Double-nuclide labeled nano-hydroxyapatite prepared based on homogeneous in-situ substitution method

Abstract

The invention belongs to the technical field of material preparation, and particularly relates to double-nuclide labeled nano-hydroxyapatite prepared based on a homogeneous in-situ substitution method. The invention firstly provides nano hydroxyapatite labeled by double nuclides, wherein the double nuclides are respectively 223Ra and 89Zr; the 223Ra is doped into the nano hydroxyapatite molecules by replacing Ca ions in a homogeneous in-situ manner; and the 89Zr is adsorbed on the nano hydroxyapatite. The invention also provides a preparation method of the hydroxyapatite. According to the invention, a radioactive element 223Ra is doped into a hydroxyapatite molecular structure through a homogeneous in-situ substitution strategy. The nuclides marked on the nano-hydroxyapatite by the method are extremely high in stability, almost do not fall off, and can stand the test of a complex physiological environment.

Description

[0001] Divisional application This application is a divisional application of a Chinese invention patent application with the application number CN202410598330.9, the application date of May 14, 2024, and the invention title of "Radioisotope 223 Ra / 89 Zr-labeled nano-hydroxyapatite and its preparation method and application". Technical field

[0002] The present invention belongs to the technical field of material preparation, and specifically relates to a dual-radionuclide-labeled nano-hydroxyapatite prepared based on the method of homogeneous in-situ substitution. Background technique

[0003] Bone tumors are malignant tumors that threaten human health. After bone tumor surgery, radiotherapy or chemotherapy is required to further remove cancer cells. Currently, the method of radionuclide internal irradiation treatment is mostly used. Radionuclide internal irradiation treatment is to introduce radionuclides into the patient's body through intravenous injection, oral administration or interstitial implantation, and then use the rays emitted by the radionuclides to irradiate tumor cells, so as to achieve the effect of killing tumor cells, and finally make the lesion shrink or disappear. Currently, the α-radionuclides widely studied in the field of nuclear medicine include 223 Ra, 225 Ac, 227 Th, 211 At, etc.

[0004] Nano-hydroxyapatite (nHA), as the main inorganic component of bone matrix, has good biocompatibility and has good application value in clinical and drug carrier research, and has been widely used in the clinical treatment of bone defects. Since Ra is in the same group as Ca and has similar physical and chemical properties, there have been schemes to introduce α-emitting radionuclides into hydroxyapatite. However, in the prior art, the adsorption method or chemical synthesis method is mostly used to label Ra onto nano-hydroxyapatite. For example, the patent with the publication number CN1972720A and the invention title of "α-emitting hydroxyapatite particles" discloses a method of contacting a solution of an α-emitting radionuclide or a β-emitting radionuclide with hydroxyapatite nanoparticles, thereby introducing the radionuclide into hydroxyapatite. The radionuclides introduced by this preparation method will continuously de-label over time, and the stability is insufficient, and it is obviously unable to cope with the long-term challenges of complex physiological environments.

[0005] In summary, it is necessary to propose new method strategies to make up for the deficiencies of the prior art. Summary of the invention

[0006] The object of the present invention is to provide a dual-radionuclide labeled nano-hydroxyapatite prepared by a homogeneous in-situ substitution method, which partially solves or alleviates the above deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0007] On the one hand, the present invention provides a dual-radionuclide labeled nano-hydroxyapatite prepared by a homogeneous in-situ substitution method, and the specific technical solution is as follows.

[0008] A dual-radionuclide labeled nano-hydroxyapatite, wherein the dual radionuclides are respectively 223 Ra and 89 Zr; the 223 Ra is doped into the nano-hydroxyapatite molecule (structure) by homogeneously in-situ substituting (partially) Ca ions; the 89 Zr is adsorbed onto the nano-hydroxyapatite; the particle size range of the dual-radionuclide labeled nano-hydroxyapatite is 200 - 300 nm.

[0009] Those skilled in the art can understand that the "doping" means that in order to improve the performance of a material or substance, a small amount of other elements or compounds are purposefully incorporated into this material or substance.

[0010] Furthermore, the nano-hydroxyapatite is synthesized from Ca(NO 3 ) 2 ·4H 2 O and (NH 4 ) 2 HPO 4 ; the calcium-phosphorus ratio of the Ca(NO 3 ) 2 ·4H 2 O and the (NH 4 ) 2 HPO 4 is 0.6.

[0011] The 223 Ra is provided by a 223 RaCl 2 solution, and the radioactivity of the 223 RaCl 2 solution is 0.3 - 0.5 MBq; the 89 Zr is provided by a 89 ZrCl 4 solution, and the radioactivity of the 89 ZrCl 4 solution is 120 - 150 MBq.

[0012] As a preference, the radioactivity of the 223 RaCl 2 solution is 0.37 MBq; the89 ZrCl 4 The radioactivity of the solution is 150 MBq.

[0013] Furthermore, the 223 RaCl 2 solution and the 89 ZrCl 4 solution are used in a ratio of 1:1.

[0014] Another aspect of the present invention is to provide a specific preparation method of the above-mentioned dual-radionuclide-labeled nano-hydroxyapatite, and the specific technical solution is as follows.

[0015] S01: Place Ca(NO 3 ) 2 ·4H 2 O in the first reaction vessel, place (NH 4 ) 2 HPO 4 in the second reaction vessel, the volume of the second reaction vessel is smaller than that of the first reaction vessel, and then place the second reaction vessel into the first reaction vessel; S02: Drop the mixed solution of the PEG solution and the 223 RaCl 2 solution into the second reaction vessel until it is full; the PEG solution is an aqueous solution of PEG6000 with a mass percentage concentration of 5-10% and a pH value of alkaline; S03: Drop the mixed solution of the PEG solution and the 223 RaCl 2 solution into the first reaction vessel until the liquid levels in the first reaction vessel and the second reaction vessel are the same, let the entire reaction system stand, and further continue to drop the mixed solution of the PEG solution and the 223 RaCl 2 solution into the first reaction vessel until the liquid level in the first reaction vessel is higher than that in the second reaction vessel, generating a liquid level difference; S04: At the liquid level difference position, calcium ions react fully with PEG to form a PEG-Ca-PEG conjugate, and the PEG-Ca-PEG conjugate further reacts with (NH 4 ) 2 HPO 4 to generate nano-hydroxyapatite, then seal the first reaction vessel and place it at room temperature to allow the nano-hydroxyapatite to incubate fully to obtain nano-hydroxyapatite labeled with 223 Ra; S05: Centrifuge the product incubated in the first reaction vessel, wash it with deionized water, and then freeze-dry the product; S06: Then add the 89 ZrCl 4 solution to the freeze-dried product. The 223 RaCl 2 solution and the 89 ZrCl 4 solution are used in a ratio of 1:1. Shake, and remove free radionuclides by centrifugation and washing with distilled water to obtain the dual-radionuclide-labeled nano-hydroxyapatite.

[0016] Furthermore, the volume of the second reaction vessel is 1 / 4 of the volume of the first reaction vessel.

[0017] As a preference, the mass percentage concentration of the PEG6000 aqueous solution is 6%.

[0018] Furthermore, the pH value range of the PEG6000 aqueous solution is 10 - 12.

[0019] As a preference, the pH value of the PEG6000 aqueous solution is 11.

[0020] Furthermore, the conditions for freeze-drying in S05 are set as follows: the temperature range is -50°C to -90°C; the pressure range is 0.01 - 0.1 MPa, and the freeze-drying time is 48 - 72 h.

[0021] Furthermore, the centrifugation operation conditions in S05 and S06 are 1000 - 2000 rpm for 3 - 10 min; the shaking operation conditions in S06 are 30°C - 37°C, 100 - 500 rpm, and 0.5 - 1 h.

[0022] Beneficial technical effects: (1) The present invention provides a method for preparing radionuclide-labeled nano-hydroxyapatite by the method of "isotopic" in-situ substitution (doping) with radioactive nuclides. Specifically, based on the 223 Ra in the nanomaterial can partially replace the position of Ca ions in hydroxyapatite and become the basis for the composition of nano-hydroxyapatite. From the implementation mode, the method of the present invention uses PEG as a template. First, the PEG solution is mixed with the 223 RaCl 2 solution. Then, the calcium ions in the reaction system react fully with PEG to form a PEG-Ca-PEG conjugate, and this PEG-Ca-PEG conjugate further reacts with (NH4) 2 HPO 4 to generate nano-hydroxyapatite. In this way, the radioactive element 223Ra is doped into the molecular structure of hydroxyapatite. The stability of the radionuclide labeled on the nano-hydroxyapatite in this way is extremely strong, and almost no de-labeling occurs, and it can withstand the test of a complex physiological environment; the radionuclide 223 Ra is slowly released and exerts a therapeutic effect, providing a lasting radiotherapy effect on target cells in the human body. Then it is labeled with 89 Zr, 89 Zr is used for in vivo tracing. Therefore, this 223 Ra / 89 Zr@nHA prepared by the present invention has strong safety, and problems can be intervened in time if there are any.

[0023] (2)This 223 Ra / 89 Zr@nHA prepared by the present invention is suitable for preparing drugs related to the treatment of malignant bone tumors. After the occurrence of bone tumors, the biomechanical strength of the bone often decreases, and the bone filling of biomaterials can enhance the biomechanical properties of bone.

[0024] (3)The reagents used in the preparation method of the present invention have low dosage and low cost, and are suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to actual scale. Obviously, the following-described drawings are some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts.

[0026] Figure 1 is a schematic diagram of the reaction system structure of the present invention; Figure 2 is a verification diagram of the dual-radionuclide-labeled nano-hydroxyapatite prepared by the present invention (A is an electron microscope image, scale bar 100 nm; B is a particle size measurement result diagram; C is an X-ray diffraction diagram; D is a Fourier transform infrared spectrum diagram); Figure 3 is the stability investigation result (A is the stability investigation of nHA with labeled radionuclides obtained by different preparation methods, where C represents the "homogeneous" in-situ substitution (doping) method, and S represents the adsorption method; B is the stability investigation of nHA with labeled radionuclides prepared with and without PEG under the "homogeneous" in-situ substitution (doping) method; C is the pH stability investigation of nHA with radionuclide labels prepared by the method of the present invention); Figure 4Results of cytotoxicity and apoptosis detection (A shows the cytotoxicity test diagram of the solution of binuclear nuclide-labeled nHA prepared by the method of the present invention and 223 RaCl 2 ; B shows the flow cytometry experimental diagram of the binuclear nuclide-labeled nHA prepared by the method of the present invention; C shows 223 RaCl 2 solution flow cytometry experimental diagram). Detailed implementation manners

[0027] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0028] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0029] As used herein, "a plurality" means two or more, that is, it includes two, three, four, five, etc.

[0030] As used in this specification, the term "about" typically represents + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0031] In this specification, certain embodiments may be disclosed in a format within a certain range. It should be understood that this description of "within a certain range" is only for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0032] Example 1 This example provides an example of a preparation method for binuclear nuclide-labeled nano-hydroxyapatite.

[0033] S01: Add Ca(NO 3 ) 2 ·4H2 O is placed at the bottom of the first reaction vessel with a volume of 100 ml, and (NH 4 ) 2 HPO 4 is placed at the bottom of the second reaction vessel with a volume of 25 ml, and then the second reaction vessel is placed into the first reaction vessel.

[0034] S02: The mixed solution of the PEG solution and 223 RaCl 2 solution is dropped into the second reaction vessel until it is full. The PEG solution is an aqueous solution of PEG6000 with a mass percentage concentration of 6.00% (the pH of the PEG solution is adjusted to 11 by adding ammonia water).

[0035] S03: The mixed solution of the PEG solution and 223 RaCl 2 solution in S02 is dropped into the first reaction vessel until the liquid levels in the first reaction vessel and the second reaction vessel are the same. Let the whole system stand for 5 min, and then continue to drop the mixed solution of the PEG solution and 223 RaCl 2 solution into the first reaction vessel until the liquid level in the first reaction vessel is 5 mm higher than that in the second reaction vessel. At this 5-mm liquid level difference, calcium ions react fully with PEG to form a PEG-Ca-PEG conjugate, and the PEG-Ca-PEG conjugate continues to react with (NH 4 ) 2 HPO 4 to generate nano-hydroxyapatite.

[0036] S04: Seal the first reaction vessel and place it at room temperature for 4 days to fully incubate the compounds in the reaction system to obtain nano-hydroxyapatite labeled with 223 Ra ( 223 Ra@nHA).

[0037] S05: Centrifuge the incubated product in the first reaction vessel, wash it with deionized water, and then lyophilize the product. Among them, the parameters of the centrifugation operation are 1500 rpm and centrifugation for 5 min. The parameters of lyophilization are: the temperature range is adjusted from -50°C to -90°C, the pressure range is 0.01 - 0.1 MPa, and the freezing time is 48 - 72 h.

[0038] S06: Then 89 ZrCl 4The solution was added to the freeze-dried product, shaken, and free nuclides were removed by centrifugation and washing with distilled water to obtain double nucleic acid labeled nanohydroxyapatite. The parameters of the centrifugation operation were 1500 rpm, centrifugation for 5 minutes, and the parameters of the shaking operation were 37°C, 200 rpm, and shaking for 1 hour.

[0039] In the above preparation method, Ca(NO 3 ) 2 ·4H 2 O and (NH 4 ) 2 HPO 4 The calcium to phosphorus ratio is 0.6.

[0040] In the above preparation method, 223 RaCl 2 The radioactivity of the solution was 0.37 MBq; 89 ZrCl 4 The radioactivity of the solution was 150 MBq and the dosage was 1 mL.

[0041] Example 2 This embodiment provides a dual-nuclide labeled nano-hydroxyapatite prepared by the method of Example 1.

[0042] The dual-nuclide labeled nano-hydroxyapatite prepared by the method of Example 1 is referred to as 223 Ra / 89 Zr nanohydroxyapatite ( 223 Ra / 89 Zr@nHA). Among them, radionuclides 223 Ra was introduced into the interior of the nano-hydroxyapatite molecule by the method of "homogeneous" in-situ substitution (doping), and then 89 Zr was used for in vivo tracing. Figure 2 , Figure 2 It was proved that PEG was successfully synthesized 223 Ra / 89 Zr nanohydroxyapatite.

[0043] Example 3 This example provides a verification of the effect of dual-nuclide labeled nano-hydroxyapatite.

[0044] 3.1 Comparative test of different synthesis methods Provided is a preparation method for labeling nuclides using adsorption means.

[0045] The specific operation is the same as that in Example 1, except that no 223 RaCl 2 The labeling and purification steps are as follows: Mix the reaction product with 223 RaCl 2 and incubate in a dry-block heater (30 min, 200 rpm, 40 °C), centrifuge three times (5 min, 1500 rpm), and remove free radionuclides by centrifugation and washing with distilled water.

[0046] 3.2 Stability experiment investigation Take the labeled 223 Ra nano-hydroxyapatite prepared in Example 1 (“homogeneous” in-situ substitution (doping) method) and the labeled 223 Ra nano-hydroxyapatite prepared in 3.1 (adsorption method). Take 3 μL (1.11 MBq) of the product to be tested and add it to 27 μL of phosphate buffer (PBS) and fetal bovine serum (FBS) respectively. After thorough and uniform mixing, store at room temperature. At different time points (depending on the half-life of different radionuclides), take samples of the above radioactive mixture with a glass capillary and develop. Remove free radioactive nuclides by centrifugation and washing with distilled water. The stability of the prepared product is defined by the following formula (1): (1); where LR is the labeling rate of the radionuclide. C 0 is the cpm value of the input radionuclide, measured by a γ-counter. C 1 is the cpm value obtained by adding the supernatant measured by a γ-counter and the distilled water after washing.

[0047] The results are as Figure 3 shown in A. Compared with the control experiment using adsorption to label the nuclide, the difference of the method of the present invention lies in using the strategy of “homogeneous” in-situ substitution (doping) of the radionuclide to 223 synthesize Ra@nHA, and the achieved effect is to significantly improve the 223 stability of Ra.

[0048] 3.3 Control experiment without using PEG template under chemical synthesis method Method: The sources of calcium and phosphorus are calcium nitrate and disodium hydrogen phosphate respectively, and the pH is adjusted to 9 using ammonium hydroxide. Add 0.25 M calcium nitrate solution to 0.15 M disodium hydrogen phosphate solution, stir until the Ca / P molar ratio is 1.67, then add 0.015 M sodium tripolyphosphate (STPP) and 223 RaCl 2. Adjust the temperature to 40 - 60 °C to form the precursor of nHA. Transfer the precipitate to a stirred hydrothermal reactor and keep it at 160 °C for 8 h continuously. Wash and filter the cooled nHA precipitate successively with deionized water and ethanol to remove free radionuclides. Finally, dry the precipitate in a laboratory oven at 50 °C for 24 h.

[0049] 3.4 Stability investigation The labeled product prepared in Example 1 223 Ra - doped nano - hydroxyapatite (prepared by "homogeneous" in - situ substitution (doping) method) with PEG and the labeled product prepared in 3.3 223 Ra - doped nano - hydroxyapatite (prepared by "homogeneous" in - situ substitution (doping) method without PEG) were tested according to the stability investigation test in 3.2.

[0050] The results are as Figure 3 shown in Figure B. Compared with the control test without using the PEG template, the difference of the method of the present invention lies in the strategy of "homogeneous" in - situ substitution (doping) of radionuclides based on the PEG template for the 223 synthesis of Ra@nHA, and the achieved effect is to significantly improve the 223 stability of Ra.

[0051] In addition, the labeled 223 Ra - doped nano - hydroxyapatite prepared in Example 1 of the present invention also has good pH stability. As Figure 3 shown in Figure C, it can maintain stability in the pH range of 7.4 - 10.

[0052] Example 4 Cytotoxicity and apoptosis detection of the dual - nuclide - labeled nano - hydroxyapatite prepared by the present invention.

[0053] 4.1 Cytotoxicity Method steps: (1) Prepare cell suspensions of human osteosarcoma cells (MG - 63 cells) and human embryonic lung fibroblasts (MRC - 5 cells) at a concentration of 2.5×10 4 cells / mL. Take three 96 - well plates for each group. Seal the outermost - circle wells with 200 µL of PBS, and add 100 µL of the above - mentioned cell suspension to each of the 6×10 wells in the middle, and let it stand for 24 h. After the standing time, remove the supernatant of the cells in the wells and add 190 µL of the corresponding cell culture medium. (2) Dilute 223 Ra / 89 Zr@nHA to 7.4 KBq / µL (determined by the dose of 223 Ra), and dilute it with a 2 - fold concentration gradient for 10 gradients. Add 10 µL of each dilution to the cells in the 96 - well plates, with three controls for each gradient. After adding 223 Ra / 89 After Zr@nHA, the 96-well plate was placed in a constant-temperature cell incubator and taken out after 96 h. The supernatant was aspirated, and then washed three times with 200 μL of PBS. (3) A control group was set with the same dose of 223 RaCl 2 solution. (4) Take the MTS cell cytotoxicity detection kit and prepare the MTS solution. The specific method is: 20% MTS + 80% cell culture medium. Add 100 μL of the above-prepared MTS solution to each well of the 96-well plate, mix well and place it in the incubator. Observe the color change every half hour. After the color is appropriate, use a spectrophotometer for detection (wavelength 490 nm). Calculate the data and fit the image through Origin software.

[0054] 4.2 Apoptosis detection Method steps: (1) Prepare resuspension solutions of MG-63 cells and MRC-5 cells at a concentration of 2.5×10 4 cells / mL. Take 2 12-well plates, add 2 mL of the above cell resuspension to each well, and place them in the cell incubator for culture. (2) After the cells adhered completely the next day, take 4 wells as a group, and add 0 KBq, 18.5 KBq, 37 KBq, and 74 KBq of 223 Ra / 89 Zr@nHA (determined by the dose of 223 Ra). After pipetting and mixing evenly, place them in the incubator for culture. (3) Take out the above 12-well plates after 96 h, transfer the supernatant to a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant. Add 1 mL of PBS for washing, transfer the washed PBS to the same 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant. Repeat three times to completely collect the cells in the supernatant. (4) Next, add 100 μL of trypsin to each well of the 12-well plate for digestion. After the digestion time is up, add 1 mL of the corresponding culture medium to terminate the digestion. After pipetting and mixing evenly, transfer them to a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant. Add 1 mL of PBS for washing, transfer the washed PBS to the same 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant. Repeat three times to completely collect the adherent cells. (5) A control group was set with the same dose of 223 RaCl 2A control group was set up for the solution. (6) Finally, an Annexin V-FITC apoptosis detection kit was taken, 500 μL of 1×Binding Buffer solution was added to the centrifuge tube, and the cells were resuspended. Under light-proof conditions, 5 μL of Annexin V-FITC staining solution and 5 μL of propidium iodide (PI) staining solution were respectively added to the tube. After pipetting and mixing evenly, the mixture was incubated at room temperature in the dark for 20 min. Immediately after the incubation was completed, it was detected using a flow cytometer (if it could not be detected immediately, it should be placed statically in the dark on ice and detected within 1 h). And the data was calculated and the image was fitted through Origin software.

[0055] The results are as Figure 4 shown. Figure 4 As shown in A, 223 Ra / 89 Ra / Zr@nHA showed dose-dependent cytotoxicity to both cell lines, and the toxicity was greater than that of 223 RaCl 2 . Secondly, compared with human embryonic lung cells MRC-5, 223 Ra / 89 Ra / Zr@nHA showed specific dose-dependent cytotoxicity to human osteosarcoma cells MG-63. After treatment at the highest dose (37 KBq) for 72 h, the survival rate of MG-63 after treatment with 223 Ra / 89 Ra / Zr@nHA was about 32.72%, which was much lower than 65.22% after treatment with 223 RaCl2. It indicated that 223 Ra / 89 Ra / Zr@nHA could effectively kill target cells.

[0056] Flow cytometry was used to analyze whether the killing effect of 223 Ra / 89 Ra / Zr@nHA and 223 RaCl 2 on MG-63 cells was related to whether it triggered programmed cell death. The results are as Figure 4 shown in B and C. Through the analysis of the apoptotic cell population (AnnexinV-FITC / PI), it was found that at high doses, 223 Ra / 89 Ra / Zr@nHA had the strongest ability to induce apoptosis, and both drugs showed dose-dependent induction of apoptosis. After treatment at the highest dose of 37 KBq for 72 h, after 223 Ra / 89The total apoptosis rate of Zr@nHA-treated MG-63 cells was approximately 30.86%, among which early apoptosis (Annexin V+ / PI-) was approximately 7.85% and late apoptosis (Annexin V+ / PI+) was approximately 23.01%. After treatment of MG-63 cells with the same dose of 223 RaCl 2 for the same period of time, the total apoptosis rate was approximately 19.41%, among which early apoptosis (Annexin V+ / PI-) was approximately 3.27% and late apoptosis (Annexin V+ / PI+) was approximately 15.75%. MG-63 cells showed more obvious apoptosis under the action of a higher dose of 223 Ra / 89 Zr@nHA. Finally, by analyzing the cell necrosis (Annexin V- / PI+) situation, it can be seen that both drugs induced cell necrosis in MG-63 cells, but the proportion was small and there was no dose-dependent and specific induction of cell necrosis. This indicates that 223 Ra / 89 Zr@nHA and 223 RaCl 2 led to cell death mainly through activating programmed cell death and inducing apoptosis.

[0057] Conclusion: 223 Ra / 89 Zr@nHA had a dose-dependent and specific cytotoxicity to bone tumor cells MG-63, and its killing effect on cells was better than that of 223 RaCl 2 solution at the same dose.

[0058] Generally speaking, doping PEG as a template improved the 223 specific activity of 223 Ra@nHA, and its specific killing effect on bone tumor cells was higher and stronger than that of the same dose of 89 Ra. And while killing tumors,

[0059] The label of

[0060] 89 Zr could achieve the purpose of real-time tracking of radioactive drugs in vivo.

[0059] It should be noted that in this article, the term "including", "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including a..." does not exclude the existence of additional identical elements in the process, method, article or device including that element.

[0060] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit of the present invention and the scope protected by the claims. All of these fall within the protection scope of the present invention.

Claims

1. A dual-nuclide labeled nano-hydroxyapatite, characterized in that: The binuclear nuclides are 223 Ra and 89 Zr; 223 Ra is doped into the nano-hydroxyapatite molecule by homogeneous in-situ substitution of Ca ions; 89 Zr is adsorbed onto the nano-hydroxyapatite; the particle size of the dual-nuclide labeled nano-hydroxyapatite is in the range of 200-300nm.

2. The dual-nuclide labeled nano-hydroxyapatite according to claim 1, characterized in that: The nano hydroxyapatite is synthesized from Ca(NO3)2·4H2O and (NH4)2HPO4; the calcium-phosphorus ratio of the Ca(NO3)2·4H2O and the (NH4)2HPO4 is 0.

6.

3. The dual-nuclide labeled nano-hydroxyapatite according to claim 1, characterized in that: Said 223 Ra 223 RaCl2 solution is provided, 223 The radioactivity of the RaCl2 solution is 0.3-0.5 MBq; 89 Zr is 89 ZrCl4 solution is provided, the 89 The radioactivity of ZrCl4 solution is 120-150 MBq.

4. The dual-nuclide labeled nano-hydroxyapatite according to claim 3, characterized in that: Said 223 RaCl2 solution and 89 The dosage ratio of ZrCl4 solution is 1:

1.

5. The method for preparing a dual-nuclide labeled nano-hydroxyapatite according to any one of claims 1 to 4, characterized in that: The following steps are involved: S01: Ca(NO3)2·4H2O is placed in a first reaction container, (NH4)2HPO4 is placed in a second reaction container, the volume of the second reaction container is smaller than the volume of the first reaction container, and then the second reaction container is placed in the first reaction container; S02: Mix PEG solution with 223 The mixed solution of RaCl2 solution is added dropwise into the second reaction container until it is full; the PEG solution is a PEG6000 aqueous solution with a mass percentage concentration of 5-10% and an alkaline pH value; S03: mixing the PEG solution with 223 The mixed solution of RaCl2 solution is added dropwise to the first reaction container until the liquid levels of the first reaction container and the second reaction container are the same, the entire reaction system is allowed to stand, and the PEG solution and the mixed solution of RaCl2 solution are further added dropwise to the first reaction container. 223 RaCl2 solution, until the liquid level in the first reaction container is higher than the liquid level in the second reaction container, resulting in a liquid level difference; S04: At the position of the liquid level difference, calcium ions react fully with PEG to form a PEG-Ca-PEG linker, and the PEG-Ca-PEG linker further reacts with (NH4)2HPO4 to generate nano-hydroxyapatite, and then the first reaction container is sealed and placed at room temperature to allow the nano-hydroxyapatite to be fully incubated to obtain the labeled 223 Nano-hydroxyapatite of Ra; S05: centrifuging the product after incubation in the first reaction container, washing it with deionized water, and then freeze-drying the product; S06: 89 ZrCl4 solution is added to the freeze-dried product. 223 RaCl2 solution and 89 The ZrCl4 solution is used in a ratio of 1:1, shaken, and free nuclides are removed by centrifugation and washing with distilled water to obtain the dual-nuclide labeled nano-hydroxyapatite.

6. The preparation method according to claim 5, characterized in that: The volume of the second reaction container is 1 / 4 of the volume of the first reaction container.

7. The preparation method according to claim 5, characterized in that: The mass percentage concentration of the PEG6000 aqueous solution is 6%.

8. The preparation method according to claim 5, characterized in that: The pH value of the PEG6000 aqueous solution ranges from 10 to 12.

9. The preparation method according to claim 5, characterized in that: The freeze drying conditions in S05 are set as follows: temperature range is -50°C to -90°C; pressure range is 0.01-0.1MPa, and freezing time is 48-72 hours.

10. The preparation method according to claim 5, characterized in that: The centrifugal operation conditions in S05 and S06 are 1000-2000 rpm, centrifugation for 3-10 min; the shaking operation conditions in S06 are 30° C.-37° C., 100-500 rpm, 0.5-1 h.

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