Manganese diphosphonate nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a diphosphonic acid manganese nanoparticle, a preparation method thereof and application of the diphosphonic acid manganese nanoparticle in tumor treatment. The preparation method comprises the following steps: dropwise adding a water-in-oil emulsion system of diphosphonate into a water-in-oil emulsion system of manganese ions in stirring to establish a coordination skeleton, curing by absolute ethyl alcohol, adding a certain proportion of fat material, performing vortex hydration by a film dispersion method, and extruding to pass through a film. The diphosphonic acid manganese nanoparticle is small in particle size and good in stability, and has a pH-sensitive release characteristic; the constructed nanoparticles have a remarkable immune regulation effect, and the ICD effect and the cytotoxic effect of oxaliplatin are enhanced by directly acting on tumor cells. The diphosphonic acid manganese nanoparticles cooperate with Oxp to effectively inhibit digestive tract tumor growth and distal metastatic tumors, the dosage of Oxp can be further reduced, and the diphosphonic acid manganese nanoparticles have good in-vivo safety.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and particularly relates to a manganese bisphosphonate nanoparticle, a preparation method thereof, and an application thereof in tumor treatment. Background Art

[0002] Immunotherapy has become the core strategy for current tumor treatment, and a variety of immunomodulatory drugs other than immune checkpoint blockers have successively entered the clinical stage. Among them, immune agonists have shown important potential in anti-tumor clinical treatment by activating key signaling pathways or cell functions of the immune system. Immune agonists mainly activate key immune activation pathways in innate immune cells such as tumor-associated macrophages and dendritic cells, as well as NK cells, including the cGAS-STING pathway, the TLR pathway, etc., enhance antigen presentation, promote the transdifferentiation of inhibitory myeloid cells, and induce anti-tumor immune responses.

[0003] For a variety of advanced unresectable tumors that have metastasized, immune agonists have shown excellent therapeutic effects by combining with antibodies such as PD-1 and PD-L1. Especially for "cold" tumors that are ineffective against single immune checkpoint blockers alone, immune agonists can effectively enhance the infiltration of cytotoxic T cells activated by antibodies such as PD-1 and PD-L1 by remodeling the inhibitory immune microenvironment in tumor tissues, significantly improving the curative effect. Currently, several immune agonists have entered the clinical trial stage.

[0004] However, the research and development of immune agonists also face many problems. Among them, after the administration of immune agonists, there may be a systemic activation of the body's innate immunity, leading to an excessive immune response, and then triggering relatively serious immune-related adverse reactions (such as cytokine storms). Therefore, how to maintain the activation of anti-tumor innate immune responses in the body while avoiding its activation of the whole body's immunity is the difficulty and challenge in the current research and development of immune agonist drugs.

[0005] Studies have shown that antigen-presenting cells can be activated by regulating the mevalonate metabolic pathway. Therefore, approved mevalonate metabolic inhibitors such as bisphosphonates (alendronate, zoledronic acid) can be used as immune metabolic regulatory molecules. Manganese ions (Mn 2+ ) can activate the cGAS-STING pathway in antigen-presenting cells (APCs) and induce the production of type I interferon (IFN-I). The combined use of the two can play an important synergistic role.

[0006] Utilizing the characteristics of two phosphate groups in bisphosphonate drugs, combining them with manganese ions (Mn 2+)They are combined through coordination, and bisphosphonate lipid nanoparticles can be prepared with the assistance of lipid materials. By using nanotechnology, targeted delivery to lymph nodes or tumor microenvironments can be achieved, thereby enhancing the targeted regulation of innate immune cells in vivo. Summary of the Invention

[0007] One object of the present invention is to provide a manganese bisphosphonate nanoparticle.

[0008] Another object of the present invention is to provide a method for preparing the manganese bisphosphonate nanoparticle.

[0009] A third object of the present invention is to apply the manganese bisphosphonate nanoparticle in combination with a cytotoxic drug capable of inducing immunogenic cell death (ICD) to the treatment of digestive tract tumors.

[0010] The above objects of the present invention are achieved by the following technical solutions:

[0011] One aspect of the present invention is to provide nanoparticles based on the coordination interaction of "bisphosphonate - manganese". Among them, the nanoparticles include a core structure and a lipid material wrapped around the outer layer of the core structure. The core structure includes manganese ions (Mn 2+ ), bisphosphonic acid, and dioleoyl phosphatidic acid (DOPA), and the outer lipid material includes dioleoyl phosphatidylcholine (DOPC), cholesterol, and methoxypolyethylene glycol 2000 - distearoylphosphoethanolamine (DSPE - mPEG2000).

[0012] The bisphosphonic acid described in the present invention is selected from any one or a mixture of more than one of zoledronic acid (Zole), sodium alendronate, sodium risedronate, and sodium pyrophosphate. Preferably, the bisphosphonic acid is a mixture of zoledronic acid and sodium pyrophosphate in a certain proportion.

[0013] The manganese ions described in the present invention refer to divalent manganese ions, selected from one or more of manganese nitrate and manganese chloride. Preferably, the manganese is manganese nitrate.

[0014] The core lipid material described in the present invention is selected from any one or a mixture of more than one of dioleoyl phosphatidic acid (DOPA), distearoyl phosphatidic acid (DSPA), and dipalmitoyl phosphatidic acid (DPPA). Preferably, the core lipid material is DOPA.

[0015] The mass ratio of manganese nitrate, zoledronic acid, sodium pyrophosphate, DOPA, DOPC, cholesterol, and DSPE - mPEG 2000 is (2 - 20):(10 - 50):(0 - 50):(10 - 40):(0 - 30):(0 - 30):(8 - 40). Preferably, the mass ratio of manganese nitrate, zoledronic acid, sodium pyrophosphate, DOPA, DOPC, cholesterol, and DSPE-mPEG2000 is (4 - 10):(10 - 20):(5 - 20):(10 - 20):(10 - 20):(5 - 20):(8 - 20). More preferably, the mass ratio of manganese nitrate, zoledronic acid, sodium pyrophosphate, DOPA, DOPC, cholesterol, and DSPE-mPEG2000 is 2:12:11:10:14:1:8.

[0016] On the other hand, the present invention provides a method for preparing the manganese bisphosphonate nanoparticles, comprising the following steps: dropping an oil-in-water emulsion system of bisphosphonate into an oil-in-water emulsion system of manganese ions to establish a coordination framework, adding a certain proportion of lipid materials after curing with absolute ethanol, and performing vortex hydration and probe sonication dispersion by the thin film dispersion method to obtain the product. Preferably, the preparation method of the present invention comprises the following steps:

[0017] (1) Slowly drop the manganese ion solution into a mixed solvent of CA-630 and cyclohexane, and stir to form an oil-in-water emulsion system of manganese. (2) Slowly drop the bisphosphonate solution and sodium pyrophosphate solution into a mixed solvent of CA-630 and cyclohexane, and then slowly drop a chloroform solution containing DOPA, and stir to form an oil-in-water emulsion system of bisphosphonate. (3) Drop the oil-in-water emulsion system of bisphosphonate into the stirring oil-in-water emulsion system of manganese ions, and continue to stir. (4) Add absolute ethanol to the reaction system for curing, and continue to stir. (5) Centrifuge the reaction solution, discard the supernatant, wash twice with absolute ethanol, add chloroform solutions of DOPC, cholesterol, and DSPE-mPEG 2000 respectively, perform vortex hydration by the thin film dispersion method, perform probe sonication dispersion, and add a glucose solution to adjust the osmotic pressure.

[0018] As a preferred technical solution, step (1) of the present invention can specifically adopt the following steps: The concentration of manganese nitrate is 23.67 mg / mL, and the volume is 30 μL. The volume of the mixed solvent of CA-630 and cyclohexane is 10 mL, wherein the volume ratio of CA-630 to cyclohexane is (30 - 40):(60 - 70). During the dropping process, continuous vigorous stirring is carried out, and after the dropping is completed, continue to stir for 30 min.

[0019] As a preferred technical solution, step (2) of the present invention may specifically adopt the following steps: the concentration of the zoledronic acid solution is 29.0 mg / mL, the pH value is adjusted to 7 - 8 using hydrochloric acid, and the added volume is 150 μL; the concentration of sodium pyrophosphate is 26.6 mg / mL, and the added volume is 150 μL; the concentration of the DOPA chloroform solution is 11.5 mg / mL, and the added volume is 300 μL; the volume of the mixed solvent is 10 mL, wherein the volume ratio of CA - 630 to cyclohexane is (30 - 40):(60 - 70). During the dropping process, continuous vigorous stirring is carried out, and after the dropping is completed, stirring is continued for 30 min.

[0020] As a preferred technical solution, step (3) of the present invention may specifically adopt the following steps: the bisphosphonate dispersion system is slowly dropped into the manganese system. During the dropping process, continuous vigorous stirring is carried out, and after the dropping is completed, stirring is continued for 30 min.

[0021] As a preferred technical solution, step (4) of the present invention may specifically adopt the following steps: 20 mL of absolute ethanol is added. During the dropping process, continuous vigorous stirring is carried out, and after the dropping is completed, stirring is continued for 20 min.

[0022] As a preferred technical solution, step (5) of the present invention may specifically adopt the following steps: the concentration of the DOPC chloroform solution is 5 mg / mL, and the added volume is 1 mL; the concentration of the cholesterol chloroform solution is 1.8 mg / mL, and the added volume is 200 μL; the concentration of the DSPE - mPEG 2000 chloroform solution is 13.5 mg / mL, and the added volume is 200 μL; the rotary evaporation conditions are a 60°C water bath, a rotation speed of 100 rpm / min, and rotary evaporation for 15 min. Immediately after the rotary evaporation is completed, 2 mL of deionized water preheated to 60°C is used, and vigorous vortex oscillation is carried out; the probe ultrasonic conditions are a power of 50 w, 3 s on, 3 s off, lasting for 5 min, and cooling is carried out in an ice - water bath.

[0023] The manganese bisphosphonate nanoparticles of the present invention have small particle size, nearly spherical appearance, basically uniform particle size distribution, good stability, and pH - sensitive release characteristics. At the same time, the materials used in the preparation of the nanoparticles have good biocompatibility and the preparation process is simple.

[0024] The present invention experimentally investigated the in vitro biological effects of the manganese bisphosphonate nanoparticles on antigen - presenting cells. The results proved that the constructed nanoparticles have significant immunomodulatory effects, can promote the maturation of DC cells, and stimulate DC cells to increase the secretion of inflammatory cytokines.

[0025] In this invention, the in vitro biological effects of manganese bisphosphonate nanoparticles combined with oxaliplatin (Oxp) on tumor cells were investigated through experiments. The results demonstrated that the constructed nanoparticles enhanced the ICD effect and cytotoxicity of Oxp by directly acting on tumor cells.

[0026] In this invention, the in vivo pharmacodynamic effects of manganese bisphosphonate nanoparticles combined with Oxp were investigated through experiments. The results demonstrated that the constructed nanoparticles could effectively inhibit the growth of digestive tract tumors and distant metastatic tumors by improving the tumor immune microenvironment and synergizing with Oxp, and could further reduce the dosage of Oxp, and had good in vivo safety. Preferably, the digestive tract tumor is colon cancer.

[0027] The abbreviated technical terms involved in this invention are further explained and described as follows:

[0028] NK cell: natural killer cell

[0029] cGAS: cyclic GMP-AMP synthase

[0030] STING: stimulator of interferon genes

[0031] TLR: Toll-like receptor

[0032] PD-1: programmed death receptor-1

[0033] PDL-1: programmed death ligand-1

[0034] Mn 2+ : manganese ion

[0035] APC: antigen-presenting cell

[0036] IFN-Ⅰ: type Ⅰ interferon

[0037] ICD: immunogenic cell death

[0038] DOPA: dioleoyl phosphatidic acid

[0039] DOPC: dioleoyl phosphatidylcholine

[0040] DSPE-mPEG 2000 : methoxypolyethylene glycol 2000-distearoylphosphoethanolamine

[0041] Zole: zoledronic acid

[0042] CA-630: polyoxyethylene octylphenol ether

[0043] DC cell: dendritic cell

[0044] Oxp: oxaliplatin

[0045] DLS: Dynamic Light Scattering

[0046] TEM: Transmission Electron Microscope

[0047] Treg cell: Regulatory T cell

[0048] CTL cell: Cytotoxic T Lymphocyte Brief Description of the Drawings

[0049] Figure 1 Schematic diagram of the preparation of the manganese bisphosphonate nanoparticles of the present invention.

[0050] Figure 2 Particle size diagram of the manganese bisphosphonate nanoparticles of the present invention.

[0051] Figure 3 Potential diagram of the manganese bisphosphonate nanoparticles of the present invention.

[0052] Figure 4 Transmission electron microscope image of the manganese bisphosphonate nanoparticles of the present invention.

[0053] Figure 5 Storage stability diagram of the manganese bisphosphonate nanoparticles of the present invention. Figure a shows the storage stability at 4°C, and Figure b shows the storage stability at room temperature.

[0054] Figure 6 In vitro release curve of Mn of the manganese bisphosphonate nanoparticles of the present invention in different pH media 2+

[0055] Figure 7 In vitro biological effects of the manganese bisphosphonate nanoparticles of the present invention on antigen-presenting cells. Figure a shows the effect of the nanoparticles (NPs) on the co-stimulatory molecule CD86 on the surface of BMDC cells, Figure b shows the effect of the nanoparticles (NPs) on CD80 / CD86 on the surface of BMDC cells, Figure c shows the effect of the nanoparticles (NPs) on the antigen-presenting complex MHC-II of BMDC cells, Figure d shows the effect of the nanoparticles (NPs) on the secretion of the inflammatory cytokine IL-1β by BMDC cells, and Figure e shows the effect of the nanoparticles (NPs) on the secretion of the inflammatory cytokine IFN-β by BMDC cells.

[0056] Figure 8 ICD effect of the combination of the manganese bisphosphonate nanoparticles of the present invention and oxaliplatin (Oxp) on colon cancer CT-26 cells. Figure a shows the effect of the nanoparticles (NPs) on the distribution of calreticulin (CRT) on the surface of CT-26 cell membranes, and Figure b shows the effect of the nanoparticles (NPs) on the distribution of high mobility group box 1 (HMGB1) protein in the nuclei of CT-26 cells.

[0057] ​Figure 9 In vitro cytotoxic effect of manganese bisphosphonate nanoparticles combined with oxaliplatin (Oxp) of the present invention on colon cancer CT-26 cells.

[0058] Figure 10 Pharmacodynamics of manganese bisphosphonate nanoparticles combined with oxaliplatin (Oxp) of the present invention on subcutaneous tumor-bearing model of colon cancer CT-26 cells. Figure a is a schematic diagram of the establishment of subcutaneous tumor-bearing mouse model and the administration scheme, Figure b is the curve of tumor volume changing with time, Figure c is the photo of the tumor dissected at the end of the experiment, and Figure d is the tumor weight graph at the end of the experiment.

[0059] Figure 11 Effect of manganese bisphosphonate nanoparticles combined with oxaliplatin (Oxp) of the present invention on lymphocyte subsets in CT-26 tumor tissues. Figure a is the analysis result of CD8 + T cells in mouse tumors, and Figure b is the ratio of the number of CD8 + T cells to CD4 + T cells, and Figure c is the analysis result of Treg cells infiltrating in mouse tumors.

[0060] Figure 12 Change of animal body weight with time during the treatment of CT-26 colon cancer with manganese bisphosphonate nanoparticles combined with oxaliplatin (Oxp) of the present invention.

[0061] Figure 13 Pharmacodynamics of manganese bisphosphonate nanoparticles combined with oxaliplatin (Oxp) of the present invention on distal metastasis model of colon cancer. Figure a is a schematic diagram of the establishment of CT-26 primary tumor and distal tumor models and the administration scheme, Figure b is the curve of primary tumor volume changing with time, Figure c is the curve of distal tumor volume changing with time, Figure d is the distal tumor weight graph at the end of the experiment, and Figure e is the photo of the distal tumor dissected at the end of the experiment.

[0062] Figure 14 Blood routine results of mice at the end of the experiment in the treatment of distal metastasis model of colon cancer with manganese bisphosphonate nanoparticles combined with oxaliplatin (Oxp) of the present invention. Figure a is the number of white blood cells in mice at the end of the experiment, Figure b is the number of lymphocytes in mice at the end of the experiment, Figure c is the number of neutrophils in mice at the end of the experiment, Figure d is the number of red blood cells in mice at the end of the experiment, Figure e is the hemoglobin concentration in mice at the end of the experiment, and Figure f is the number of platelets in mice at the end of the experiment.

[0063] Figure 15 Liver function results of mice at the end of the experiment in the treatment of distal metastasis model of colon cancer with manganese bisphosphonate nanoparticles combined with oxaliplatin (Oxp) of the present invention. Figure a is alanine aminotransferase in the blood of mice at the end of the experiment, Figure b is aspartate aminotransferase in the blood of mice at the end of the experiment, Figure c is total protein in the blood of mice at the end of the experiment, Figure d is albumin in the blood of mice at the end of the experiment, and Figure e is globulin in the blood of mice at the end of the experiment.

[0064] Figure 16 Results of renal function of mice at the experimental endpoint of the model of distal metastasis of colon cancer treated with manganese bisphosphonate nanoparticles combined with oxaliplatin (Oxp). Panel a shows creatinine in the blood of mice at the experimental endpoint, panel b shows blood urea nitrogen in mice at the experimental endpoint, and panel c shows uric acid in the blood of mice at the experimental endpoint. Detailed implementation manners

[0065] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. For the experimental methods without specific conditions noted in the description of the embodiments, they are generally carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturer. Those skilled in the art should understand that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention. Example 1 Preparation of manganese bisphosphonate nanoparticles (1) Preparation of the water-in-oil emulsion system of manganese: Slowly drop the manganese nitrate solution into the mixed solvent of CA-630 and cyclohexane, and stir to form the water-in-oil emulsion system of manganese. Among them, the concentration of manganese nitrate is 23.67 mg / mL, the volume is 30 μL, the volume of the mixed solvent of CA-630 and cyclohexane is 10 mL; the volume ratio of CA-630 to cyclohexane is 35:65, continuously stir vigorously during the dropping process, and continue to stir for 30 min after the dropping is completed; (2) Water-in-oil emulsion system of bisphosphonate: Slowly drop the zoledronic acid solution and sodium pyrophosphate solution into the stirring mixed solvent of CA-630 and cyclohexane, and then slowly drop the chloroform solution containing dioleoyl phosphatidic acid (DOPA) into the mixed solvent, and stir to form the water-in-oil emulsion system of bisphosphonate. Among them, the concentration of the zoledronic acid solution is 29.0 mg / mL, the pH value is adjusted to 7-8 with hydrochloric acid, and the volume of the added zoledronic acid solution is 150 μL; the concentration of sodium pyrophosphate is 26.6 mg / mL, and the added volume is 150 μL; the concentration of the DOPA chloroform solution is 11.5 mg / mL, and the added volume is 300 μL; the volume of the mixed solvent of CA-630 and cyclohexane is 10 mL, among which the volume ratio of CA-630 to cyclohexane is 35:65, continuously stir vigorously during the dropping process, and continue to stir for 30 min after the dropping is completed; (3) The water-in-oil emulsion system of bisphosphonate was dropped into the water-in-oil emulsion system of manganese ions under stirring. During the dropping process, continuous vigorous stirring was carried out, and after the dropping was completed, stirring was continued for 30 min; (4) 20 mL of absolute ethanol was added to the reaction system for solidification. During the dropping process, continuous vigorous stirring was carried out, and after the dropping was completed, stirring was continued for 20 min; (5) The reaction solution was centrifuged, the supernatant was discarded, and it was washed twice with absolute ethanol. Chloroform solutions containing DOPC, cholesterol, and DSPE-mPEG 2000 were added respectively. After vortex hydration by the thin-film dispersion method and probe ultrasonic dispersion, a glucose solution was added to adjust the osmotic pressure. Among them, the concentration of the chloroform solution of DOPC was 5 mg / mL, and the added volume was 1 mL; the concentration of the chloroform solution of cholesterol was 1.8 mg / mL, and the added volume was 200 μL; the concentration of the chloroform solution containing DSPE-mPEG 2000 was 13.5 mg / mL, and the added volume was 200 μL; the rotary evaporation conditions were a water bath at 60 °C, a rotation speed of 100 rpm / min, and rotary evaporation for 15 min. Immediately after the rotary evaporation was completed, 2 mL of deionized water preheated at 60 °C was added and vigorously shaken and vortexed; the probe ultrasonic conditions were a power of 50 w, turned on for 3 s and turned off for 3 s, lasting for 5 min, and cooled in an ice-water bath. The schematic diagram of the specific preparation process is shown in Figure 1 ; Particle size characterization of manganese bisphosphonate nanoparticles in Example 2 1. Test materials: Manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test method: The particle size and polydispersity coefficient of manganese bisphosphonate nanoparticles were measured by dynamic light scattering (DLS). The nanoparticles were redispersed by resuspension with PBS solution, and the parameters of the Malvern Zetasizer Nano ZS laser particle size analyzer were set as follows: the laser beam wavelength was 633 nm, and the angle between the incident beam and the scattered beam was 90°. Each sample was measured for 7 cycles, the equilibration time was set to 10 seconds, and the measurement was repeated 3 times. The measurement temperature was set to 25 °C, and 3 samples were measured in parallel for each group. 3. Test results: The particle size distribution diagram of manganese bisphosphonate nanoparticles measured by dynamic light scattering (DLS) showed a single peak. The average particle size was 87.08 ± 2.30 nm, and the polydispersity coefficient was 0.27 ± 0.01. The results are shown in Figure 2 . Potential characterization of manganese bisphosphonate nanoparticles in Example 3 1. Test materials: Manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test method: After diluting the prepared manganese bisphosphonate nanoparticle sample 10 times with ultrapure water, take 1 mL and add it to the potentiometric cup, and use a Malvern Zetasizer Nano ZS laser particle size analyzer to measure the Zeta potential. Each sample is measured for 9 cycle times, the sample equilibrium time is 120 s, and the measurement temperature is set at 25 °C. 3. Test results: The Zeta potential of the nanoparticles is -32.70 ± 2.08 mV, and the results are shown in Figure 3 . Transmission electron microscopy image of manganese bisphosphonate nanoparticles in Example 4 1. Test materials: Manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test method: Characterize the morphology of manganese bisphosphonate nanoparticles by transmission electron microscopy (TEM). Use ultrapure water as the dispersion medium to dilute the manganese bisphosphonate nanoparticles to a lipid concentration of 0.5 mg / mL. Take a drop of the nanoparticle solution and drop it on the PARA film. Place the copper grid coated with a carbon film on the test solution (film side up). After 10 min, take out the copper grid and blot the excess liquid from the edge of the copper grid with filter paper. Then, place the copper grid on the phosphotungstic acid staining solution in the same way for about 90 s, and also blot the excess liquid. After drying the copper grid, put it into the TEM for observation. 3. Test results: The nanoparticles are spherical in appearance, and the particle size is about 50 - 70 nm, and the results are shown in Figure 4 . Characterization of the encapsulation efficiency of manganese bisphosphonate nanoparticles in Example 5 1. Test materials: Manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test method: Quantitatively sample 100 μL of the prepared manganese bisphosphonate nanoparticle sample and transfer it to an ultrafiltration centrifugal tube with a cut-off molecular weight of 3 kD. Centrifuge at 14000 g and 25 °C for 15 min, collect 50 μL of the ultrafiltrate, add 1 mL of concentrated HNO3 and digest it overnight at room temperature, then dilute it to 10 mL with pure water, and use an inductively coupled plasma mass spectrometer (ICP-MS) to measure the Mn content in the ultrafiltrate as the free Mn mass in the nanoparticles. Then continue to use ICP-MS to measure the total Mn mass in the nanoparticle solution, and further calculate the encapsulation efficiency of manganese based on this. 3. Test results: The encapsulation efficiency of manganese in manganese bisphosphonate nanoparticles is about 75.1 ± 2.3%. Characterization of the stability of manganese bisphosphonate nanoparticles in Example 6 1. Test materials: Manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test method: Divide the same prepared sample of manganese bisphosphonate nanoparticles into two portions, and place them at 4°C and room temperature for 15 days respectively. During this period, use a Zetasizer Nano ZS laser particle size analyzer to measure their particle size every day, and measure in parallel three times each time. 3. Test results: After observing for 15 days at normal temperature and 4°C respectively, the particle size of the manganese bisphosphonate nanoparticles did not change significantly, and no obvious aggregation phenomenon occurred, proving its good stability. The results are shown in Figure 5 . Example 7 In vitro release characterization of manganese bisphosphonate nanoparticles 1. Test materials: The manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test method: Quantitatively sample 0.5 mL of the prepared manganese bisphosphonate nanoparticle sample and add it to a dialysis bag (cut-off molecular weight of 3.5 kD). Clamp both ends of the dialysis bag with dialysis clips, and place it in a conical flask containing 40 mL of release medium (PBS phosphate buffer with pH values of 7.4 and 5.5 respectively). After sealing, place it in a shaker at 37°C and oscillate at a rate of 100 rpm. At 1, 2, 4, 6, 12, 24, and 48 h, aspirate 500 μL of the release solution, and at the same time supplement 500 μL of fresh release medium to the conical flask and continue oscillating for release. After the release is completed, loosen the dialysis clips clamped at both ends of the dialysis bag to fully mix the preparation solution with the release medium, continue oscillating for 0.5 h, stir evenly, and aspirate 500 μL of the release solution as the drug release amount at infinite time. Use ICP-MS to measure the mass of Mn in the release samples at each time point and calculate the drug release rate. 3. Test results: The nanoparticles showed different release characteristics in different pH environments. They were relatively stable at normal physiological pH, while they had sensitive release ability in the acidic environment of tumor tissues. The results are shown in Figure 6 . Example 8 Immunostimulatory effect of manganese bisphosphonate nanoparticles on BMDC cells 1. Test materials: The manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test method: Seed BMDC cells in a 24-well cell culture plate at a density of 8×10 5 cells per well, with every 4 wells as a group. Add Mn(500 μM)+Zole(70 μM) and manganese bisphosphonate nanoparticles (Mn 2+The concentration was 500 μM). After culturing the cells for another 24 h, the cell suspension was collected, centrifuged at 2000 rpm for 5 min, and the cell culture medium was collected. The contents of interleukin-1β (IL-1β) and interferon-β (IFN-β) secreted by the cells were detected using an IL-1β and IFN-β ELISA kit according to the steps in the manufacturer's instructions. The above cells were resuspended in 100 μL of PBS solution, and specific-labeled antibodies were added thereto: 1.25 μL of APC-labeled anti-mouse CD11c antibody, 2.5 μL of PE / Cyanine7-labeled anti-mouse CD80 antibody, 2 μL of FITC-labeled anti-mouse CD86 antibody, and 1.25 μL of PE-labeled anti-mouse MHC II antibody. At the same time, single-labeled tube samples and blank control tube samples were also set as controls. After incubating the above samples in the dark on ice for 20 min, the cells were washed 3 times with PBS solution to remove excess antibodies and non-specific binders. Subsequently, the cells were resuspended in 300 μL of PBS solution and filtered through a 300-mesh cell strainer to remove possible cell clumps and impurities. Finally, the treated cells were detected using a flow cytometer. 3. Test results: Compared with the group co-administered with Mn 2+ and Zole, the manganese bisphosphonate nanoparticle group could further upregulate the expression of co-stimulatory molecules CD86 and CD80 / CD86 and the antigen presentation complex MHC-II on the surface of BMDC cells, and significantly increase the secretion of inflammatory cytokines IL-1β and IFN-β. The above results indicate that the nanoparticles have a significant immunomodulatory effect, and the results are shown in Figure 7 . Example 9 Induction of ICD effect of manganese bisphosphonate nanoparticles combined with oxaliplatin on CT-26 cells 1. Test materials: Manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test method: CT-26 cells were seeded into a confocal-specific eight-well chamber at a density of 2 × 10 4 cells per well. After culturing for 48 h, manganese bisphosphonate nanoparticles (NPs, Mn 2+Concentrations of 500 μM), Oxp (100 μM), and Oxp + NPs, where the concentrations of each component in the combination group were the same as those in the single-drug groups. Another control group (only adding cell culture medium) was set up and cultured for another 24 h. After the incubation, cells were washed three times with cold PBS, then treated with 200 μL of 4% paraformaldehyde for 20 min, and then washed three times again with cold PBS. After washing, the cells were placed in an immunological rapid blocking solution containing Triton for 15 min of blocking treatment, and then incubated overnight at 4 °C in the dark with the working solution of Anti-Calreticulin or Anti-HMGB1 primary antibody. After the incubation, the cells were washed five times with cold PBS to remove the excess primary antibody, and then continued to be incubated for 1 h in the dark with Alexa 647-labeled goat anti-rabbit IgG H&L. Washed five times again with cold PBS, and then stained with Hoechst 33342 for 15 min to label the cell nuclei. Finally, the distribution of cell CRT or HMGB1 was observed by a laser scanning confocal microscope. 3. Test results: When manganese bisphosphonate nanoparticles (NPs) were combined with Oxp, the fluorescence intensity of CRT on the cell membrane surface in the combination group increased significantly, and the fluorescence intensity of HMGB1 in the cell nucleus decreased further. The above results indicate that manganese bisphosphonate nanoparticles (NPs) can further enhance the ICD effect of Oxp on tumor cells. The results are shown in Figure 8 . Example 10 In vitro cytotoxic effect of manganese bisphosphonate nanoparticles combined with oxaliplatin on CT-26 cells 1. Test materials: Manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test method: CT-26 cells were taken and prepared into a cell suspension of 6×10 4 cells / mL with RPMI-1640 culture medium, and then inoculated into 96-well plates at 180 μL / well, with six replicates. Incubated in a carbon dioxide incubator for 24 h until the cells adhered and grew. Then, 20 μL of different concentrations of drugs or manganese bisphosphonate nanoparticles (NPs) were added to the corresponding cell culture wells respectively. Another six replicates were set for the control group and the blank reference wells. The 96-well plates were incubated in a carbon dioxide incubator for 24 h. After the incubation, 100 μL of cell culture medium containing 10% Alamar Blue was added to each well and incubated at 37 °C for another 2.5 h. After the incubation, the absorbance value of each well was measured at 530 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the average cell survival rate of each group of cells was calculated. 3. Test results: When manganese bisphosphonate nanoparticles (NPs) were combined with Oxp, the toxic effect on CT-26 cells was further enhanced; and with the increase of the dosing concentration of NPs, the killing effect on cells gradually increased. The results are shown in Figure 9 . Experimental Example 11 Pharmacodynamics of Manganese Bisphosphonate Nanoparticles Combined with Oxaliplatin on Subcutaneous Tumor-bearing Model of Colon Cancer CT-26 Cells 1. Test Materials: Manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test Methods: (1) Establishment of Tumor-bearing Mouse Model: CT26 (murine colon cancer cells) cells were cultured in RPMI-1640 medium containing 10% FBS. Before use, the cells were digested with 0.25% trypsin digestion solution, centrifuged at 1000 rpm for 3 min, the supernatant was taken out, and the cells were resuspended in serum-free RPMI-1640 medium. The cell concentration was adjusted to 1.0×10 5 cells / 100 μL, and then the cell suspension was inoculated subcutaneously into the right axilla of each BALB / c female mouse (6 - 8 weeks old, body weight 18 - 22 g) at a volume of 100 μL per mouse using a 1 mL syringe. (2) Drug Administration Scheme: 7 days after inoculation, the subcutaneous tumor volume of the mice grew to approximately 50 mm 3 , and the mice were randomly divided into 5 groups with 6 mice in each group. The following 5 groups of preparations were intravenously injected on the 8th, 13th, and 18th days after tumor inoculation: ① 5% glucose injection (glucose), ② low-concentration oxaliplatin injection (Oxp 2.5 mg / kg), ③ oxaliplatin injection (Oxp 5 mg / kg), ④ low-concentration oxaliplatin injection (Oxp 2.5 mg / kg), ⑤ oxaliplatin injection (Oxp 5 mg / kg); meanwhile, the fourth and fifth groups were intravenously injected with manganese bisphosphonate nanoparticles (NPs, Mn 2+ 0.5 mg / kg) on the 8th day after tumor inoculation. (3) Monitoring Indicators: The signs and behavioral activities of the mice were monitored, and the long and short diameters of the tumors in each group were measured using vernier calipers to calculate the tumor volume V (V = [length × (width) 2 / 2), and a tumor volume-time change graph was plotted; the body weight of the mice was measured every 1 - 2 days, and a body weight-time change curve was plotted. (4) Endpoint Detection Indicators: The animals were sacrificed on the 18th day after tumor inoculation, the tumors were dissected, and the tumor weights were weighed. The tumor tissues of each group of mice were gently minced with ophthalmic scissors, placed in a tissue grinder, homogenization rinsing solution was added, ground, passed through a 70 μm cell sieve, centrifuged at 2000 rpm for 5 min, and the tumor cells were collected. The leukocytes in the tumor cell suspension were separated using a mouse tumor-infiltrating tissue leukocyte separation kit, washed twice with PBS and then counted, and the cell suspension was diluted to 10 7cells / mL. Take 100 μL and place it in a 1.5 mL centrifuge tube. Add the required fluorescently labeled flow antibodies. At the same time, set up single-label tube samples and blank control tube samples, and incubate in the dark on ice for 20 min. Wash the cells 3 times with PBS, and finally resuspend the cells with 300 μL of PBS. After filtering through a 300-mesh cell sieve, perform flow cytometry detection. 3. Test results: (1) The tumor volume-time change curve is shown in Figure 10 b, and the anatomical results of the terminal tumors are shown in Figure 10 c. The results show that after systemic delivery of manganese bisphosphonate nanoparticles (NPs), the therapeutic effect of oxaliplatin (Oxp) can be significantly improved. The tumor volume in the group treated with nanoparticles combined with oxaliplatin is significantly smaller than that in the group treated with oxaliplatin alone. (2) The results of the tumor weight at the experimental endpoint are shown in Figure 10 d. The results show that after systemic delivery of manganese bisphosphonate nanoparticles (NPs), the growth of tumors can be significantly inhibited. The tumor weight in the group treated with nanoparticles combined with oxaliplatin is significantly lower than that in the group treated with oxaliplatin alone. (3) The changes in lymphocyte subsets in tumor tissues at the experimental endpoint are shown in Figure 11 . The results show that in the tumor tissues of the group treated with manganese bisphosphonate nanoparticles (NPs) combined with oxaliplatin, the ratio of CD8 + T cells and CD8 + / CD4 + T cells increased significantly, and the ratio of regulatory T cells (Treg cells) decreased significantly, indicating that manganese bisphosphonate nanoparticles (NPs) can increase the infiltration of cytotoxic T lymphocytes (CTL cells) in tumor tissues and reduce Treg cells, thereby significantly improving the tumor immune microenvironment. (4) The mouse body weight-time change curve is shown in Figure 12 , and the results show that after systemic delivery of manganese bisphosphonate nanoparticles (NPs), no significant decrease in animal body weight was observed. Pharmacodynamics of Manganese Bisphosphonate Nanoparticles Combined with Oxaliplatin on Colon Cancer Distant Metastasis Model in Test Example 12 1. Test materials: Manganese bisphosphonate nanoparticles prepared in Example 1. 2. Test methods: (1) Establishment of primary tumor and distant tumor models: CT-26 cells were cultured in RPMI-1640 medium containing 10% FBS. Before use, the cells were digested with digestive fluid, centrifuged at 1000 rpm for 4 min, the supernatant was taken out, and the cells were resuspended in serum-free RPMI-1640 medium. The cell concentration was adjusted to 1×10 5cells / 50 μL, and then use a 1 mL syringe to inoculate the cell suspension into the right axillary subcutaneous area of each BALB / c mouse at a liquid volume of 50 μL per mouse to construct a primary tumor. 10 days after inoculation, when the volume of the primary tumor grows to approximately 100 mm 3 , inoculate 1×10 5 cells / 50 μL subcutaneously in the axillary area on the other side (left side) of the mouse to construct a distant tumor. (2) Drug administration plan: 9 days after inoculation, randomly divide the mice into 5 groups with 6 mice in each group. Inject the following 5 groups of drugs intravenously on the 9th, 14th, and 19th days after the primary tumor inoculation: ① 5% glucose injection (glucose), ② low-concentration oxaliplatin injection (Oxp 2.5 mg / kg), ③ oxaliplatin injection (Oxp 5 mg / kg), ④ low-concentration oxaliplatin injection (Oxp 2.5 mg / kg), ⑤ oxaliplatin injection (Oxp 5 mg / kg); meanwhile, on the 9th day after the primary tumor inoculation, inject manganese bisphosphonate nanoparticles (NPs, Mn 2+ 0.5 mg / kg) intravenously in groups ④ and ⑤. (3) Monitoring indicators: Monitor the signs and behavioral activities of the mice, and use vernier calipers to measure the long and short diameters of the tumors in each group, calculate the volumes V of the primary tumor and the distant tumor (V = [length × (width) 2 / 2), and draw a tumor volume-time change graph. (4) Endpoint detection indicators: Sacrifice the animals on the 24th day after the primary tumor inoculation, take 100 μL and 200 μL of venous blood from the orbital cavities of each mouse respectively, and perform a blood routine examination using a fully automatic three-class hematology analyzer. The detection indicators include white blood cells (White blood cells, WBC), red blood cells (Red blood cells, RBC), platelets (Platelet, PLT), lymphocytes (Lymphocyte, LYM), neutrophils (Granulocyte, GRA), hemoglobin (Hemoglobin, HGB); perform a blood biochemical examination using a fully automatic biochemical analyzer. The liver function indicators include aspartate aminotransferase (Aspartate Aminotransferase, AST), alanine transaminase (Alanine Transaminase, ALT), total protein (Total Protein, TP), albumin (Albumin, ALB), globulin (Globulin, GLB), and the kidney function indicators include creatinine (Creatinine, CREA), blood urea nitrogen (Blood Urea Nitrogen, BUN), and uric acid (Uric Acid, UA). 3. Experimental results: (1) The volume-time change curve of the primary tumor is shown in Figure 13 b. The results show that the combined use of manganese bisphosphonate nanoparticles (NPs) and Oxp has a better effect on inhibiting the growth of the primary tumor. (2) The volume-time change curve of the distal tumor is shown in Figure 13 c. The results of the distal tumor weight at the experimental endpoint are shown in Figure 13 d. The anatomical results of the distal tumor at the endpoint are shown in Figure 13 e. The results show that the combined use of manganese bisphosphonate nanoparticles (NPs) and Oxp exhibits a synergistic antitumor therapeutic effect and can inhibit the distally metastatic tumors. (3) The blood routine results of the mice at the experimental endpoint are shown in Figure 14 . The results show that manganese bisphosphonate nanoparticles (NPs) have no obvious effect on the blood routine indexes of mice. (4) The liver function indexes of the mice at the experimental endpoint are shown in Figure 15 . The results show that manganese bisphosphonate nanoparticles (NPs) have no obvious effect on the liver function indexes of mice. (5) The kidney function indexes of the mice at the experimental endpoint are shown in Figure 16 . The results show that manganese bisphosphonate nanoparticles (NPs) have no obvious effect on the kidney function indexes of mice. Example 13. Preparation of Manganese Bisphosphonate Nanoparticles 1. Prescription mass ratio 2. Preparation method: (1) Preparation of the water-in-oil emulsion system of manganese: Slowly drop the manganese nitrate solution into the mixed solvent of CA-630 and cyclohexane and stir to form the water-in-oil emulsion system of manganese. Among them, the concentration of manganese nitrate is 23.67 mg / mL, the volume is 300 μL, and the volume of the mixed solvent is 10 mL; The volume ratio of CA-630 to cyclohexane is 30:70. Keep stirring vigorously during the dropping process and continue to stir for 30 min after the dropping is completed. (2) Water-in-oil emulsion system of bisphosphonate: Slowly drop the zoledronic acid solution and sodium pyrophosphate solution into the stirring In a mixed solvent of CA-630 and cyclohexane, a chloroform solution of dioleoylphosphatidic acid (DOPA) was then slowly added dropwise, and stirred to form a water-in-oil emulsion system of bisphosphonate. Among them, the concentration of zoledronic acid solution was 29.0 mg / mL, the pH value was adjusted to 7-8 with hydrochloric acid, and the added volume was 600 μL; the concentration of sodium pyrophosphate was 26.6 mg / mL, and the added volume was 600 μL; the concentration of DOPA chloroform solution was 11.5 mg / mL, and the added volume was 1.2 mL; the volume of the mixed solvent was 10 mL, among which the volume ratio of CA-630 to cyclohexane was 30:70. During the dropping process, continuous vigorous stirring was carried out, and after the dropping was completed, stirring was continued for 30 min. (3) The water-in-oil emulsion system of bisphosphonate was added dropwise to the water-in-oil emulsion system of manganese ions under stirring. During the dropping process, continuous vigorous stirring was carried out, and after the dropping was completed, stirring was continued for 30 min. (4) 20 mL of absolute ethanol was added to the reaction system for solidification. During the dropping process, continuous vigorous stirring was carried out, and after the dropping was completed, stirring was continued for 20 min. (5) The reaction solution was centrifuged, the supernatant was discarded, and it was washed twice with absolute ethanol. Chloroform solutions of DOPC, cholesterol, and DSPE-mPEG 2000 were respectively added. After vortex hydration by the thin film dispersion method, probe sonication was carried out, and a glucose solution was added to adjust the osmotic pressure. Among them, the concentration of the DOPC chloroform solution was 5 mg / mL, and the added volume was 2 mL; the concentration of the cholesterol chloroform solution was 8.0 mg / mL, and the added volume was 1.2 mL; the concentration of the DSPE-mPEG 2000 chloroform solution was 13.5 mg / mL, and the added volume was 1 mL; the rotary evaporation conditions were a water bath at 60 °C, a rotation speed of 100 rpm / min, and rotary evaporation for 15 min. Immediately after the rotary evaporation was completed, 2 mL of deionized water preheated at 60 °C was used for vigorous shaking and vortexing; the probe sonication conditions were a power of 50 w, 3 s on, 3 s off, for 5 min, and the temperature was lowered in an ice-water bath to obtain the product. Preparation of manganese bisphosphonate nanoparticles in Example 14 1. Prescription mass ratio 2. Preparation method: (1) Preparation of the water-in-oil emulsion system of manganese: The manganese nitrate solution was slowly added dropwise to a mixed solvent of CA-630 and cyclohexane, and stirred to form a water-in-oil emulsion system of manganese. Among them, the concentration of manganese nitrate was 23.67 mg / mL, the volume was 30 μL, and the volume of the mixed solvent was 10 mL; the volume ratio of CA-630 to cyclohexane was 40:60. During the dropping process, continuous vigorous stirring was carried out, and after the dropping was completed, stirring was continued for 30 min. (2) Water-in-oil emulsion system of bisphosphonate: Slowly drop the zoledronic acid solution into the mixed solvent of CA-630 and cyclohexane under stirring, and then slowly drop the chloroform solution of dioleoyl phosphatidic acid (DOPA) to form a water-in-oil emulsion system of bisphosphonate by stirring. Among them, the concentration of the zoledronic acid solution is 29.0 mg / mL, the pH value is adjusted to 7 - 8 with hydrochloric acid, and the added volume is 150 μL; the concentration of the DOPA chloroform solution is 11.5 mg / mL, and the added volume is 300 μL; the volume of the mixed solvent is 10 mL, where the volume ratio of CA-630 to cyclohexane is 40:60. Keep stirring vigorously during the dropping process, and continue stirring for 30 min after the dropping is completed. (3) Drop the water-in-oil emulsion system of bisphosphonate into the water-in-oil emulsion system of manganese ions under stirring. Keep stirring vigorously during the dropping process, and continue stirring for 30 min after the dropping is completed. (4) Add 20 mL of absolute ethanol to the reaction system for solidification. Keep stirring vigorously during the dropping process, and continue stirring for 20 min after the dropping is completed. (5) Centrifuge the reacted solution, discard the supernatant, wash it twice with absolute ethanol, add the chloroform solutions of DOPC and DSPE-mPEG respectively, after vortex hydration by the thin film dispersion method, disperse it by probe sonication, and add glucose solution to adjust the osmotic pressure. Among them, the concentration of the DOPC chloroform solution is 5 mg / mL, and the added volume is 1 mL; the concentration of the DSPE-mPEG chloroform solution is 13.5 mg / mL, and the added volume is 200 μL; the rotary evaporation conditions are a water bath at 60 °C, a rotation speed of 100 rpm / min, and rotary evaporation for 15 min. Immediately after the rotary evaporation is completed, use 2 mL of deionized water preheated at 60 °C to shake it vigorously by vortex; the probe sonication conditions are a power of 50 w, 3 s on, 3 s off, for 5 min, and cool it down in an ice-water bath to obtain the product. 2000 (5) Centrifuge the reacted solution, discard the supernatant, wash it twice with absolute ethanol, add the chloroform solutions of DOPC and DSPE-mPEG respectively, after vortex hydration by the thin film dispersion method, disperse it by probe sonication, and add glucose solution to adjust the osmotic pressure. Among them, the concentration of the DOPC chloroform solution is 5 mg / mL, and the added volume is 1 mL; the concentration of the DSPE-mPEG 2000 chloroform solution is 13.5 mg / mL, and the added volume is 200 μL; the rotary evaporation conditions are a water bath at 60 °C, a rotation speed of 100 rpm / min, and rotary evaporation for 15 min. Immediately after the rotary evaporation is completed, use 2 mL of deionized water preheated at 60 °C to shake it vigorously by vortex; the probe sonication conditions are a power of 50 w, 3 s on, 3 s off, for 5 min, and cool it down in an ice-water bath to obtain the product. Preparation of manganese bisphosphonate nanoparticles in Example 15 1. Prescription mass ratio 2. Preparation method: (1) Preparation of the water-in-oil emulsion system of manganese: Slowly drop the manganese nitrate solution into the mixed solvent of CA-630 and cyclohexane, and stir to form a water-in-oil emulsion system of manganese. Among them, the concentration of manganese nitrate is 23.67 mg / mL, the volume is 150 μL, and the volume of the mixed solvent is 10 mL; the volume ratio of CA-630 to cyclohexane is 35:65. Keep stirring vigorously during the dropping process, and continue stirring for 30 min after the dropping is completed. (2) Water-in-oil emulsion system of bisphosphonate: The zoledronic acid solution and sodium pyrophosphate solution were slowly added dropwise to the stirred mixed solvent of CA-630 and cyclohexane, and then the dicholoyl phosphatidic acid (DOPA) chloroform solution was slowly added dropwise, and stirred to form a water-in-oil emulsion system of bisphosphonate. Among them, the concentration of the zoledronic acid solution was 29.0 mg / mL, the pH value was adjusted to 7-8 with hydrochloric acid, and the added volume was 300 μL; the concentration of sodium pyrophosphate was 26.6 mg / mL, and the added volume was 300 μL; the concentration of the DOPA chloroform solution was 11.5 mg / mL, and the added volume was 600 μL; the volume of the mixed solvent was 10 mL, among which the volume ratio of CA-630 to cyclohexane was 35:65. During the dropping process, continuous vigorous stirring was carried out, and after the dropping was completed, stirring was continued for 30 min. (3) The water-in-oil emulsion system of bisphosphonate was added dropwise to the stirred water-in-oil emulsion system of manganese ions. During the dropping process, continuous vigorous stirring was carried out, and after the dropping was completed, stirring was continued for 30 min. (4) 20 mL of absolute ethanol was added to the reaction system for solidification. During the dropping process, continuous vigorous stirring was carried out, and after the dropping was completed, stirring was continued for 20 min. (5) The reacted solution was centrifuged, the supernatant was discarded, and it was washed 2 times with absolute ethanol. Cholesterol and DSPE-mPEG 2000 chloroform solutions were respectively added. After vortex hydration by the thin film dispersion method and probe sonication dispersion, a glucose solution was added to adjust the osmotic pressure. Among them, the concentration of the cholesterol chloroform solution was 1.8 mg / mL, and the added volume was 1 mL; the concentration of the DSPE-mPEG 2000 chloroform solution was 13.5 mg / mL, and the added volume was 600 μL; the rotary evaporation conditions were a water bath at 60 °C, a rotation speed of 100 rpm / min, and rotary evaporation for 15 min. Immediately after the rotary evaporation was completed, 2 mL of deionized water preheated at 60 °C was added and vigorously shaken and vortexed; the probe sonication conditions were a power of 50 w, turned on for 3 s and turned off for 3 s, lasting for 5 min, and cooled in an ice-water bath to obtain the product. Preparation of manganese bisphosphonate nanoparticles in Example 16 1. Formulation mass ratio 2. Preparation method: (1) Preparation of the water-in-oil emulsion system of manganese: The manganese nitrate solution was slowly added dropwise to the mixed solvent of CA-630 and cyclohexane, and stirred to form a water-in-oil emulsion system of manganese. Among them, the concentration of manganese nitrate was 23.67 mg / mL, the volume was 75 μL, and the volume of the mixed solvent was 10 mL; The volume ratio of CA-630 to cyclohexane is 35:65. During the dropping process, continuous vigorous stirring is carried out, and after the dropping is completed, stirring continues for 30 min. (2) Oil-in-water emulsion system of bisphosphonate: The zoledronic acid solution and sodium pyrophosphate solution are slowly dropped into the stirred mixed solvent of CA-630 and cyclohexane, and then the chloroform solution of dioleoylphosphatidic acid (DOPA) is slowly dropped, and stirred to form an oil-in-water emulsion system of bisphosphonate. Among them, the concentration of the zoledronic acid solution is 29.0 mg / mL, the pH value is adjusted to 7-8 with hydrochloric acid, and the added volume is 300 μL; the concentration of sodium pyrophosphate is 26.6 mg / mL, and the added volume is 450 μL; the concentration of the DOPA chloroform solution is 11.5 mg / mL, and the added volume is 900 μL; the volume of the mixed solvent is 10 mL, among which the volume ratio of CA-630 to cyclohexane is 35:65. During the dropping process, continuous vigorous stirring is carried out, and after the dropping is completed, stirring continues for 30 min. (3) The oil-in-water emulsion system of bisphosphonate is dropped into the stirred oil-in-water emulsion system of manganese ions. During the dropping process, continuous vigorous stirring is carried out, and after the dropping is completed, stirring continues for 30 min. (4) 20 mL of absolute ethanol is added to the reaction system for curing. During the dropping process, continuous vigorous stirring is carried out, and after the dropping is completed, stirring continues for 20 min. (5) The reaction solution is centrifuged, the supernatant is discarded, and washed twice with absolute ethanol. The chloroform solutions of DOPC, cholesterol, and DSPE-mPEG 2000 are respectively added. After vortex hydration by the thin film dispersion method and probe sonication dispersion, a glucose solution is added to adjust the osmotic pressure. Among them, the concentration of the DOPC chloroform solution is 5 mg / mL, and the added volume is 500 μL; the concentration of the cholesterol chloroform solution is 1.8 mg / mL, and the added volume is 2 mL; the concentration of the DSPE-mPEG 2000 chloroform solution is 13.5 mg / mL, and the added volume is 400 μL; the rotary evaporation conditions are a 60 °C water bath, a rotation speed of 100 rpm / min, and rotary evaporation for 15 min. Immediately after the rotary evaporation is completed, 2 mL of deionized water preheated at 60 °C is used for vigorous shaking and vortexing; the probe sonication conditions are a power of 50 w, 3 s on, 3 s off, for 5 min, and cooling in an ice-water bath to obtain the product. The above is only the preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited by the above embodiments. Without departing from the principle of the present invention, several changes, modifications, substitutions, combinations, and simplifications can be made, all of which should be equivalent replacement methods, and these should also be regarded as the protection scope of the present invention.

Claims

1. A manganese bisphosphonate nanoparticle, characterized in that, The nanoparticles comprise a core structure and a lipid material coating the outer layer of the core structure. The core structure comprises manganese ions (Mn 2+ ), bisphosphonic acid, and dioleoyl phosphatidic acid (DOPA). The outer lipid material comprises dioleoyl phosphatidylcholine (DOPC), cholesterol, and methoxypolyethylene glycol 2000-distearoyl phosphatidylethanolamine (DSPE-mPEG2000).

2. The manganese bisphosphonate nanoparticles according to claim 1, wherein Among them, the bisphosphonic acid includes any one or a mixture of more than one of zoledronic acid, sodium alendronate, sodium risedronate, and sodium pyrophosphate, preferably a mixture of zoledronic acid and sodium pyrophosphate in a certain proportion; wherein, the manganese ion refers to a divalent manganese ion, selected from one or more of manganese nitrate and manganese chloride; wherein, the core lipid material is selected from any one or a mixture of more than one of dioleoyl phosphatidic acid, distearoyl phosphatidic acid, and dipalmitoyl phosphatidic acid.

3. The manganese bisphosphonate nanoparticles according to claim 1, wherein the mass ratio of manganese nitrate, zoledronic acid, sodium pyrophosphate, dioleoyl phosphatidic acid, dioleoyl phosphatidylcholine, cholesterol, and methoxypolyethylene glycol 2000-distearoylphosphoethanolamine is (2-20):(10-50):(0-50):(10-40):(0-30):(0-30):(8-40).

4. The preparation method of the manganese bisphosphonate nanoparticles according to claim 1, characterized in that, comprises the following steps: Drop the water-in-oil emulsion system of bisphosphonate into the water-in-oil emulsion system of manganese ions to establish a coordination skeleton, solidify with absolute ethanol, add a certain proportion of lipid materials, and vortex and hydrate by the thin film dispersion method, and then disperse with a probe ultrasonic wave to obtain.

5. The preparation method of the manganese bisphosphonate nanoparticles according to claim 1, characterized in that, comprises the following steps: (1) Slowly drop the manganese ion solution into the mixed solvent of CA-630 and cyclohexane, and stir to form an oil-in-water emulsion system of manganese; (2) Slowly drop the bisphosphonate solution and sodium pyrophosphate solution into a mixed solvent of CA-630 and cyclohexane, and then slowly drop a chloroform solution containing DOPA, and stir to form a water-in-oil emulsion system of bisphosphonate; (3) Drop the water-in-oil emulsion system of bisphosphonate into the stirring water-in-oil emulsion system of manganese ions, and continue stirring; (4) Add absolute ethanol to the reaction system for solidification, and continue stirring; (5) Centrifuge the reacted solution, discard the supernatant, wash with absolute ethanol, and add chloroform solutions of DOPC, cholesterol, and DSPE-mPEG respectively. After vortex hydration by the thin film dispersion method, probe sonicate and add glucose solution to adjust the osmotic pressure to obtain the product. 2000 That is how it is obtained.

6. The preparation method according to claim 5, wherein In step (1), the concentration of manganese nitrate is 23.67 mg / mL, the volume is 30 μL, and the volume of the mixed solvent is 10 mL, where the volume ratio of CA-630 to cyclohexane is (30 - 40):(60 - 70).

7. The preparation method according to claim 5, wherein In step (2), the concentration of the zoledronic acid solution is 29.0 mg / mL, the pH value is adjusted to 7 - 8 using hydrochloric acid, and the added volume is 150 μL; the concentration of sodium pyrophosphate is 26.6 mg / mL, and the added volume is 150 μL; the concentration of the DOPA chloroform solution is 11.5 mg / mL, and the added volume is 300 μL; the volume of the mixed solvent is 10 mL, where the volume ratio of CA - 630 to cyclohexane is (30 - 40):(60 - 70).

8. The preparation method according to claim 5, wherein In step (3): Slowly drop the bisphosphonate dispersion system into the manganese system, continuously stir vigorously during the dropping process, and continue stirring for 30 min after the dropping is completed; In step (4): Add 20 mL of absolute ethanol, continuously stir vigorously during the dropping process, and continue stirring for 20 min after the dropping is completed; In step (5): The concentration of the DOPC chloroform solution is 5 mg / mL, and the added volume is 1 mL; the concentration of the cholesterol chloroform solution is 1.8 mg / mL, and the added volume is 200 μL; the concentration of the DSPE-mPEG 2000 chloroform solution is 13.5 mg / mL, and the added volume is 200 μL.

9. Use of the manganese bisphosphonate nanoparticles according to any one of claims 1-8 in the preparation of a drug for immunomodulation, and / or promoting the maturation of DC cells, and / or stimulating DC cells to increase the secretion of inflammatory cytokines.

10. Use of the manganese bisphosphonate nanoparticles according to any one of claims 1-8 in combination with oxaliplatin in the preparation of a drug for treating digestive tract tumors.

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