 mg / ml; 4-6 ml of sodium citrate solution with a concentration of 10-16 mM and 4-6 ml of CuCl2 with a concentration of 10-16 mM are mixed to obtain solution B; 4-6 ml of Na2S•9H2O with a concentration of 10-16 mM is dropped; In step (1), the dropping speed of solution B into solution A cannot be faster than 1 ml / min; In step (1), the dropping speed of Na2S•9H2O cannot be faster than 1 ml / min; In step (2), 40-60 μl of TEA, 0.2-0.5 g of CTAB and 50-63 mg of NaSal are dissolved to obtain solution C; the mixture of 1-2 ml of TEOS and 1-2 ml of BTESPTS is dropped into solution C; In step (5), the added mass of oxidized dextran is greater than that of MSN@Cu2S.

2. Use according to claim 1, characterized in that, In step (2), the dropping speed of the mixture of TEOS and BTESPTS cannot be faster than 1 ml / min.

3. Use according to claim 1, characterized in that, In step (4), the dextran concentration is 25-100 mg / ml, the sodium periodate concentration is 25-100 mg / ml, and the glycerol concentration is 25-100 mg / ml.

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