Manganese-doped calcium phosphate material, preparation method and application thereof

Abstract

The application discloses a manganese-doped calcium phosphate material and a preparation method and application thereof, and belongs to the field of nanometer materials. 2+ The manganese-doped calcium phosphate material comprises calcium phosphate nanoclusters and manganese ions distributed in the calcium phosphate nanoclusters. 4+ The manganese-doped calcium phosphate material has a nanocluster morphology, a particle size of less than 10 nm, and good suspension in a liquid phase environment. The material has glutathione responsiveness, good biocompatibility, photothermal performance and antibacterial property, and can be applied to preparation of an antibacterial agent, an antitumor material, a nuclear magnetic imaging agent or a chemotherapy sensitizer. The preparation method of the manganese-doped calcium phosphate nanocluster material of the application adopts polyacrylic acid as a size control agent, and does not introduce impurity ions. The polyacrylic acid can not only regulate a calcium phosphate mineralization process by complexing Ca 2+ ions in raw materials, but also obtain a smaller-size nanocluster structure calcium phosphate material through a steric hindrance effect.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a manganese-doped calcium phosphate material, its preparation method, and its application. Background Technology

[0002] Cancer poses a serious threat to human health, and conventional treatments (such as surgical resection and chemotherapy) often have significant side effects. Near-infrared photothermal energy conversion technology is a novel cancer treatment method with fewer side effects, and it has attracted considerable attention in recent years. Photothermal therapy (PTT) is a treatment method that uses materials with high photothermal conversion efficiency to convert light energy into heat energy under light irradiation, causing localized high temperatures in tumor cells, thereby killing cancer cells. However, relying solely on photothermal therapy cannot precisely locate the tumor, limiting its effectiveness in detecting and treating tumors. Imaging technology can overcome this deficiency, providing information about the tumor's location, shape, and size. It can also determine the enrichment of photothermal therapeutic agents in tumor tissue, evaluating treatment efficacy. Therefore, organically combining photothermal therapy and imaging technology is beneficial for improving the effectiveness of cancer treatment.

[0003] Magnetic resonance imaging (MRI) is an imaging technique that uses the differences in water content among different tissues of a living organism and the variations in the chemical environment of hydrogen nuclei in the water to create images. MRI has attracted widespread attention due to the absence of ionizing damage caused by radiation and its advantages such as high resolution and high sensitivity. Although MRI has high spatial resolution, the sensitivity of MRI imaging alone is still not high, thus requiring the use of contrast agents to enhance signal contrast and improve the resolution of soft tissue images. MRI contrast agents are generally paramagnetic gadolinium or manganese-based contrast agents. Small-molecule gadolinium contrast agents generally have low relaxation rates, resulting in low contrast intensity, while nanoscale manganese-based contrast agents show better clinical application prospects. Therefore, it is necessary to develop a novel manganese-based nanomaterial that can be used as both an MRI contrast agent and for photothermal therapy.

[0004] Calcium phosphate is a high-quality biomaterial, structurally and functionally similar to the inorganic components of human bone, exhibiting excellent biocompatibility and not causing rejection reactions in vivo. It is used as a non-ceramic artificial bone material, demonstrating significant advantages in repairing bone defects. The bioabsorbability, unique biocompatibility, biodegradability, chemical stability, and low toxicity of calcium phosphate nanoparticles allow for effective cell delivery. As a targeted drug carrier, it can penetrate damaged or diseased tissues, making it an ideal carrier material for drugs. However, the particle size of common calcium phosphate nanoparticles is mostly between 100nm and 500nm; larger sizes can affect the later biological functions of calcium phosphate. Therefore, how to prepare small-sized manganese-based calcium phosphate nanomaterials and simultaneously apply them to magnetic resonance imaging contrast agents and photothermal therapy is a problem that needs to be solved. Summary of the Invention

[0005] To overcome the problems existing in the prior art, one objective of this invention is to provide a manganese-doped calcium phosphate material. A second objective is to provide a method for preparing this manganese-doped calcium phosphate material. A third objective is to provide the application of this manganese-doped calcium phosphate material in the degradation of methylene blue. A fourth objective is to provide the application of this manganese-doped calcium phosphate material in the preparation of antibacterial agents. A fifth objective is to provide the application of this manganese-doped calcium phosphate material in the preparation of antitumor materials. A sixth objective is to provide the application of this manganese-doped calcium phosphate material in the preparation of nuclear magnetic resonance imaging agents. A seventh objective is to provide the application of this manganese-doped calcium phosphate material in the preparation of chemotherapy sensitizers.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides a manganese-doped calcium phosphate material, comprising calcium phosphate nanoclusters and manganese ions distributed within the calcium phosphate nanoclusters; the manganese ions include Mn 2+ and Mn 4+ .

[0008] Preferably, in the manganese ions, Mn 2+ The proportion is 40-70%, Mn 4+ The proportion is 30% to 60%.

[0009] Preferably, the manganese-doped calcium phosphate material is spherical with a particle size of 1–10 nm.

[0010] Preferably, the longitudinal relaxation rate r1 of the manganese-doped calcium phosphate material is 0.89-1.18 mM. -1 s -1 .

[0011] The second aspect of the present invention provides a method for preparing the manganese-doped calcium phosphate material described in the first aspect, comprising the following steps: first, preparing a mixed solution of calcium salt, manganese salt and polyacrylic acid, then adding phosphate to the mixed solution, reacting, and obtaining the manganese-doped calcium phosphate material.

[0012] Preferably, the calcium salt is selected from calcium chloride, calcium nitrate, or a combination thereof.

[0013] Preferably, the manganese salt is selected from one or more of manganese chloride, manganese sulfate, and manganese nitrate.

[0014] Preferably, the phosphate is selected from one or more of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0015] Preferably, the polyacrylic acid has a molecular weight of 1.5-2.0 kDa.

[0016] Preferably, the molar ratio of the manganese salt to the sum of the calcium salt and manganese salt is (0.01 to 0.2):1.

[0017] Preferably, the total concentration of the calcium and manganese salts in the mixed solution is 0.01–0.1 mol / L.

[0018] Preferably, the molar ratio of the phosphate to the sum of the calcium and manganese salts is 1:(1.5-2).

[0019] Preferably, the concentration of the polyacrylic acid in the mixed solution is 5–35 mg / mL.

[0020] More preferably, the concentration of the polyacrylic acid in the mixed solution is 10–30 mg / mL.

[0021] Preferably, the solvent of the mixed solution is water.

[0022] Preferably, the specific steps include the following:

[0023] 1) First, prepare a mixed solution of calcium salt and manganese salt, then add polyacrylic acid solution to the above mixed solution, adjust the pH of the mixed solution to 7-8 and disperse it evenly;

[0024] 2) Add phosphate to the above mixture and disperse it evenly. Adjust the pH of the mixture to 10-12 and react at 15-40℃ for 1-3 hours to obtain the manganese-doped calcium phosphate material.

[0025] More preferably, the polyacrylic acid solution is added dropwise.

[0026] More preferably, in step 2), the product is collected by dialysis using a dialysis bag after the reaction. Even more preferably, the dialysis bag has a molecular weight cutoff of 5000.

[0027] The third aspect of the present invention provides the application of the manganese-doped calcium phosphate material described in the first aspect in the degradation of methylene blue.

[0028] The fourth aspect of this invention provides the application of the manganese-doped calcium phosphate material described in the first aspect in the preparation of antibacterial agents.

[0029] The fourth aspect of this invention provides the application of the manganese-doped calcium phosphate material described in the first aspect in the preparation of anti-Escherichia coli agents.

[0030] The fifth aspect of this invention provides the application of the manganese-doped calcium phosphate material described in the first aspect in the preparation of antitumor materials.

[0031] Preferably, the manganese-doped calcium phosphate material is used in the preparation of anti-breast cancer cell tumor-bearing materials.

[0032] The sixth aspect of the present invention provides the application of the manganese-doped calcium phosphate material described in the first aspect in the preparation of nuclear magnetic resonance imaging agents.

[0033] The seventh aspect of the present invention provides the application of the manganese-doped calcium phosphate material described in the first aspect in the preparation of chemotherapeutic sensitizers.

[0034] Preferably, the manganese-doped calcium phosphate material is used in the preparation of antibiotic drug sensitizers. More preferably, the manganese-doped calcium phosphate material is used in the preparation of azithromycin sensitizers.

[0035] The beneficial effects of this invention are:

[0036] (I) This invention provides a manganese-doped calcium phosphate material with the following characteristics: 1) The material exhibits a nanocluster morphology with a particle size within 10 nm, which is beneficial for cell phagocytosis and exerts a size effect when applied in antitumor materials; 2) The material exhibits good suspension properties in a liquid environment, and suspensions prepared using this material are stable and have strong fluidity, which is beneficial for subsequent preparation of various drugs for application in magnetic imaging, antibacterial, and antitumor fields; 3) The material is glutathione responsive, and the valence state of manganese ions in the material can be reduced from +4 to +2, enhancing the material's... 4) Manganese-doped calcium phosphate nanoclusters have good biocompatibility, photothermal properties and antibacterial properties, and can be used to prepare antibacterial agents and antitumor materials, and then applied to biomedical fields such as clinical antibacterial and tumor treatment diagnosis; 5) Manganese-doped calcium phosphate nanoclusters have high longitudinal relaxation rate and rapid tumor regional accumulation effect, and can be used as nuclear magnetic resonance imaging agents in the field of clinical diagnosis; 6) Manganese-doped calcium phosphate nanoclusters have chemosensitizing effect on drugs, and can be used to prepare chemosensitizers.

[0037] (II) The preparation method of the manganese-doped calcium phosphate nanoclusters of the present invention uses polyacrylic acid as a size control agent, which does not introduce impurity ions. The low molecular weight polyacrylic acid can not only complex the Ca in the raw materials... 2+ This invention can regulate the calcium phosphate mineralization process and also obtain smaller-sized nanocluster calcium phosphate materials through steric hindrance. The preparation method of this invention is simple, the obtained product has a uniform size distribution, the reaction conditions are mild, and no complicated instruments or equipment are required. Attached Figure Description

[0038] Figure 1 The X-ray photoelectron spectrum of the manganese-doped calcium phosphate nanoclusters obtained in Example 1 is shown below.

[0039] Figure 2 The particle size distribution and transmission electron microscopy image of the manganese-doped calcium phosphate nanoparticles obtained in Example 1 are shown.

[0040] Figure 3 The X-ray photoelectron spectrum of the manganese-doped calcium phosphate nanoclusters obtained in Example 2 is shown below.

[0041] Figure 4 Here is a scanning electron microscope image of the manganese-doped calcium phosphate nanoparticles obtained in Comparative Example 2;

[0042] Figure 5 The image shows the antibacterial effect of the manganese-doped calcium phosphate nanoclusters obtained in Example 2 on Escherichia coli.

[0043] Figure 6 The images show the T1-weighted images and relaxation rate curves of the manganese-doped calcium phosphate nanoclusters obtained in Examples 1-2 and the nanoclusters of Example 2 after treatment with glutathione.

[0044] Figure 7 The images show MRI images of tumor-bearing mice with manganese-doped calcium phosphate nanomaterials obtained in Example 2 and Control Example 2.

[0045] Figure 8 The heating and cooling curves of the manganese-doped calcium phosphate nanoclusters obtained in Example 3 and the linear relationship between the negative natural logarithm of the cooling temperature and time are shown.

[0046] Figure 9 The absorption spectra of the manganese-doped calcium phosphate nanoclusters obtained in Example 3 and the degraded methylene blue after glutathione treatment of the nanoclusters in Example 3 are shown.

[0047] Figure 10 The cytotoxicity (A) and blood compatibility (B) of the manganese-doped calcium phosphate nanoclusters obtained in Example 3 are shown.

[0048] Figure 11Tumor volume inhibition diagrams of manganese-doped calcium phosphate nanomaterials obtained in Example 2 and Control Example 1;

[0049] Figure 12 The diagram shows the synergistic relationship between manganese-doped calcium phosphate nanoclusters obtained in Example 3 and azithromycin for chemosensitization, where A is the Fa-CI trend diagram and B is the Fa-DRI trend diagram. Detailed Implementation

[0050] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0051] The polyacrylic acid used in the following examples has an Mw of 1.8 kDa.

[0052] Example 1

[0053] This embodiment provides a method for preparing manganese-doped calcium phosphate nanoclusters. The specific preparation method is as follows:

[0054] S1. Add calcium chloride dihydrate and manganese chloride dihydrate to water to prepare a mixed solution. The total concentration of calcium chloride and manganese chloride in the mixed solution is 0.0334 mol / L, and the molar ratio of manganese salt to the total of calcium and manganese salts (i.e., Mn / (Ca+Mn)) is 0.01:1. Stir with a magnetic stirrer for 1 hour. Add 15 mL of polyacrylic acid to the above mixed solution and adjust the pH to 7 with sodium hydroxide solution. The concentration of polyacrylic acid in the solution is 10 mg / mL. Stir for 1 hour.

[0055] S2. Prepare an aqueous solution of disodium hydrogen phosphate dodecahydrate, PO4 3- At a concentration of 0.02 mol / L, 20 mL of disodium hydrogen phosphate aqueous solution was quickly poured into 20 mL of the above mixed aqueous solution at room temperature. The molar ratio of phosphate to the total calcium and manganese salts (i.e., P / (Ca+Mn)) was 1:1.67. The pH was adjusted to 10 with sodium hydroxide solution, and then stirring was continued for 1 hour. The concentration of polyacrylic acid in the final cluster suspension was 5 mg / mL. The final manganese-doped calcium phosphate product was obtained by dialysis using a dialysis bag (molecular weight cutoff 5000).

[0056] The obtained material was subjected to XPS testing to determine the valence state of manganese ions, and the results are as follows: Figure 1 The results showed that Mn in the material 2+ The proportion was 68.58%, Mn 4+ The proportion was 31.42%.

[0057] The prepared material was subjected to transmission electron microscopy and particle size analysis, and the results are as follows: Figure 2 The results showed that the prepared manganese-doped calcium phosphate nanoclusters had an approximately spherical morphology and uniform size distribution, with an average particle size of 5 nm and good dispersibility.

[0058] Example 2

[0059] This embodiment provides a method for preparing manganese-doped calcium phosphate nanoclusters. The specific preparation method is as follows:

[0060] S1. Add calcium chloride dihydrate and manganese chloride dihydrate to water to prepare a mixed solution. The total concentration of calcium chloride and manganese chloride in the mixed solution is 0.0334 mol / L, and the molar ratio of manganese salt to the total of calcium and manganese salts (i.e., Mn / (Ca+Mn)) is 0.2:1. Stir with a magnetic stirrer for 3 hours. Add 15 mL of polyacrylic acid to the above mixed solution and adjust the pH to 8 with sodium hydroxide solution. The concentration of polyacrylic acid in the solution is 30 mg / mL. Stir for 3 hours.

[0061] S2. Prepare an aqueous solution of disodium hydrogen phosphate dodecahydrate, PO4 3- At a concentration of 0.02 mol / L, 20 mL of disodium hydrogen phosphate aqueous solution was quickly poured into 20 mL of the above mixed aqueous solution at room temperature. The molar ratio of phosphate to the total calcium and manganese salts (i.e., P / (Ca+Mn)) was 1:1.67. The pH was adjusted to 12 with sodium hydroxide solution, and then stirring was continued for 3 hours. The concentration of polyacrylic acid in the final cluster suspension was 15 mg / mL. The final manganese-doped calcium phosphate product was obtained by dialysis using a dialysis bag (molecular weight cutoff 5000).

[0062] The obtained material was subjected to XPS testing to determine the valence state of manganese ions, and the results are as follows: Figure 3 The results showed that Mn in the material 2+ The proportion was 42.77%, Mn 4+ The proportion is 57.23%.

[0063] Example 3

[0064] This embodiment provides a method for preparing manganese-doped calcium phosphate nanoclusters. The specific preparation method is as follows:

[0065] S1. Add calcium chloride dihydrate and manganese chloride dihydrate to water to prepare a mixed solution. The total concentration of calcium chloride and manganese chloride in the mixed solution is 0.0334 mol / L, and the molar ratio of manganese salt to the sum of calcium salt and manganese salt (i.e., Mn / (Ca+Mn)) is 0.2:1. Stir with a magnetic stirrer for 1 hour. Add 15 mL of polyacrylic acid to the above mixed solution and adjust the pH to 7 with sodium hydroxide solution. The concentration of polyacrylic acid in the solution is 15 mg / mL. Stir for 1 hour.

[0066] S2. Prepare an aqueous solution of disodium hydrogen phosphate dodecahydrate, PO4 3- At a concentration of 0.02 mol / L, 20 mL of disodium hydrogen phosphate aqueous solution was quickly poured into 20 mL of the above mixed aqueous solution at room temperature. The molar ratio of phosphate to the total calcium and manganese salts (i.e., P / (Ca+Mn)) was 1:1.67. The pH was adjusted to 11 with sodium hydroxide solution, and then the mixture was stirred for 1 hour. The final manganese-doped calcium phosphate product was obtained by dialysis using a dialysis bag (molecular weight cutoff 5000).

[0067] Compare with Example 1

[0068] This comparative example provides a method for preparing calcium phosphate materials, the specific preparation method of which is as follows:

[0069] S1. Prepare a calcium chloride dihydrate solution, controlling the total Ca concentration to 0.0334 mol / L. Add 15 mL of polyacrylic acid solution to the above solution and adjust the pH to 7.2 with sodium hydroxide solution. Stir for 30 min. The concentration of polyacrylic acid in the solution is 15 mg / mL.

[0070] S2. Prepare an aqueous solution of disodium hydrogen phosphate dodecahydrate, PO4 3- With a concentration of 0.02 mol / L, and a molar ratio of Ca / P = 1.67, 20 mL of disodium hydrogen phosphate aqueous solution was quickly poured into 20 mL of the above aqueous solution at room temperature, and the pH was adjusted to 12 with sodium hydroxide solution. After stirring and mixing evenly, the reaction was carried out for 2 hours. The final manganese-doped calcium phosphate product was obtained by dialysis using a dialysis bag (molecular weight cutoff 5000).

[0071] Compare with Example 2

[0072] This comparative example provides a method for preparing manganese-doped calcium phosphate materials. The specific preparation method is as follows:

[0073] S1. Add calcium chloride dihydrate and manganese chloride dihydrate to water to prepare a mixed solution. The total concentration of calcium chloride and manganese chloride in the mixed solution is 0.0334 mol / L. The molar ratio of manganese salt to the total of calcium salt and manganese salt (i.e., Mn / (Ca+Mn)) is 0.2:1. Stir with a magnetic stirrer for 1 hour.

[0074] S2. Prepare an aqueous solution of disodium hydrogen phosphate dodecahydrate, PO4 3- At a concentration of 0.02 mol / L, 20 mL of disodium hydrogen phosphate aqueous solution was quickly poured into 20 mL of the above mixed aqueous solution at room temperature. The molar ratio of phosphate to the total calcium and manganese salts (i.e., P / (Ca+Mn)) was 1:1.67. The pH was adjusted to 12 with sodium hydroxide solution, and then stirring was continued for 2 hours. The final manganese-doped calcium phosphate product was obtained by dialysis using a dialysis bag (molecular weight cutoff 5000).

[0075] The prepared material was examined by scanning electron microscopy, and the results are as follows: Figure 4 The results showed that the prepared manganese-doped calcium phosphate nanoparticles had a short rod-shaped morphology and a particle size of 100-200 nm.

[0076] Experimental Analysis

[0077] 1. Antibacterial

[0078] The antibacterial activity of the nanocluster material prepared in Example 2 was tested using *Escherichia coli* as the experimental subject. First, the sterilized material was added to a 24-well bacterial culture plate, and 200 μL of a 10⁻⁶ concentration was inoculated onto the surface of each plate. 7 CFU / mL of bacteria were collected and incubated at 37°C for 24 hours. Then, the centrifuge tubes were shaken, and a certain amount of the bacterial suspension was diluted 100-fold. 100 μL of the diluted bacterial solution was added dropwise to a nutrient agar plate, spread evenly with a spreader, and incubated upside down at 37°C for 18 hours. The bacterial colonies were then photographed and counted using a digital camera and ImageJ software. The results are as follows: Figure 5 As shown, with manganese ion doping into the calcium phosphate material, the cluster exhibits a significant inhibitory effect on Escherichia coli, achieving an antibacterial rate of 81%. After irradiating the material with a near-infrared laser for 10 minutes, the inhibition rate of the cluster against E. coli increases to 98%.

[0079] 2. Magnetic imaging

[0080] In vitro magnetic imaging was performed on the nanoclusters prepared in Examples 1 and 2. Figure 6 The display material exhibits significantly enhanced magnetic signals and a marked positive contrast effect, with a longitudinal relaxation rate r1 ranging from 0.89 to 1.18 Mm. -1 s -1 The nanocluster material prepared in Example 2 was treated with glutathione, which further enhanced the magnetic signal and positive contrast effect of the material, and increased the longitudinal relaxation rate r1 to 1.31 mM. -1 s -1 This indicates that the nanocluster material prepared by the present invention has glutathione reducing properties and can improve its own magnetic imaging performance.

[0081] In vivo magnetic imaging was performed on the materials prepared in Example 2 and Control Example 2. Figure 7 The nanoclusters of Example 2 showed a significant positive contrast effect in the tumor area 20 minutes after intravenous injection into tumor-bearing mice, while the nanoparticles of Control Example 2 did not show a significant contrast effect at the same time point. This indicates that the nanoclusters prepared by the present invention can achieve tumor accumulation in a faster manner than nanoparticles.

[0082] 3. Photothermal

[0083] The photothermal conversion efficiency of the material prepared in Example 3 was tested by exposing the material to a near-infrared laser (808nm laser, 1W / cm²). 2 The photothermal conversion efficiency (η) is calculated using the following formula: (The image is then captured by a thermal imaging camera, and its temperature rise curve is recorded.)

[0084]

[0085] In this formula, h represents the heat transfer coefficient; A represents the surface area of ​​the container; ΔTmax represents the maximum steady-state temperature at which the nanoparticles increase in temperature; and Q represents the maximum surface area of ​​the container. dis : Light scattering absorbed by water and sample cell; I: Laser power (1W / cm²) 2 A808: Absorbance of the nanoparticle at 808 nm; Figure 8 The nanocluster material prepared in Example 2 can reach a maximum temperature of 61°C after being irradiated with a near-infrared laser, and the photothermal conversion efficiency of the material is 24-31%.

[0086] 4. Catalysis

[0087] The catalytic ability of the material prepared in Example 3 was tested by a methylene blue degradation experiment. The material was added to a buffer solution containing 25 mM NaHCO3, methylene blue (MB, 0.01 mg / mL), and 10 mM H2O2 and stirred continuously. After a certain reaction time, the reaction mixture was centrifuged to collect the supernatant, and the absorbance at 664 nm was measured using a UV-Vis-NIR spectrophotometer. Figure 9 The results showed a significant decrease in the absorbance of methylene blue at 664 nm within just 3 minutes, indicating that the methylene blue was partially degraded by the nanocluster material. Further treatment of the nanocluster material prepared in Example 3 with glutathione showed that the methylene blue was completely degraded by the material within 3 minutes, demonstrating that the nanocluster material prepared in this invention has excellent catalytic performance and can be further enhanced by glutathione reduction in the tumor microenvironment.

[0088] 5. Biocompatibility

[0089] Using mammalian bone marrow mesenchymal stem cells (BMSCs) and mouse erythrocytes as experimental subjects, the cytotoxicity and blood compatibility of the materials prepared in Example 3 were tested. The cytotoxicity test method was as follows: cells were placed in a 96-well plate (10... 5Cells were cultured in wells (U / well) at 37°C in a 5% CO2 incubator to a suitable cell density. They were then incubated with different concentrations (0, 50, 100, 250, 500, and 1000 μg / mL) of material for 24 and 48 hours, respectively. The culture medium was discarded, and 100 μL of 10% CCK8 solution was added. After incubation for 1 hour, the absorbance (OD) at 450 nm was measured using a microplate reader. Blood compatibility was assessed by diluting mouse red blood cells (RBCs) obtained from BALB / c mice with a certain amount of PBS, followed by centrifugation at 2000 rpm for 10 minutes to separate RBCs from serum. After washing at least four times, the RBCs were diluted with 10 mL of PBS. Subsequently, 200 μL of the diluted RBC suspension was mixed with 1 mL of PBS (negative control), deionized water (positive control), or different concentrations of material (0, 100, 250, 500, and 1000 μg / mL). After incubating at 37°C for 2 hours, the mixture was centrifuged at 12,000 rpm for 10 minutes. All the obtained supernatant was added to a 96-well plate, and the absorbance at 570 nm was measured using a microplate reader. The hemolysis rate was calculated using the formula: Hemolysis rate % = [(OD sample - OD negative) / (OD positive – OD negative)] × 100%.

[0090] Test results are as follows Figure 10 A. The results showed that the cell viability of the nanoclusters at various concentrations within 24 and 48 hours was not significantly different from the control, indicating that the materials synthesized in the embodiments of this invention have no significant cytotoxicity to normal mammalian cells. Furthermore, Figure 10 B indicates that the hemolysis rate of the material at each concentration was not significantly different from that of the control. The nanocluster materials synthesized in the embodiments of the present invention did not produce significant cell hemolysis when applied to normal mammalian cells.

[0091] 6. Anti-tumor

[0092] First, a 4T1 breast cancer cell-bearing mouse model was constructed. The materials from Example 2 and Control Example 1 were injected into the mice via tail vein at a concentration of 5 mg / mL, with a volume of 100 μL. One hour after injection of the material from Example 2, the tumor area was irradiated with a near-infrared laser. After 14 days, as... Figure 11 As shown, the manganese-doped calcium phosphate nanoclusters in Example 2 exhibited excellent antitumor effects. Compared with the blank group and Control Example 1, tumor growth was effectively inhibited, and after near-infrared laser irradiation, the tumors were almost completely eliminated, indicating that the nanoclusters prepared by this invention can achieve effective antitumor capabilities.

[0093] 7. Chemotherapy sensitization

[0094] The chemotherapy sensitization ability of the material prepared in Example 3 was tested, using manganese-doped nanoclusters to sensitize azithromycin chemotherapy drugs in the treatment of 4T1 breast cancer cells as an example. Figure 12 A is the inhibition rate-combination index (Fa-CI) plot. The plot shows that as drug concentration and inhibition rate increase, the CI value decreases. When Fa is greater than 0.23, CI below 1 indicates a synergistic effect. This corresponds to a significant synergistic effect in 4T1 cells when the concentrations of azithromycin and the nanoclusters from Example 3 are greater than 0.38 μM and greater than 189.29 μg / mL, respectively. The dose-reduction index (DRI) curve shows... Figure 12 B) Due to the chemosensitizing effect of nanoclusters on azithromycin, combination therapy can reduce the drug toxicity caused by excessive use of azithromycin in clinical treatment for most patients with Fa, while still achieving the same therapeutic effect.

[0095] The preparation method of manganese-doped calcium phosphate nanoclusters in this invention is simple, with mild reaction conditions and no need for complex instruments or equipment. Furthermore, the calcium phosphate-based nanoclusters prepared by this invention exhibit good biocompatibility and antibacterial and antitumor capabilities, and also demonstrate nuclear magnetic resonance imaging (NMR) efficacy, making them widely applicable in biomedical fields such as clinical antibacterial and tumor treatment and diagnosis.

[0096] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A manganese-doped calcium phosphate material, characterized in that, It includes calcium phosphate nanoclusters and manganese ions distributed within the calcium phosphate nanoclusters; the manganese ions include Mn 2+ and Mn 4+ ; The manganese-doped calcium phosphate material is prepared by a method comprising the following steps: first, calcium salt, manganese salt and polyacrylic acid are prepared into a mixed solution, then phosphate is added to the mixed solution and reacted to obtain the manganese-doped calcium phosphate material.

2. The manganese-doped calcium phosphate material according to claim 1, characterized in that, In the manganese ions, Mn 2+ The proportion is 40-70%, Mn 4+ The proportion is 30-60%.

3. The manganese-doped calcium phosphate material according to claim 1, characterized in that, The manganese-doped calcium phosphate material is spherical with a particle size of 1~10 nm.

4. The method for preparing the manganese-doped calcium phosphate material according to any one of claims 1 to 3, characterized in that, The process includes the following steps: first, calcium salt, manganese salt, and polyacrylic acid are prepared into a mixed solution, then phosphate is added to the mixed solution and reacted to obtain the manganese-doped calcium phosphate material.

5. The method for preparing manganese-doped calcium phosphate material according to claim 4, characterized in that, The calcium salt is selected from one or a combination of calcium chloride, calcium nitrate, and calcium salt. And / or, the manganese salt is selected from one or more of manganese chloride, manganese sulfate, and manganese nitrate; And / or, the phosphate is selected from one or more of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate; And / or, the polyacrylic acid has a molecular weight of 1.5~2.0 kDa.

6. The method for preparing manganese-doped calcium phosphate material according to claim 4, characterized in that, The molar ratio of the manganese salt to the sum of the calcium and manganese salts is (0.01~0.2):1; And / or, the molar ratio of the phosphate to the sum of the calcium and manganese salts is 1:(1.5~2); And / or, the concentration of the polyacrylic acid in the mixed solution is 5~35 mg / mL.

7. The method for preparing manganese-doped calcium phosphate material according to claim 4, characterized in that, Specifically, the steps include the following: 1) First, prepare a mixed solution of calcium salt and manganese salt, then add polyacrylic acid solution to the above mixed solution, adjust the pH of the mixed solution to 7-8 and disperse it evenly; 2) Add phosphate to the above mixture and disperse it evenly. Adjust the pH of the mixture to 10-12 and react at 15-40℃ for 1-3 h to obtain the manganese-doped calcium phosphate material.

8. The use of the manganese-doped calcium phosphate material according to any one of claims 1 to 3 in the degradation of methylene blue.

9. The use of the manganese-doped calcium phosphate material according to any one of claims 1 to 3 in the preparation of antibacterial agents or antitumor materials.

10. The use of the manganese-doped calcium phosphate material according to any one of claims 1 to 3 in the preparation of nuclear magnetic resonance imaging agents or chemotherapeutic sensitizers.

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