Preparation method and application of acidic degradable hybrid nano-composite for treating osteosarcoma

Abstract

The invention discloses a preparation method and application of an acidic degradable hybrid nano composite for treating osteosarcoma. The preparation method comprises the following steps: 1, preparing hybrid silicon dioxide / hydroxyapatite nano particles MH; 2, dispersing MH into absolute ethyl alcohol, adding ultrapure water, ammonia water and 3-aminopropyltriethoxysilane, heating and stirring to react, centrifuging and washing to obtain MH-NH2; 3, dispersing the MH-NH2 into ultrapure water, adding DATS, stirring and reacting at room temperature, centrifuging and washing to obtain MHD; and 4, dispersing the MHD into ultrapure water, adding a carboxymethyl chitosan solution under a stirring condition, reacting, centrifuging and washing to obtain the nano-composite C-coated MHD. According to the nano-composite disclosed by the invention, the calcium overload performance of the hybrid silicon dioxide / hydroxyapatite nano-particles is combined with H2S damaged DNA released by hydrophobic drug diallyl trisulfide, so that the death of cancer cells is accelerated, and the purpose of treating osteosarcoma is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a preparation method and application of an acidic degradable hybrid nanocomposite for treating osteosarcoma. Background Art

[0002] Osteosarcoma is a malignant bone tumor that mainly occurs in adolescents and is often accompanied by the formation of tumor-like bone matrix, which seriously threatens the patient's life safety. At present, the standardized clinical treatment of osteosarcoma includes surgery and combined neoadjuvant chemotherapy. However, due to the complex anatomical structure of bones, the invasive tumor has no obvious boundary with the surrounding tissue and functional reconstruction is required. It is difficult to remove bone tumors without damaging the bone matrix during surgery. In addition, the systemic toxicity of chemotherapeutic drugs has prompted researchers to explore new strategies to solve this difficult problem. Therefore, advanced treatments need to be developed.

[0003] Gas therapy is an emerging treatment that destroys cancer cells by increasing the level of specific gases at the tumor site. Hydrogen sulfide (H2S) is a colorless gas that is considered a biological gas transfer molecule together with nitric oxide (NO) and carbon monoxide (CO). High concentrations (about 200mmol / L) of H2S can cause DNA damage and exert its anti-tumor effects. Therefore, the clever use of H2S at a certain concentration is a promising cancer treatment method. However, direct use of H2S releasers for treatment has certain limitations, including rapid clearance, poor distribution and side effects, and it is difficult to accurately control the release concentration of H2S. Therefore, developing strategies for remote and precise control of H2S production with high spatiotemporal precision is crucial to optimize the therapeutic effect of H2S.

[0004] Mesoporous silica (MSNs) is a nanomaterial with a highly ordered mesoporous channel structure, large pore size, and high pore volume, which makes them a very promising carrier for drug delivery systems. Compared with other nanocarriers, MSNs have a highly controllable structure and adjustable pore size, which allows the loading and release of drugs to be precisely controlled; secondly, their surface is easy to be chemically modified, which can be used to achieve targeted delivery and selective delivery of specific drugs; finally, they have good chemical stability, high biocompatibility and low toxicity, which allows them to exist stably in the body and complete drug delivery tasks. Therefore, MSNs have been widely studied and are considered to be a very promising carrier for drug delivery systems. However, MSNs tend to accumulate in large quantities in the liver and spleen, causing organ damage. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of an acid-degradable hybrid nanocomposite for treating osteosarcoma in view of the deficiencies of the above-mentioned prior art. The nanocomposite of the present invention has good biocompatibility, powerful therapeutic effect and good pH-responsive biodegradability, can be degraded in the acidic tumor microenvironment, enhances the efficacy of systemic circulation, tumor targeting and intracellular delivery, and through the combination of gas therapy and calcium overload strategy, enables it to achieve an ideal synergistic therapeutic effect at a lower drug dose, and has excellent application prospects in the treatment of osteosarcoma.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a preparation method of an acid-degradable hybrid nanocomposite for treating osteosarcoma, characterized by including the following steps:

[0007] Step 1: Dissolve cetyltrimethylammonium bromide and triethanolamine in ultrapure water, stir evenly, adjust the pH value of the solution to 9-11 with ammonia water, heat to 40°C-85°C, and stir and react for 1h-3h; then add calcium nitrate tetrahydrate to the solution after stirring and reacting, and react for 15min-30min; then add a silicon source and disodium hydrogen phosphate dodecahydrate, heat to 85°C-105°C and react for 10h-24h, and then centrifuge; wash the precipitate obtained by centrifugation with anhydrous ethanol and water to obtain hybrid silica / hydroxyapatite nanoparticles MH.

[0008] Step 2: Disperse the MH obtained in Step 1 in anhydrous ethanol, add ultrapure water, ammonia water and 3-aminopropyltriethoxysilane, heat to 30°C-65°C and stir and react for 12h-48h, centrifuge, and wash the precipitate with anhydrous ethanol and ultrapure water to obtain amino-functionalized MH nanoparticles MH-NH2.

[0009] Step 3: Disperse the MH-NH2 obtained in Step 2 in ultrapure water, add DATS, stir and react at room temperature for 12h-24h, centrifuge, and wash the precipitate with ultrapure water to obtain MH nanoparticles MHD loaded with DATS.

[0010] Step 4: Disperse the MHD obtained in Step 3 in ultrapure water, add a carboxymethyl chitosan solution under stirring conditions, react for 1h-5h, centrifuge, and wash the precipitate with water to obtain an acid-degradable hybrid nanocomposite C@MHD for treating osteosarcoma.

[0011] The above-mentioned preparation method of an acid-degradable hybrid nanocomposite for treating osteosarcoma is characterized in that the hybrid silica / hydroxyapatite nanoparticles MH obtained in Step 1 have a particle size of 50nm-80nm.

[0012] The preparation method of the above-mentioned acidic degradable hybrid nanocomposite for treating osteosarcoma is characterized in that the addition amount of calcium nitrate tetrahydrate in step one is such that the concentration of calcium nitrate tetrahydrate in the reaction system is 20 mM to 100 mM.

[0013] The preparation method of the above-mentioned acidic degradable hybrid nanocomposite for treating osteosarcoma is characterized in that the addition amount of disodium hydrogen phosphate dodecahydrate in step one is such that the concentration of disodium hydrogen phosphate dodecahydrate in the reaction system is 2 mM to 15 mM.

[0014] The preparation method of the above-mentioned acidic degradable hybrid nanocomposite for treating osteosarcoma is characterized in that the mass ratio of MH to 3-aminopropyltriethoxysilane in step two is 1:(1 - 8).

[0015] The preparation method of the above-mentioned acidic degradable hybrid nanocomposite for treating osteosarcoma is characterized in that the mass ratio of MH-NH2 to DATS in step three is 1:(0.2 - 1).

[0016] The preparation method of the above-mentioned acidic degradable hybrid nanocomposite for treating osteosarcoma is characterized in that the mass ratio of MHD to carboxymethyl chitosan in step four is 1:(0.5 - 2).

[0017] Furthermore, the present invention provides a nanocomposite C@MHD prepared by the above preparation method.

[0018] The above-mentioned nanocomposite C@MHD is characterized in that the particle size of the nanocomposite C@MHD is 65 nm to 105 nm.

[0019] Even further, the present invention provides an application of the above-mentioned nanocomposite C@MHD in the preparation of an osteosarcoma therapeutic agent.

[0020] The present invention has the following advantages compared with the prior art:

[0021] 1. The nanocomposite of the present invention uses hybrid silica / hydroxyapatite nanoparticles MH as a carrier, which has the advantages of stable structural properties, easy synthesis, adjustable particle and pore sizes, strong drug loading capacity, and acid responsiveness, and loads the H2S releasing agent DATS. Finally, acid-responsive carboxymethyl chitosan is modified on the surface of the nanoparticles, so that it changes from a negative potential to a positive potential under acidic conditions, selectively decomposes and releases H2S in the acidic tumor microenvironment, improves the delivery efficiency of the nanocomposite, greatly reduces the metabolic toxicity and side effects of DATS, and ultimately improves its therapeutic effect on cancer cells.

[0022] 2. The present invention innovatively synthesizes a H2S-promoting Ca 2+The released acid-degradable hybrid nanocomposite generates H2S, which can not only inhibit the activity of catalase, leading to an increase in intracellular hydrogen peroxide concentration and enhanced oxidative stress, but also promote the influx of Ca 2+ influx, and synergistically generate Ca 2+ to trigger Ca 2+ overload; in addition, excessive Ca 2+ retention in tumor cells leads to mitochondrial dysfunction and simultaneously inhibits energy synthesis, and can play a synergistic anti-tumor role through combined gas therapy and calcium overload to achieve synergistic cancer treatment.

[0023] 3. The nanocomposite of the present invention damages DNA by releasing H2S from the hydrophobic drug diallyl trisulfide (DATS) in combination with the calcium overload performance of hybrid silica / hydroxyapatite nanoparticles (MH), accelerating cancer cell death, thereby achieving the purpose of treating osteosarcoma.

[0024] 4. The nanocomposite of the present invention has good biocompatibility, strong therapeutic effects and good pH-responsive biodegradability, can degrade in the acidic tumor microenvironment, enhances the efficacy of systemic circulation, tumor targeting and intracellular delivery, and through combined gas therapy and calcium overload strategies, enables it to achieve an ideal synergistic therapeutic effect at a lower drug dose, and has excellent application prospects in the treatment of osteosarcoma.

[0025] 5. The positive charge of the nanocomposite of the present invention promotes its accumulation on the negatively charged mitochondrial membrane, optimizes mitochondrial localization, and the released H2S can not only inhibit the activity of catalase, but also promote calcium influx, thereby synergistically damaging mitochondria, amplifying oxidative stress and reducing intracellular energy supply. In addition, H2S can effectively relieve tumor hypoxia, thereby achieving an effective anti-tumor effect.

[0026] The following further describes the technical solutions of the present invention in detail with reference to the accompanying drawings and embodiments. Description of the Drawings

[0027] Figure 1 is the nitrogen adsorption-desorption isotherm of MH, MHD and C@MHD synthesized in Example 1 of the present invention.

[0028] Figure 2 is the transmission electron microscope photograph of MH, MHD and C@MHD nanoparticles synthesized in Example 1 of the present invention.

[0029] Figure 3 is the X-ray diffraction pattern of MH and MSNs nanoparticles in Example 1 of the present invention and the X-ray diffraction pattern of standard HAP (No. 09-0432).

[0030] Figure 4The degradation process of the C@MHD nanocomposite synthesized in Example 1 of the present invention under different conditions.

[0031] Figure 5 The H2S generation performance of the C@MHD nanocomposite synthesized in Example 1 of the present invention.

[0032] Figure 6 The killing effect of the C@MHD nanocomposite synthesized in Example 5 of the present invention on HOS cells.

[0033] Figure 7 The effect of the C@MHD nanocomposite synthesized in Example 5 of the present invention on the cell viability of NIH-3T3 cells. Detailed implementation manners

[0034] The present invention will be specifically described below through examples. They are only used for further illustration of the present invention and should not be construed as limiting the protection scope of the present invention. For the experimental methods without specific conditions in the examples, they are usually carried out according to the conventional conditions and the conditions described in the manuals, or according to the conditions recommended by the manufacturers; the equipment, materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0035] Example 1

[0036] This example provides an acidic degradable hybrid nanocomposite C@MHD for the treatment of osteosarcoma, and the preparation method includes:

[0037] Step 1: Dissolve 1 g of cetyltrimethylammonium bromide and 0.4 g of triethanolamine in 25 mL of ultrapure water, stir evenly, adjust the pH of the mixture to 9 with ammonia water, heat to 85 °C, and stir and react for 1 h; then add calcium nitrate tetrahydrate to make the concentration of calcium nitrate tetrahydrate in the reaction system 50 mM, and react for 15 min; then add 1 mL of tetraethyl orthosilicate and disodium hydrogen phosphate dodecahydrate to make the concentration of disodium hydrogen phosphate dodecahydrate in the reaction system 5 mM, heat to 95 °C and react for 20 h, centrifuge, and wash the precipitate with absolute ethanol and water 3 times respectively to obtain hybrid silica / hydroxyapatite nanoparticles MH;

[0038] Step 2: Disperse 1 mg of MH obtained in Step 1 in 10 mL of absolute ethanol, add 0.1 mL of ultrapure water, 0.2 mL of ammonia water and 2 mg of 3-aminopropyltriethoxysilane, heat to 40 °C and stir and react for 24 h, centrifuge, and wash the precipitate with absolute ethanol and ultrapure water 3 times respectively to obtain amino-functionalized MH nanoparticles MH-NH2;

[0039] Step 3: Disperse 1 mg of MH-NH2 obtained in Step 2 into 3 mL of ultrapure water, add 0.8 mg of DATS, stir and react at room temperature for 24 h, centrifuge, and wash the precipitate 5 times with ultrapure water to obtain MH nanoparticles MHD loaded with DATS;

[0040] Step 4: Disperse 1 mg of MHD obtained in Step 3 into 5 mL of ultrapure water, add a carboxymethyl chitosan solution containing 1.5 mg of carboxymethyl chitosan under stirring conditions. The concentration of the carboxymethyl chitosan solution is 100 μg / mL, react for 2 h, centrifuge, and wash the precipitate 5 times with water to obtain an acidic degradable hybrid nanocomposite C@MHD for the treatment of osteosarcoma.

[0041] Performance evaluation

[0042] Figure 1 Figure 1 shows the nitrogen adsorption-desorption isotherms of MH, MHD, and C@MHD synthesized in Example 1. It can be seen from the figure that the synthesized MH nanoparticles have a large specific surface area. With further modification, the specific surface areas of MHD and C@MHD nanoparticles gradually decrease.

[0043] Figure 2 Figure 2 is the transmission electron microscope photos of MH, MHD, and C@MHD nanoparticles in Example 1. According to Figure 2 It can be seen that the particle size of MH nanoparticles is 50 nm - 80 nm, and the particle size of C@MHD nanoparticles is 65 - 105 nm.

[0044] Figure 3 Figure 3 is the X-ray diffraction pattern of MH and MSNs nanoparticles in Example 1 and the X-ray diffraction pattern of standard HAP (No. 09-0432). The test method includes: using XRD to test and analyze the crystal structure of the freeze-dried sample powder, and the results are as Figure 3 shown. According to Figure 3 It can be seen that the stronger characteristic diffraction peaks in the MH nanoparticles match the diffraction peaks of No. 09-0432 in the XRD standard card (JCPDS), confirming the successful preparation of MH nanoparticles.

[0045] Figure 4 Figure 4 shows the degradation process of C@MHD synthesized in Example 1 under different conditions. The test method includes: dispersing 1 mg of C@MHD into phosphate buffer solutions with different pH values (7.4, 6.5, 5.5, or 4.5), placing the solution in a water bath at 37 °C and gently shaking. Take out a small amount of the solution and centrifuge at a given time. Redisperse the precipitate in water and observe with a scanning electron microscope, and the results are as Figure 4 shown. According to Figure 4It can be seen that the structural morphology of C@MHD remains basically unchanged at pH = 7.4, significantly ruptures in acidic buffer solution, and is significantly degraded within 12 h at pH = 4.5 and completely degraded, confirming that C@MHD not only has high biosafety in blood circulation but also helps the specific release of Ca 2+ and H2S in the acidic tumor microenvironment, thereby treating malignant tumors.

[0046] Figure 5 For the H2S generation performance of C@MHD synthesized in Example 1. The test method includes: taking C@MHD solutions with different concentrations (10, 25, 50, 100, 150 μg / mL), adding 1 mM glutathione solution, mixing evenly and then adding 5 μM WSP-1. Finally, the fluorescence intensity of the sample solution is detected by a fluorescence spectrophotometer, with the excitation wavelength being 465 nm and the emission wavelength being 515 nm. The results are as Figure 5 shown. According to Figure 5 it can be seen that as the concentration of C@MHD increases, the fluorescence intensity significantly increases, indicating that more H2S substances are generated.

[0047] Example 2

[0048] This example provides an acidic degradable hybrid nanocomposite C@MHD for treating osteosarcoma. The preparation method includes:

[0049] Step 1: Dissolve 1 g of cetyltrimethylammonium bromide and 0.15 g of triethanolamine in 25 mL of ultrapure water, stir evenly, adjust the pH of the mixture to 11 with ammonia water, heat to 60 °C, and stir and react for 1.5 h; then add calcium nitrate tetrahydrate to make the concentration of calcium nitrate tetrahydrate in the reaction system 20 mM, and react for 30 min; then add 1 mL of tetraethyl orthosilicate and disodium hydrogen phosphate dodecahydrate to make the concentration of disodium hydrogen phosphate dodecahydrate in the reaction system 10 mM, heat to 105 °C and react for 24 h, centrifuge, and wash the precipitate with anhydrous ethanol and water 3 times respectively to obtain hybrid silica / hydroxyapatite nanoparticles MH;

[0050] Step 2: Disperse 1 mg of MH obtained in Step 1 in 10 mL of anhydrous ethanol, add 0.1 mL of ultrapure water, 0.2 mL of ammonia water and 1 mg of 3-aminopropyltriethoxysilane, heat to 30 °C and stir and react for 12 h, centrifuge, and wash the precipitate with anhydrous ethanol and ultrapure water 3 times respectively to obtain amino-functionalized MH nanoparticles MH-NH2;

[0051] Step 3: Disperse 1 mg of MH-NH2 obtained in Step 2 in 3 mL of ultrapure water, add 0.2 mg of DATS, stir and react at room temperature for 18 h, centrifuge, and wash the precipitate with ultrapure water 5 times to obtain MH nanoparticles MHD loaded with DATS;

[0052] Step 4: Disperse 1 mg of the MHD obtained in Step 3 into 5 mL of ultrapure water. Under stirring conditions, add a carboxymethyl chitosan solution containing 0.5 mg of carboxymethyl chitosan. The concentration of the carboxymethyl chitosan solution is 100 μg / mL. React for 5 h, then centrifuge. Wash the precipitate 5 times with water to obtain an acidic degradable hybrid nanocomposite C@MHD for treating osteosarcoma.

[0053] The physicochemical properties of the C@MHD nanoparticles in this example are basically the same as those in Example 1.

[0054] Example 3

[0055] This example provides an acidic degradable hybrid nanocomposite C@MHD for treating osteosarcoma. The preparation method includes:

[0056] Step 1: Dissolve 0.8 g of cetyltrimethylammonium bromide and 0.3 g of triethanolamine in 25 mL of ultrapure water, stir evenly, adjust the pH of the mixture to 9.5 with ammonia water, heat to 40 °C, and stir and react for 3 h; then add calcium nitrate tetrahydrate to make the concentration of calcium nitrate tetrahydrate in the reaction system 100 mM, and react for 20 min; then add 1 mL of tetraethyl orthosilicate and disodium hydrogen phosphate dodecahydrate to make the concentration of disodium hydrogen phosphate dodecahydrate in the reaction system 2 mM, heat to 85 °C and react for 15 h, then centrifuge. Wash the precipitate 3 times with anhydrous ethanol and water respectively to obtain hybrid silica / hydroxyapatite nanoparticles MH.

[0057] Step 2: Disperse 1 mg of the MH obtained in Step 1 into 10 mL of anhydrous ethanol, add 0.1 mL of ultrapure water, 0.2 mL of ammonia water and 8 mg of 3-aminopropyltriethoxysilane, heat to 65 °C and stir and react for 30 h, then centrifuge. Wash the precipitate 3 times with anhydrous ethanol and ultrapure water respectively to obtain amino-functionalized MH nanoparticles MH-NH2.

[0058] Step 3: Disperse 1 mg of the MH-NH2 obtained in Step 2 into 3 mL of ultrapure water, add 1 mg of DATS, stir and react at room temperature for 12 h, then centrifuge. Wash the precipitate 5 times with ultrapure water to obtain MH nanoparticles MHD loaded with DATS.

[0059] Step 4: Disperse 1 mg of the MHD obtained in Step 3 into 5 mL of ultrapure water. Under stirring conditions, add a carboxymethyl chitosan solution containing 2 mg of carboxymethyl chitosan. The concentration of the carboxymethyl chitosan solution is 100 μg / mL. React for 1 h, then centrifuge. Wash the precipitate 5 times with water to obtain an acidic degradable hybrid nanocomposite C@MHD for treating osteosarcoma.

[0060] The physicochemical properties of the C@MHD nanoparticles in this example are basically the same as those in Example 1.

[0061] Example 4

[0062] This example provides an acidic degradable hybrid nanocomposite C@MHD for treating osteosarcoma, and the preparation method includes:

[0063] Step 1: Dissolve 0.6 g of cetyltrimethylammonium bromide and 0.1 g of triethanolamine in 25 mL of ultrapure water, stir evenly, adjust the pH of the mixture to 10.5 with ammonia water, heat to 75 °C, and stir and react for 1.8 h; then add calcium nitrate tetrahydrate to make the concentration of calcium nitrate tetrahydrate in the reaction system 75 mM, and react for 15 min; then add 1 mL of tetraethyl orthosilicate and disodium hydrogen phosphate dodecahydrate to make the concentration of disodium hydrogen phosphate dodecahydrate in the reaction system 15 mM, heat to 90 °C and react for 10 h, centrifuge, and wash the precipitate with absolute ethanol and water 3 times respectively to obtain hybrid silica / hydroxyapatite nanoparticles MH;

[0064] Step 2: Disperse 1 mg of MH obtained in Step 1 into 10 mL of absolute ethanol, add 0.1 mL of ultrapure water, 0.2 mL of ammonia water and 5 mg of 3-aminopropyltriethoxysilane, heat to 50 °C and stir and react for 48 h, centrifuge, and wash the precipitate with absolute ethanol and ultrapure water 3 times respectively to obtain amino-functionalized MH nanoparticles MH-NH2;

[0065] Step 3: Disperse 1 mg of MH-NH2 obtained in Step 2 into 3 mL of ultrapure water, add 0.4 mg of DATS, stir and react at room temperature for 12 h, centrifuge, and wash the precipitate with ultrapure water 5 times to obtain MH nanoparticles MHD loaded with DATS;

[0066] Step 4: Disperse 1 mg of MHD obtained in Step 3 into 5 mL of ultrapure water, add 1.4 mg of a 100 μg / mL carboxymethyl chitosan solution under stirring conditions, react for 4 h, centrifuge, and wash the precipitate with water 5 times to obtain an acidic degradable hybrid nanocomposite C@MHD for treating osteosarcoma.

[0067] The physicochemical properties of the C@MHD nanoparticles in this example are basically the same as those in Example 1.

[0068] Example 5

[0069] This example provides an acidic degradable hybrid nanocomposite C@MHD for treating osteosarcoma, and the preparation method includes:

[0070] Step 1: Dissolve 1 g of cetyltrimethylammonium bromide and 0.4 g of triethanolamine in 25 mL of ultrapure water, stir evenly, adjust the pH of the mixture to 10 with ammonia water, heat to 70 °C, and stir and react for 2.5 h; then add calcium nitrate tetrahydrate to make the concentration of calcium nitrate tetrahydrate in the reaction system 60 mM, and react for 20 min; then add 1 mL of tetraethyl orthosilicate and disodium hydrogen phosphate dodecahydrate to make the concentration of disodium hydrogen phosphate dodecahydrate in the reaction system 12 mM, heat to 95 °C and react for 22 h, centrifuge, and wash the precipitate with anhydrous ethanol and water 3 times respectively to obtain hybrid silica / hydroxyapatite nanoparticles MH;

[0071] Step 2: Disperse 1 mg of MH obtained in Step 1 into 10 mL of anhydrous ethanol, add 0.1 mL of ultrapure water, 0.2 mL of ammonia water and 6 mg of 3-aminopropyltriethoxysilane, heat to 45 °C and stir and react for 35 h, centrifuge, and wash the precipitate with anhydrous ethanol and ultrapure water 3 times respectively to obtain amino-functionalized MH nanoparticles MH-NH2;

[0072] Step 3: Disperse 1 mg of MH-NH2 obtained in Step 2 into 3 mL of ultrapure water, add 1.7 mg of DATS, stir and react at room temperature for 24 h, centrifuge, and wash the precipitate with ultrapure water 5 times to obtain DATS-loaded MH nanoparticles MHD;

[0073] Step 4: Disperse 1 mg of MHD obtained in Step 3 into 5 mL of ultrapure water, and add a carboxymethyl chitosan solution containing 1.8 mg of carboxymethyl chitosan under stirring conditions. The concentration of the carboxymethyl chitosan solution is 100 μg / mL, react for 4 h, centrifuge, and wash the precipitate with water 5 times to obtain an acidic degradable hybrid nanocomposite C@MHD for the treatment of osteosarcoma.

[0074] Performance evaluation

[0075] Figure 6 The killing effect of the C@MHD nanocomposites with different concentrations synthesized in Example 5 on HOS cells. The test method includes: inoculate HOS cells onto a 96-well plate, after growing for 24 h, add different concentrations of C@MHD nanocomposites respectively, and continue to culture for 24 h; discard the culture medium, add 100 μL of a tetramethyl azo salt solution with a concentration of 1 mg / mL to each well, and continue to culture for 4 h; discard the tetramethyl azo salt solution, add 150 μL of dimethyl sulfoxide to each well, and detect the absorbance value at 490 nm with an enzyme-linked immunosorbent assay instrument. The results are as Figure 6 shown. According to Figure 6 it can be seen that the cell survival rate gradually decreases with the increase of the C@MHD concentration; when the C@MHD concentration is 200 μg / mL, the cell survival rate is 47.20%. The above experiments show that C@MHD can effectively kill HOS cells.

[0076] Figure 7 Effect of the C@MHD nanocomposite synthesized in Example 5 on the cell viability of mouse fibroblast NIH-3T3 cells. The test method includes: inoculating mouse fibroblast NIH-3T3 cells onto a 96-well plate, allowing them to grow for 24 h, then adding C@MHD nanocomposites at different concentrations and continuing to culture for 24 h, 48 h, and 72 h; discarding the culture medium, adding 100 μL of a 1 mg / mL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to each well, and continuing to culture for 4 h; discarding the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution, adding 150 μL of dimethyl sulfoxide to each well, and measuring the absorbance value at 490 nm using an enzyme-linked immunosorbent assay reader. The results are as Figure 7 shown. According to Figure 7 visible, C@MHD has no obvious effect on cell viability in the concentration range of 0 - 400 μg / mL, indicating that the C@MHD nanocomposite has good biocompatibility at the cellular level.

[0077] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent structural changes made to the above embodiments based on the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A preparation method of an acid-degradable hybrid nanocomposite for treating osteosarcoma, characterized in that, It includes the following steps: Step 1: Dissolve cetyltrimethylammonium bromide and triethanolamine in ultrapure water, stir evenly, adjust the pH value of the solution to 9 - 11 with ammonia water, heat to 40°C - 85°C, and stir and react for 1h - 3h; then add calcium nitrate tetrahydrate to the solution after stirring and reacting, and react for 15min - 30min; then add a silicon source and disodium hydrogen phosphate dodecahydrate, heat to 85°C - 105°C and react for 10h - 24h, and then centrifuge; wash the precipitate obtained by centrifugation with anhydrous ethanol and water to obtain hybrid silica / hydroxyapatite nanoparticles MH; Step 2: Disperse the MH obtained in Step 1 in anhydrous ethanol, add ultrapure water, ammonia water and 3-aminopropyltriethoxysilane, heat to 30°C - 65°C and stir and react for 12h - 48h, centrifuge, and wash the precipitate with anhydrous ethanol and ultrapure water to obtain amino-functionalized MH nanoparticles MH-NH2; Step 3: Disperse the MH-NH2 obtained in Step 2 in ultrapure water, add DATS, stir and react at room temperature for 12h - 24h, centrifuge, and wash the precipitate with ultrapure water to obtain MH nanoparticles MHD loaded with DATS; Step 4: Disperse the MHD obtained in Step 3 in ultrapure water, add a carboxymethyl chitosan solution under stirring conditions, react for 1h - 5h, centrifuge, and wash the precipitate with water to obtain an acidic degradable hybrid nanocomposite C@MHD for treating osteosarcoma.

2. The preparation method of an acid-degradable hybrid nanocomposite for treating osteosarcoma according to claim 1, characterized in that, The particle size of the hybrid silica / hydroxyapatite nanoparticles MH obtained in Step 1 is 50nm - 80nm.

3. The preparation method of an acidic degradable hybrid nanocomplex for treating osteosarcoma according to claim 1, characterized in that The addition amount of the calcium nitrate tetrahydrate in Step 1 is such that the concentration of calcium nitrate tetrahydrate in the reaction system is 20mM - 100mM.

4. The preparation method of an acidic degradable hybrid nanocomposite for treating osteosarcoma according to claim 1, characterized in that, The addition amount of the disodium hydrogen phosphate dodecahydrate in Step 1 is such that the concentration of disodium hydrogen phosphate dodecahydrate in the reaction system is 2mM - 15mM.

5. The preparation method of an acid-degradable hybrid nanocomposite for treating osteosarcoma according to claim 1, characterized in that, The mass ratio of MH to 3-aminopropyltriethoxysilane in Step 2 is 1:(1 - 8).

6. The preparation method of an acidic degradable hybrid nanocomposite for treating osteosarcoma according to claim 1, characterized in that, The mass ratio of MH-NH2 to DATS in Step 3 is 1:(0.2 - 1).

7. The preparation method of an acid-degradable hybrid nanocomposite for treating osteosarcoma according to claim 1, characterized in that, The mass ratio of MHD to carboxymethyl chitosan in Step 4 is 1:(0.5 - 2).

8. A nanocomposite C@MHD prepared by the preparation method according to any one of claims 1 - 7.

9. The nano - composite C@MHD according to claim 10, characterized in that, The particle size of the nanocomposite C@MHD is 65nm - 105nm.

10. Use of the nanocomposite C@MHD according to claim 10 in the preparation of an osteosarcoma therapeutic agent.

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