Modified Prussian blue nano particle and preparation method and application thereof

Abstract

The invention relates to the technical field of tumor nano medicine, and discloses a modified Prussian blue nanoparticle as well as a preparation method and application thereof. The modified prussian blue nano particles are hafnium-doped prussian blue nano particles with the surfaces sequentially subjected to tannic acid coating and manganese ion modification, and when the modified prussian blue nano particles are applied to preparation of osteosarcoma radiotherapy drugs through triple mechanisms of hafnium-enhanced radiotherapy sensitization, prussian blue mediated photothermal therapy and Mn < 2 + > activated STING immune pathway, the sensitivity of the modified prussian blue nano particles is greatly improved. In-vitro tests prove that the compound can eradicate tumor cells through triple mechanisms of ROS outbreak, DNA damage and apoptosis, in-vivo tests show that the compound can completely inhibit in-situ osteosarcoma and eliminate lung metastasis, and the curative effect of the compound is remarkably superior to that of a single therapy; negative charges are endowed through tannic acid coating, efficient tumor targeting after intravenous administration is achieved, and meanwhile excellent biocompatibility is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor nanomedicine, and in particular to modified Prussian blue nanoparticles and a preparation method and application thereof. Background Art

[0002] Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, with an annual global incidence of approximately 3.4 cases per million people. Through multidisciplinary comprehensive treatment and various chemotherapy regimens, the survival rate of patients undergoing surgical resection has increased from 10%-20% to 60%-70%, and this proportion has remained stable for the past two decades. For patients with recurrence, metastasis, or difficult surgical resection, available treatments include chemotherapy, radiotherapy, and emerging targeted therapies and immunotherapy. Among them, radiotherapy is one of the most commonly used methods in oncology, and precision radiotherapy is usually the first choice for treating local lesions. However, radiotherapy is not sensitive enough to osteosarcoma. Studies have shown that the local control rate of radiotherapy can reach 79.6%, but the overall control rate is only 30%. Therefore, how to improve the sensitivity of osteosarcoma cells to radiotherapy is a difficult problem that needs to be solved urgently.

[0003] In recent years, radiotherapy sensitization has become an important research direction in the field of tumor nanomedicine. High atomic number (high Z) elements can enhance the radiation energy deposition inside the tumor, thereby increasing sensitivity to radiotherapy. This discovery is expected to overcome the current limitation of radiotherapy insensitivity to osteosarcoma. Among them, hafnium oxide, as a radiotherapy sensitizer, has entered the clinical trial stage and has promoted an increase in the pathological complete remission rate in locally advanced soft tissue sarcoma. However, existing hafnium oxide preparations are limited to intratumoral injection and are difficult to target intrabone tumors. Therefore, there is an urgent need to develop intravenously injectable, well-targeted hafnium radiotherapy sensitizers. Summary of the Invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems in the prior art. To this end, one of the purposes of the present invention is to provide a modified Prussian blue nanoparticle.

[0005] A second object of the present invention is to provide a method for preparing the modified Prussian blue nanoparticles.

[0006] A third object of the present invention is to provide a radiotherapy sensitizer.

[0007] A fourth object of the present invention is to provide modified Prussian blue nanoparticles or applications of radiotherapy sensitizers.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A first aspect of the present invention provides a modified Prussian blue nanoparticle, wherein the modified Prussian blue nanoparticle is a hafnium-doped Prussian blue nanoparticle whose surface is sequentially coated with tannic acid and modified with manganese ions.

[0010] In some embodiments of the present invention, the modified Prussian blue nanoparticles have a particle size of 40-60 nm and a hydrated particle size of 95-140 nm.

[0011] In some preferred embodiments of the present invention, the modified Prussian blue nanoparticles have a particle size of 45-55 nm and a hydrated particle size of 105-130 nm.

[0012] In some embodiments of the present invention, the modified Prussian blue nanoparticles are prepared from the following raw materials: potassium ferrocyanide, a nanoparticle surface stabilizer, acid, a hafnium salt, tannic acid, a manganese salt, and water.

[0013] In some embodiments of the present invention, the nanoparticle surface stabilizer is selected from polyvinyl pyrrolidone, polyethylene glycol or sodium citrate.

[0014] In some preferred embodiments of the present invention, the nanoparticle surface stabilizer is polyvinylpyrrolidone (PVP).

[0015] In some embodiments of the present invention, the hafnium salt is selected from hafnium chloride, hafnium nitrate or hafnium sulfate.

[0016] In some preferred embodiments of the present invention, the hafnium salt is hafnium chloride (HfCl 4 ).

[0017] In some embodiments of the present invention, the manganese salt is selected from manganese chloride, manganese acetate or manganese sulfate.

[0018] In some preferred embodiments of the present invention, the manganese salt is manganese chloride (MnCl2).

[0019] In some embodiments of the present invention, the acid solution is selected from hydrochloric acid, nitric acid or acetic acid.

[0020] In some preferred embodiments of the present invention, the acid solution is hydrochloric acid (HCl).

[0021] In some embodiments of the present invention, the concentration of the acid solution is 0.4-0.6 mol / L.

[0022] The second aspect of the present invention provides a method for preparing the modified Prussian blue nanoparticles according to the first aspect of the present invention, comprising the following steps:

[0023] S1, mixing potassium ferrocyanide, a nanoparticle surface stabilizer, and an acid solution, adding a hafnium salt, and heating the mixture for reaction to obtain hafnium-doped Prussian blue nanoparticles;

[0024] S2, dispersing the hafnium-doped Prussian blue nanoparticles in water, and adding tannic acid to obtain tannic acid-coated hafnium-doped Prussian blue nanoparticles;

[0025] S3. Dispersing the tannic acid-coated hafnium-doped Prussian blue nanoparticles in water, adding manganese salt, and obtaining the modified Prussian blue nanoparticles.

[0026] In some embodiments of the present invention, in step S1, the mass ratio of the potassium ferrocyanide, the nanoparticle surface stabilizer and the hafnium salt is (70-105):1:(22-88).

[0027] In some preferred embodiments of the present invention, in step S1, the mass ratio of the potassium ferrocyanide, the nanoparticle surface stabilizer and the hafnium salt is (80-95):1:(80-88).

[0028] In some embodiments of the present invention, in step S1, after the potassium ferrocyanide, the nanoparticle surface stabilizer and the acid solution are mixed, a stirring operation is further included.

[0029] In some embodiments of the present invention, the stirring speed is 800-1200 rpm and the stirring time is 10-20 min.

[0030] In some embodiments of the present invention, in step S1, the heating reaction temperature is 100-140° C. and the time is 20-30 hours.

[0031] In some preferred embodiments of the present invention, in step S1, the heating reaction temperature is 110-130° C. and the time is 20-25 hours.

[0032] In some embodiments of the present invention, in step S1, the heating reaction is carried out in a hydrothermal reactor.

[0033] In some embodiments of the present invention, in step S1, the heating reaction is carried out in an oil bath.

[0034] In some embodiments of the present invention, in step S1, after the heating reaction is completed, the steps of standing, solid-liquid separation, solid phase collection, and washing are further included.

[0035] In some embodiments of the present invention, the standing time is 20-30 hours.

[0036] In some embodiments of the present invention, the solid-liquid separation method includes centrifugation, and the centrifugation speed is 16000-24000 rpm and the time is 10-20 min.

[0037] In some embodiments of the present application, the washing reagent comprises ethanol and water.

[0038] In some embodiments of the present application, in step S2, the mass ratio of tannic acid to hafnium-doped Prussian blue nanoparticles is (30-50):1.

[0039] In some preferred embodiments of the present application, in step S2, the mass ratio of tannic acid to hafnium-doped Prussian blue nanoparticles is (35-45):1.

[0040] In some embodiments of the present application, in step S2, the concentration of the aqueous solution of hafnium-doped Prussian blue nanoparticles dispersed in water is 0.8-1.2 mg / mL.

[0041] In some embodiments of the present application, in step S2, the dispersion process is assisted by ultrasonic.

[0042] In some embodiments of the present application, in step S2, the tannic acid (TA) participates in the reaction in the form of a tannic acid aqueous solution, and the concentration of the tannic acid aqueous solution is 8-12 mg / mL.

[0043] In some embodiments of the present application, in step S2, the aqueous solution of hafnium-doped Prussian blue nanoparticles is added dropwise to the tannic acid aqueous solution.

[0044] In some embodiments of the present application, the dropwise addition process is assisted by stirring, and the stirring speed is 400-600 rpm, and the stirring time is 20-40 min.

[0045] In some embodiments of the present application, after the dropwise addition is completed, the operation of solid-liquid separation to collect the solid phase and washing is further included.

[0046] In some embodiments of the present application, the solid-liquid separation method comprises centrifugation, and the centrifugation speed is 10000-14000 rpm, and the centrifugation time is 5-15 min.

[0047] In some embodiments of the present application, the washing reagent comprises water.

[0048] In some embodiments of the present application, in step S3, the mass ratio of manganese salt to tannic acid-coated hafnium-doped Prussian blue nanoparticles is (30-50):1.

[0049] In some preferred embodiments of the present application, in step S3, the mass ratio of manganese salt to tannic acid-coated hafnium-doped Prussian blue nanoparticles is (35-45):1.

[0050] In some embodiments of the present application, the concentration of the tannic acid-coated hafnium-doped Prussian blue nanoparticle dispersion in the aqueous solution obtained by dispersing the tannic acid-coated hafnium-doped Prussian blue nanoparticles in water is 0.8-1.2 mg / mL.

[0051] In some embodiments of the present application, the dispersion process in step S3 is assisted by ultrasonic.

[0052] In some embodiments of the present application, in step S3, the manganese salt participates in the reaction in the form of an aqueous manganese salt solution, and the concentration of the aqueous manganese salt solution is 8-12 mg / mL.

[0053] In some embodiments of the present application, the tannic acid-coated hafnium-doped Prussian blue nanoparticle aqueous solution is added to the aqueous manganese salt solution in a dropwise manner.

[0054] In some embodiments of the present application, the dropwise process is assisted by stirring at a speed of 400-600 rpm for 20-40 min.

[0055] In some embodiments of the present application, after the dropwise process is completed, the operation of solid-liquid separation to collect the solid phase and washing is further included.

[0056] In some embodiments of the present application, the solid-liquid separation is performed by centrifugation at a speed of 10000-14000 rpm for 5-15 min.

[0057] In some embodiments of the present application, the washing reagent comprises water.

[0058] The basic principle of the present application is described as follows:

[0059] The tannic acid-coated hafnium-doped Prussian blue nanoparticles provided by the present application have the following advantages: ① Prussian blue (PB) is a photothermal agent that can be efficiently converted into heat energy (photothermal effect) under near-infrared light (808 nm) irradiation, directly killing tumor cells by local heating (>42℃). Since hypoxia is one of the main reasons for radiotherapy resistance, heating can increase blood flow, thereby improving the hypoxic microenvironment of tumors and enhancing the effect of radiotherapy. In the present application, potassium ferricyanide is used as a Prussian blue precursor, and a nanoparticle surface stabilizer such as polyvinylpyrrolidone is added to prevent nanoparticle aggregation and improve biocompatibility; ② Hafnium (Hf) is a high-atomic-number (Z=72) element that can significantly enhance the energy deposition of X-rays in tumor tissues (photoelectric effect and Compton scattering), thereby improving the killing effect of radiotherapy on tumor cells. In the nanoparticles, hafnium exists in a doped form, avoiding the toxicity of free hafnium ions while maintaining the radiosensitization activity; ③ Tannic acid is a polyphenolic compound rich in phenolic hydroxyl groups, which can chelate with metal ions (such as Fe 3+ 、Mn 2+) form a stable metal-phenol network, wrap hafnium-doped Prussian blue nanoparticles to form a negatively charged surface, thereby improving blood circulation stability and avoiding rapid clearance by the immune system; 4) The activation of the cyclic guanosine monophosphate-adenosine monophosphate synthetase (cGAS)-STING signaling pathway is crucial for the production of systemic anti-tumor immunity after radiotherapy, and Mn 2+ is a key activator of the STING pathway, can promote the maturation and antigen presentation of dendritic cells, and stimulate T cell-mediated anti-tumor immunity. In tannic acid-coated nanoparticles, Mn 2+ is loaded in the tannic acid network through coordination, released in the tumor microenvironment, thereby activating the cGAS-STING pathway, inducing immunogenic cell death (ICD), and synergizing with radiotherapy and photothermal therapy to inhibit orthotopic tumors and reduce lung metastasis.

[0060] The third aspect of the present application provides a radiotherapy sensitizer comprising the modified Prussian blue nanoparticles of the first aspect of the present application.

[0061] The fourth aspect of the present application provides the use of the modified Prussian blue nanoparticles of the first aspect of the present application or the radiotherapy sensitizer of the third aspect of the present application in the preparation of a radiotherapy drug for osteosarcoma.

[0062] Compared with the prior art, the present application has the following beneficial effects:

[0063] 1) The modified Prussian blue nanoparticles provided by the present application have three mechanisms of enhancing radiotherapy sensitization, Prussian blue-mediated photothermal therapy, and Mn 2+ activating the STING immune pathway, which can significantly improve the anti-tumor effect when used in the preparation of a radiotherapy drug for osteosarcoma. In vitro experiments have verified that it can eradicate tumor cells through the ROS burst, DNA damage, and apoptosis triple mechanisms, and in vivo experiments have shown that it can completely inhibit orthotopic osteosarcoma and eliminate lung metastasis, with a significantly better therapeutic effect than single therapy.

[0064] 2) The modified Prussian blue nanoparticles provided by the present application are coated with tannic acid to impart a negative charge, achieving efficient tumor targeting after intravenous administration, while also having excellent biocompatibility.

[0065] 3) The preparation method of the modified Prussian blue nanoparticles provided by the present application is stable, efficient, safe, and has potential for clinical transformation. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 The transmission electron micrographs of HfPB, HPTA, and HPTM in Example 1 are shown in Figure 1.

[0067] Figure 2 The particle size graphs of HfPB, HPTA, and HPTM in Example 1 are shown in Figure 2.

[0068] Figure 3 Elemental mapping of modified Prussian blue nanoparticles in Example 1;

[0069] Figure 4 Zeta potential (A) and hydrated particle size (B) of HfPB, HPTA and HPTM in Example 1;

[0070] Figure 5 XPS mapping of modified Prussian blue nanoparticles in Example 1;

[0071] Figure 6 Near-infrared photoexcitation photothermal mapping (A) and temperature curve (B) of modified Prussian blue nanoparticles in Example 1;

[0072] Figure 7 Cytotoxicity test and hemolysis test results of modified Prussian blue nanoparticles in Example 1;

[0073] Figure 8 Active oxygen detection results of modified Prussian blue nanoparticles in Example 1 after near-infrared light and X-ray irradiation;

[0074] Figure 9 Apoptosis detection results of modified Prussian blue nanoparticles in Example 1 after near-infrared light and X-ray irradiation;

[0075] Figure 10 DNA damage detection results of modified Prussian blue nanoparticles prepared in Example 1 after near-infrared light and X-ray irradiation;

[0076] Figure 11 Cell clonality detection results of modified Prussian blue nanoparticles prepared in Example 1 after near-infrared light and X-ray irradiation;

[0077] Figure 12 Imaging of mice at different time points after injection of modified Prussian blue nanoparticles prepared in Example 1 in a mouse subcutaneous tumor model in the left dorsal scapular region;

[0078] Figure 13 Statistical results of average luminescence intensity of mouse back tumors at different time points;

[0079] Figure 14 Imaging of ex vivo tumors at different time points after injection of modified Prussian blue nanoparticles prepared in Example 1 in a mouse subcutaneous tumor model;

[0080] Figure 15 Statistical results of average luminescence intensity of ex vivo tumors at different time points;

[0081] Figure 16The photothermal image (A) and temperature curve (B) of the modified Prussian blue nanoparticles prepared in Example 1 in the treatment of mouse tibial osteosarcoma model;

[0082] Figure 17 This is the bioluminescence imaging of the modified Prussian blue nanoparticles prepared in Example 1 in the treatment of mouse tibial osteosarcoma model. DETAILED DESCRIPTION

[0083] The present invention is further described in detail below by way of specific examples. Unless otherwise specified, the raw materials, reagents, or devices used in the examples are all commercially available or can be obtained by conventional methods. Unless otherwise specified, all experiments or testing methods are conventional methods in the art.

[0084] Example 1

[0085] In this embodiment, modified Prussian blue nanoparticles are prepared in the following steps:

[0086] S11. Add 264 mg of potassium ferricyanide and 3 g of polyvinyl pyrrolidone to 0.5 mol / L hydrochloric acid, stir thoroughly at 1000 rpm for 15 min, until a light yellow clear solution is formed; add 264 mg of hafnium chloride and stir thoroughly, then place the mixture in a hydrothermal reactor, heat to 120° C. in an oil bath for 24 h, stop heating, let stand for 24 h, take out the product and place it in a high-speed centrifuge tube, centrifuge at 20,000 rpm for 15 min to obtain a precipitate, wash the precipitate three times with ethanol and water each, to obtain hafnium-doped Prussian blue nanoparticles (HfPB);

[0087] S21, re-ultrasonic-dispersing the hafnium-doped Prussian blue nanoparticles in deionized water to form a 1 mg / mL dark blue solution, and preparing a 10 mg / mL tannic acid aqueous solution;

[0088] S22, taking 1 mL of the hafnium-doped Prussian blue nanoparticle aqueous solution and slowly dropping it into 4 mL of the tannic acid aqueous solution while stirring at a stirring speed of 500 rpm for 30 min, taking out the mixture, and centrifuging it at a speed of 12000 rpm for 10 min, collecting the solid phase and washing it three times with deionized water to obtain tannic acid-coated hafnium-doped Prussian blue nanoparticles (HPTA);

[0089] S31, re-ultrasonic-dispersing the tannic acid-coated hafnium-doped Prussian blue nanoparticles in deionized water to form a 1 mg / mL dark blue solution, and preparing a 10 mg / mL manganese chloride aqueous solution;

[0090] S32, 1 mL of tannic acid-coated hafnium-doped Prussian blue nanoparticle aqueous solution was slowly dropped into 4 mL of manganese chloride aqueous solution, stirring at a speed of 500 rpm, and stirring for 30 min. The mixture was taken out and centrifuged at a speed of 12000 rpm for 10 min. The solid phase was collected and washed with deionized water for 3 times to obtain modified Prussian blue nanoparticles (HPTM).

[0091] Example 2

[0092] In this example, a modified Prussian blue nanoparticle was prepared, and the steps were as follows:

[0093] S11, 264 mg of potassium ferricyanide and 3 g of polyvinylpyrrolidone were added to 0.5 mol / L hydrochloric acid, and stirred at a speed of 1000 rpm for 15 min until a light yellow clear solution was formed. 66 mg of hafnium chloride was added and stirred thoroughly, and then the mixture was placed in a hydrothermal reactor and heated to 120°C in an oil bath for 24 h. The heating was stopped, and the product was left to stand for 24 h. The product was taken out and placed in a high-speed centrifuge tube and centrifuged at a speed of 20000 rpm for 15 min to obtain a precipitate. The precipitate was washed with ethanol and water for three times to obtain hafnium-doped Prussian blue nanoparticles (HfPB);

[0094] S21, the hafnium-doped Prussian blue nanoparticles were ultrasonically dispersed in deionized water to form a deep blue solution of 1 mg / mL. A tannic acid aqueous solution of 5 mg / mL was prepared;

[0095] S22, 1 mL of hafnium-doped Prussian blue nanoparticle aqueous solution was slowly dropped into 6 mL of tannic acid aqueous solution, stirring at a speed of 500 rpm, and stirring for 30 min. The mixture was taken out and centrifuged at a speed of 12000 rpm for 10 min. The solid phase was collected and washed with deionized water for 3 times to obtain tannic acid-coated hafnium-doped Prussian blue nanoparticles (HPTA);

[0096] S31, the tannic acid-coated hafnium-doped Prussian blue nanoparticles were ultrasonically dispersed in deionized water to form a deep blue solution of 1 mg / mL. A manganese chloride aqueous solution of 12.5 mg / mL was prepared;

[0097] S32, 1 mL of tannic acid-coated hafnium-doped Prussian blue nanoparticle aqueous solution was slowly dropped into 3 mL of manganese chloride aqueous solution, stirring at a speed of 500 rpm, and stirring for 30 min. The mixture was taken out and centrifuged at a speed of 12000 rpm for 10 min. The solid phase was collected and washed with deionized water for 3 times to obtain modified Prussian blue nanoparticles (HPTM).

[0098] 1. Characterization of modified Prussian blue nanoparticles (HPTM) and intermediate products (HfPB, HPTA) prepared in Example 1:

[0099] Figure 1 TEM images of HfPB, HPTA and HPTM in Example 1, Figure 2 Particle size distribution of HfPB, HPTA and HPTM in Example 1, obtained from Figure 1 and Figure 2 It can be seen that the hafnium-doped Prussian blue nanoparticles (HfPB) are spherical, with a particle size of about 50 nm. The particle size of the intermediate product, tannic acid-coated hafnium-doped Prussian blue nanoparticles (HPTA), and the modified Prussian blue nanoparticles (HPTM) obtained after modification with manganese ions does not increase significantly, indicating that the surface coating layer formed is thin and uniform. The modified Prussian blue nanoparticles have good particle monodispersity and no agglomeration, meeting the particle size requirement (less than 200 nm) for intravenous injection.

[0100] Figure 3 Elemental distribution of modified Prussian blue nanoparticles in Example 1, obtained from Figure 3 It can be seen that the three elements, iron (Fe), hafnium (Hf) and manganese (Mn), are uniformly distributed in the modified Prussian blue nanoparticles, confirming that Hf is successfully doped into the Prussian blue nanoparticle framework, and Mn 2+ is then loaded on the surface through tannic acid coordination.

[0101] Figure 4 Zeta potential (A) and hydration particle size (B) of HfPB, HPTA and HPTM in Example 1, obtained from Figure 4 It can be seen that the surface of Hf 4+ / Fe 3+ doped Prussian blue nanoparticles (HfPB) is positively charged. After tannic acid coating, the tannic acid-coated hafnium-doped Prussian blue nanoparticles (HPTA) are negatively charged, indicating that the phenolic hydroxyl groups of tannic acid are dissociated and successfully coated on the surface of hafnium-doped Prussian blue nanoparticles. The modified Prussian blue nanoparticles (HPTM) after modification with manganese ions have enhanced negative charge, indicating that the coordination of Mn 2+ with tannic acid further exposes negative charge groups, i.e., manganese ions are successfully adsorbed on the surface. The hydration particle size of hafnium-doped Prussian blue nanoparticles (HfPB) is 87.0 nm, and the hydration particle sizes of tannic acid-coated hafnium-doped Prussian blue nanoparticles (HPTA) and modified Prussian blue nanoparticles (HPTM) are 103.0 nm and 118.53 nm, respectively, indicating that tannic acid coating and manganese ion adsorption will gradually increase the hydration particle size of hafnium-doped Prussian blue nanoparticles, but the hydration particle size is still within the ideal range (less than 150 nm).

[0102] Figure 5The XPS graph of the modified Prussian blue nanoparticles in Example 1 is shown in the figure, Figure 5 (A) in the figure is a characteristic absorption peak of Mn and Fe elements, Figure 5 (B) in the figure is a characteristic absorption peak of Hf element, Fe 3+ The characteristic peak is consistent with the Prussian blue nanoparticles, Mn 2+ The appearance of the characteristic peak proves that the manganese ions are successfully loaded, Hf 4+ The characteristic peak indicates that hafnium is successfully doped, that is, the modified Prussian blue nanoparticles are successfully synthesized.

[0103] 2. The modified Prussian blue nanoparticles (HPTM) prepared in Example 1 were subjected to near-infrared light excitation test:

[0104] The HPTM solutions with concentrations of 50 μg / mL, 100 μg / mL and 200 μg / mL were prepared respectively with phosphate buffered saline (PBS), and the photothermal effect of HPTM was verified by infrared thermometer under 808 nm near-infrared light irradiation for 5 min.

[0105] Figure 6 The near-infrared light excitation photothermal graph (A) and temperature curve (B) of the modified Prussian blue nanoparticles in Example 1 are shown in the figure, Figure 6 It can be seen that with the increase of the concentration of HPTM solution and the irradiation time, the picture recorded by the temperature meter after irradiation of HPTM is brighter, and the temperature rise is more obvious. When the concentration of HPTM solution is 200 μg / mL, the temperature can be raised to above 60℃ after 808 nm near-infrared light irradiation for 5 min, which indicates that the modified Prussian blue nanoparticles (HPTM) provided by the application have excellent photothermal conversion efficiency.

[0106] 3. The modified Prussian blue nanoparticles (HPTM) prepared in Example 1 were subjected to cytotoxicity test and hemolysis test:

[0107] The HPTM solutions with concentrations of 0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL and 800 μg / mL were prepared respectively with 10% fetal bovine serum high glucose DMEM medium; MC3T3, hFOB1.19, 293T and RAW264.7 cells were inoculated in 96-well plates at a density of 3500 cells per well, and after overnight adhesion, different concentrations of HPTM solution were co-incubated with cells for 24 h, and 10% CCK8 solution was added, and the absorption peak at 450 nm was recorded on the enzyme marker, and the cell viability was calculated to detect the cytotoxicity of HPTM.

[0108] HPTM solution with concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL were prepared with phosphate buffered saline (PBS) respectively. The eyeball blood of Balb / c mice was extracted, and the eyeball blood of Balb / c mice was mixed with HPTM solution with different concentrations, and then incubated at 37℃ for 4h. Centrifugation was carried out at a speed of 3000 rpm for 15 min, and the supernatant was recorded by taking photos. The absorbance (OD) at 542 nm was recorded by an enzyme-labeled instrument, and the hemolysis rate (%) was calculated. PBS (0% hemolysis) was used as a negative control, and deionized water (100% hemolysis) was used as a positive control.

[0109] Figure 7 The results of the cell toxicity test and hemolysis test of the modified Prussian blue nanoparticles in Example 1 are shown in Table 1, wherein, Figure 7 (A), (B), (C) and (D) in Table 1 are the results of the cell toxicity test of HPTM on MC3T3, hFOB1.19, 293T and RAW264.7 cells, respectively, Figure 7 (E) and (F) in Table 1 are the hemolysis test photos and hemolysis rate statistics of HPTM, respectively, and Figure 7 It can be seen from Table 1 that even if the concentration of HPTM is continuously increased, the cell viability of MC3T3, hFOB1.19, 293T and RAW264.7 cells does not appear to be significantly decreased, and the cell viability can always be maintained above 80%, indicating that the cell toxicity of HPTM is low; when the concentration of HPTM is less than or equal to 200 μg / mL, the hemolysis rate is always less than 10%, and no hemolysis can be observed by naked eye, and the biological safety is high.

[0110] 4. Detection of reactive oxygen species after near-infrared light and X-ray irradiation of the modified Prussian blue nanoparticles (HPTM) prepared in Example 1:

[0111] K7M2RR cells resistant to radiotherapy were selected, and 250,000 cells per well were inoculated in a six-well plate and cultured for 24 h. Then, 50 μg / mL HfPB (denoted as HP) and HPTM were used for treatment for 12 h, followed by exposure to near-infrared (NIR) irradiation (808 nm, 1 W / cm 2 , 5 min) and radiotherapy (RT, 4 Gy). After 6 h, the cells were incubated with 5 μmol / L 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) in serum-free DMEM for 20 min at 37℃ in the dark, and the fluorescence was observed using a fluorescence microscope (Nikon, Japan).

[0112] Figure 8 The results of the detection of reactive oxygen species after near-infrared light and X-ray irradiation of the modified Prussian blue nanoparticles in Example 1 are shown in Table 2, wherein, Figure 8It can be seen that HPTM can significantly increase the level of reactive oxygen species (ROS) in tumor cells under the combined action of near-infrared light and X-ray radiotherapy (HPTM+NIR+RT), showing the strongest green fluorescence signal. This result reveals the triple synergistic mechanism of HPTM: hafnium (Hf) enhances the energy deposition of radiotherapy to produce ·OH free radicals, photothermal heating accelerates the diffusion of ROS, and Mn 2+ It may catalyze the Fenton-like reaction to further amplify oxidative stress.

[0113] 5. Apoptosis detection of modified Prussian blue nanoparticles (HPTM) prepared in Example 1 after near-infrared light and X-ray irradiation:

[0114] K7M2RR cells were seeded into a six-well plate at a density of 200,000 per well and cultured for 24 h, then 50 μg / mL HfPB (HP) and HPTM were added and incubated for 12 h, and near-infrared (NIR) and X-ray irradiation (RT) was performed. After 48-72 h, dead cells in the supernatant were collected, and adherent cells were separated by trypsin digestion without EDTA. The cells were washed three times with ice phosphate buffer (PBS) and mixed with 1× loading buffer containing 5 μmol / L fluorescein isothiocyanate (FITC) and 10 μmol / L propidium iodide (PI) under light-protected conditions for another 30 min. Finally, the proportion of apoptotic cells was analyzed using a flow cytometer (Beckman, USA).

[0115] Figure 9 For the apoptosis detection results of modified Prussian blue nanoparticles in Example 1 after near-infrared light and X-ray irradiation, it can be seen that Figure 9 It can be seen that the apoptosis rate of the HPTM+NIR+RT group is significantly higher than that of the single treatment group (HPTM+NIR and HPTM+RT), confirming that the combined therapy can maximize the induction of tumor cell programmed death. Apoptosis is mainly triggered by the ROS-mediated mitochondrial pathway and DNA damage, and the activated STING pathway may further promote immunogenic cell death (ICD). 2+

[0116] 6. DNA damage detection of modified Prussian blue nanoparticles (HPTM) prepared in Example 1 after near-infrared light and X-ray irradiation:

[0117] To evaluate the DNA damage caused by radiotherapy, cell immunofluorescence technology and comet assay were used for analysis. γH2A.x is a commonly used indicator of DNA damage. For cell immunofluorescence, K7M2RR cells were seeded into a six-well plate at a density of 4×10 4 ​The cells were seeded at a density of 100 cells / well in a 24-well plate (with cell slides in advance) and cultured for 24 h. Subsequently, 50 μg / mL of HfPB (HP) and HPTM were added for pretreatment for 12 h. The cells were then subjected to near-infrared (808 nm, 1 W / cm 2 , irradiation for 5 min) and radiotherapy (RT, 4 Gy). After 24 h, the slides were taken out and fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, blocked with 5% goat serum for 1 h, and incubated with the primary antibody of γH2A.x at 4 °C overnight. After washing three times with PBS, the cells were incubated with the secondary antibody labeled with FITC (green) for 2 h, and then the cell nuclei were stained with DAPI (blue). After sealing, the cells were observed under a confocal microscope.

[0118] The comet assay can assess DNA damage by detecting the tail formed by DNA electrophoresis in agar. For the comet assay, cell treatment is the same as for γH2A.x immunofluorescence. After cell treatment, an agar gel is prepared. First, 100 μL of 0.7% normal melting point agarose is boiled and spread on a matte glass slide. The gel is allowed to solidify at 4°C for 15 minutes to form the first layer of gel. 80 μL of 0.7% low melting point agarose is mixed with 20 μL of a cell suspension in PBS (15,000 cells) and spread on the first layer of gel. The gel is allowed to solidify at 4°C for 15 minutes to form the second layer of gel. 100 μL of preheated 0.7% normal melting point agarose is added to form the third layer of gel. The gel is then spread evenly and solidified at 4°C for 30 minutes. The gel was washed three times with PBS, placed in a pre-cooled lysis buffer ice bath for 1.5 h, washed three times with PBS, immersed in alkaline electrophoresis buffer for 30 min, and electrophoresed at 25 V for 20 min. Finally, the gel slide was immersed in 1:10000 diluted YeaRed nucleic acid dye (Yisheng Bio, China) for staining for 15 min, and images were collected under a fluorescence microscope after adding a coverslip.

[0119] Figure 10 The results of DNA damage detection of the modified Prussian blue nanoparticles prepared in Example 1 after irradiation with near-infrared light and X-rays are shown, wherein: Figure 10 (A) is the immunofluorescence result of γH2A.x. Figure 10 (B) in the figure is the comet test result. Figure 10 It can be seen that the HPTM combined treatment group (HPTM+NIR+RT) caused the most severe DNA double-strand breaks (dense green fluorescence foci and the longest comet tail), which was attributed to the Hf 4+ Enhanced radiotherapy energy deposition works synergistically with ROS to cause irreversible genomic damage, thereby completely inhibiting tumor cell proliferation and providing direct evidence for radiosensitization.

[0120] 7. Cell clone detection after near-infrared light and X-ray irradiation of the modified Prussian blue nanoparticles (HPTM) prepared in Example 1:

[0121] Cell colony formation assay is a common method to detect cell proliferation ability, which is used to evaluate the inhibition ability of radiotherapy on cells. After K7M2RR cells were irradiated by near-infrared (808 nm, 1 W / cm 2 , 5 min) and radiotherapy (RT, 4 Gy), the cells were digested by trypsin and adjusted to a density of 500 cells per well, then inoculated into 12-well plates and continued to culture for 7-10 days. After washing the cells with PBS, they were fixed with 4% paraformaldehyde for 15 min, then stained with 0.1% crystal violet for 20 min. After washing with water, the cells were dried and photographed.

[0122] Figure 11 The results of cell colony detection after near-infrared light and X-ray irradiation of the modified Prussian blue nanoparticles prepared in Example 1 are shown in Figure 11 It can be seen that there is almost no tumor cell colony formation in the HPTM+NIR+RT group, indicating that combined treatment can completely block the cell proliferation ability. This result is directly related to the ROS burst ( Figure 8 ) and DNA damage ( Figure 10 ), which verifies the potential of HPTM to eradicate tumor cells in vitro models and lays a theoretical foundation for its in vivo efficacy.

[0123] 8. Targeted tumor lesion test of the modified Prussian blue nanoparticles (HPTM) prepared in Example 1:

[0124] In order to evaluate whether HPTM can effectively target tumor lesions, a subcutaneous osteosarcoma model was established using 143B human osteosarcoma cells. After the cells were cultured, digested, and separated, they were resuspended in PBS. Four-week-old Balb / c nude mice were selected, and 100 μL of cell suspension containing 2 million cells was injected subcutaneously into the left dorsal scapular region of the mice. One week later, mice with tumors about 500 mm 3 in size were selected and randomly divided into two groups, and the two groups received tail vein injection of IR780 or HPTM-IR780 (IR780 concentration was 2 mg / kg), respectively. After injection, imaging was performed at different time points, and the luminescence intensity was quantitatively analyzed.

[0125] Figure 12 The imaging of the left dorsal scapular region of mice at different time points after injection of the modified Prussian blue nanoparticles prepared in Example 1 into the subcutaneous tumor model of mice, Figure 13 The average luminescence intensity statistical results of the tumor on the back of the mice at different time points, Figure 14 The imaging of the ex vivo tumor at different time points after injection of the modified Prussian blue nanoparticles prepared in Example 1 into the subcutaneous tumor model of mice, Figure 15 The average luminescence intensity statistical results of the ex vivo tumor at different time points, Figure 12- Figure 15It can be seen that HPTM can be efficiently enriched in the tumor site, and the signal intensity is much higher than that of free IR780. This targeting ensures the effective accumulation of therapeutic ingredients in the local tumor, reduces systemic side effects, and provides a basis for the precise implementation of photothermal and radiotherapy.

[0126] 9. Investigate the temperature changes of the modified Prussian blue nanoparticles (HPTM) prepared in Example 1 in the treatment of mouse tibial osteosarcoma model:

[0127] An orthotopic tumor model with lung metastasis was established using fluorescein-labeled K7M2RR-Luc cells. After K7M2RR-Luc cells were cultured to the required number, they were digested with trypsin, resuspended in PBS, and injected into the right tibia of 3-week-old Balb / c mice at a concentration of 20 μL per mouse and a total of 100,000 cells. After 10 days, the mice were photographed and bioluminescence was recorded using a live imaging system (PerkinElmer, USA), and randomly divided into seven groups, with five mice in each group: control group, HPTM group, HP+NIR+RT group, RT group, HPTM+NIR group, HPTM+RT group, and HPTM+NIR+RT group. HP (100 μL, 5 mg / kg) and HPTM (100 μL, 5 mg / kg) were given and irradiated under near-infrared (NIR) (808 nm, 1.5 W / cm 2 The cells were incubated for an additional 12 hours before treatment with 5 min of RT (6 Gy). Temperature changes induced by the photothermal reaction were recorded using an infrared thermometer. Bioluminescence imaging was performed by intraperitoneal injection of 100 μL of luciferin potassium salt (Yeasen, China) at the beginning (day 0) and end (day 14) of the study.

[0128] Figure 16 The photothermal image (A) and temperature curve (B) of the modified Prussian blue nanoparticles prepared in Example 1 in the treatment of mouse tibial osteosarcoma model. Figure 16 It can be seen that the HPTM+NIR+RT group can still rapidly increase temperature in vivo, and the effect is better than that of the unmodified HP+NIR+RT group, indicating that the tannic acid coating and Mn 2+ The loading did not weaken the photothermal performance of Prussian blue. Heating the temperature not only directly killed the tumor but also relieved hypoxia to enhance radiotherapy sensitivity, which was consistent with the in vitro data.

[0129] Figure 17 This is the bioluminescent imaging of the modified Prussian blue nanoparticles prepared in Example 1 in the treatment of mouse tibial osteosarcoma model. Figure 17 It can be seen that on the 14th day, the HPTM+NIR+RT group almost completely suppressed the in situ osteosarcoma and eliminated lung metastasis. This therapeutic effect was attributed to the triple synergy: direct photothermal ablation, radiotherapy sensitization and killing, and Mn 2+The systemic anti-tumor response triggered by the activated STING immune pathway fully demonstrates the clinical translation potential of HPTM as a multifunctional nanoplatform.

Claims

1. A modified Prussian blue nanoparticle, characterized in that: The modified Prussian blue nanoparticles are hafnium-doped Prussian blue nanoparticles whose surfaces are sequentially coated with tannic acid and modified with manganese ions.

2. The modified Prussian blue nanoparticles according to claim 1, characterized in that The modified Prussian blue nanoparticles have a particle size of 40-60 nm and a hydrated particle size of 95-140 nm.

3. The modified Prussian blue nanoparticles according to claim 1, characterized in that The modified Prussian blue nanoparticles are prepared from the following raw materials: potassium ferrocyanide, a nanoparticle surface stabilizer, acid solution, a hafnium salt, tannic acid, a manganese salt and water.

4. The modified Prussian blue nanoparticles according to claim 3, characterized in that The nanoparticle surface stabilizer is selected from polyvinyl pyrrolidone, polyethylene glycol or sodium citrate; and / or, the hafnium salt is selected from hafnium chloride, hafnium nitrate or hafnium sulfate; And / or, the manganese salt is selected from manganese chloride, manganese acetate or manganese sulfate.

5. The method for preparing modified Prussian blue nanoparticles according to claim 3 or 4, characterized in that: The following steps are involved: S1, mixing potassium ferrocyanide, a nanoparticle surface stabilizer, and an acid solution, adding a hafnium salt, and heating the mixture for reaction to obtain hafnium-doped Prussian blue nanoparticles; S2, dispersing the hafnium-doped Prussian blue nanoparticles in water, and adding tannic acid to obtain tannic acid-coated hafnium-doped Prussian blue nanoparticles; S3. Dispersing the tannic acid-coated hafnium-doped Prussian blue nanoparticles in water, adding manganese salt, and obtaining the modified Prussian blue nanoparticles.

6. The preparation method according to claim 5, characterized in that In step S1, the mass ratio of the potassium ferrocyanide, the nanoparticle surface stabilizer and the hafnium salt is (70-105):1:(22-88); And / or, the heating reaction temperature is 100-140° C. and the time is 20-30 hours.

7. The preparation method according to claim 5, characterized in that In step S2, the mass ratio of the tannic acid to the hafnium-doped Prussian blue nanoparticles is (30-50):

1.

8. The preparation method according to claim 5, characterized in that In step S3, the mass ratio of the manganese salt to the tannic acid-coated hafnium-doped Prussian blue nanoparticles is (30-50):

1.

9. A radiotherapy sensitizer, characterized in that The invention comprises the modified Prussian blue nanoparticles according to any one of claims 1 to 4.

10. Use of the modified Prussian blue nanoparticles according to any one of claims 1 to 4, or the radiotherapy sensitizer according to claim 9, in the preparation of radiotherapy drugs for osteosarcoma.

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