Bimodal-labeled nano-hydroxyapatite prepared based on homogeneous in situ replacement method

Abstract

The application belongs to the technical field of material preparation, and particularly relates to a kind of double-nuclide labeled nano-hydroxyapatite based on homogeneous in-situ substitution method. 223 Ra and 89 Zr;The 223 Ra is doped into the nano-hydroxyapatite molecule by homogeneous in-situ substitution of Ca ion;The 89 Zr is adsorbed to the nano-hydroxyapatite.The application also provides a preparation method of the hydroxyapatite.The application realizes the doping of radioactive element 223 Ra into the molecular structure of hydroxyapatite by homogeneous in-situ substitution strategy.In this way, the stability of the nuclide labeled on the nano-hydroxyapatite is extremely strong, almost no label will be removed, and can withstand the test of complex physiological environment.

Description

[0001] Divisional application

[0002] This application is based on application number CN202410598330.9, filed on May 14, 2024, entitled "Radioactive Isotopes". 223 Ra / 89 This is a divisional application of the Chinese invention patent application for "Zr-labeled nano-hydroxyapatite, its preparation method and application". Technical Field

[0003] This invention belongs to the field of materials preparation technology, specifically relating to a dual-nucleolabeled nano-hydroxyapatite prepared based on the homogeneous in-situ substitution method. Background Technology

[0004] Bone tumors are malignant tumors that threaten human health. Post-operative radiotherapy or chemotherapy is needed to further eliminate cancer cells. Currently, internal radionuclide therapy is commonly used. Internal radionuclide therapy involves introducing a radionuclide into the patient's body via intravenous injection, oral administration, or interstitial placement. The emitted rays from the radionuclide then irradiate the tumor cells, killing them and ultimately shrinking or eliminating the lesions. Alpha nuclides, which are currently the most widely studied in the field of nuclear medicine, include... 223 Ra, 225 Ac, 227 Th, 211 At, etc.

[0005] Nano-hydroxyapatite (nHA), as a major inorganic component of bone matrix, possesses good biocompatibility and has significant application value in clinical and drug carrier research, and is already widely used in the clinical treatment of bone defects. Since Ra is in the same group as Ca and has similar physicochemical properties, there are existing methods for introducing α-emitting radionuclides into hydroxyapatite. However, current technologies mostly employ adsorption or chemical synthesis methods to label Ra onto nano-hydroxyapatite. For example, patent CN1972720A, entitled "α-Emitting Hydroxyapatite Microparticles," discloses a method of contacting α-emitting or β-emitting radionuclides in solution with hydroxyapatite nanoparticles to introduce radionuclides into hydroxyapatite. However, the radionuclides introduced by this preparation method tend to delabel over time, lacking stability and clearly unable to withstand the long-term challenges of complex physiological environments.

[0006] In conclusion, it is necessary to propose new methods and strategies to compensate for the shortcomings of existing technologies. Summary of the Invention

[0007] The purpose of this invention is to provide a dual-nucleolabeled nano-hydroxyapatite prepared by the homogeneous in-situ substitution method, which partially solves or alleviates the above-mentioned deficiencies in the prior art. The specific technical solution adopted by this invention is as follows.

[0008] One aspect of the present invention is to provide a dual-nucleolabeled nano-hydroxyapatite prepared based on the homogeneous in-situ substitution method, the specific technical solution of which is as follows.

[0009] A dual-nucleoside labeled nano-hydroxyapatite, wherein the dual nuclides are respectively 223 Ra and 89 Zr; the 223 Ra is incorporated into the nano-hydroxyapatite molecules (structure) through homogeneous in-situ substitution (partial) of Ca ions; 89 Zr is adsorbed onto the nano-hydroxyapatite; the particle size range of the dual-nucleolabeled nano-hydroxyapatite is 200-300 nm.

[0010] Those skilled in the art will understand that "doping" refers to the intentional incorporation of small amounts of other elements or compounds into a material or substance in order to improve its properties.

[0011] Furthermore, the nano-hydroxyapatite is synthesized from Ca(NO3)2·4H2O and (NH4)2HPO4; the calcium-to-phosphorus ratio of Ca(NO3)2·4H2O and (NH4)2HPO4 is 0.6.

[0012] The 223 Ra by 223 RaCl2 solution is provided, the 223 The radioactivity of the RaCl2 solution is 0.3-0.5 MBq; 89 Zr by 89 ZrCl4 solution is provided, the 89 The radioactivity of ZrCl4 solution is 120-150 MBq.

[0013] As a preferred option, the 223 The radioactivity of the RaCl2 solution is 0.37 MBq; 89 The radioactivity of the ZrCl4 solution is 150 MBq.

[0014] Furthermore, the aforementioned 223 RaCl2 solution and the 89 The ratio of ZrCl4 solution used is 1:1.

[0015] Another aspect of the present invention is to provide a specific preparation method for the above-mentioned dual-nucleolabeled nano-hydroxyapatite, the specific technical solution of which is as follows.

[0016] S01: Place Ca(NO3)2·4H2O in the first reaction vessel, place (NH4)2HPO4 in the second reaction vessel, the volume of the second reaction vessel is smaller than the volume of the first reaction vessel, and then place the second reaction vessel into the first reaction vessel;

[0017] S02: Mix the PEG solution with... 223 The RaCl2 solution mixture is added dropwise to 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 an alkaline pH value.

[0018] S03: Mix the PEG solution with... 223 The RaCl2 solution mixture was added dropwise to the first reaction vessel until the liquid levels in both the first and second reaction vessels were equal. The entire reaction system was then allowed to stand. Further dropwise addition of the PEG solution and... 223 The RaCl2 solution mixture is prepared until the liquid level in the first reaction vessel is higher than the liquid level in the second reaction vessel, creating a liquid level difference.

[0019] S04: At the liquid level difference position, calcium ions react fully with PEG to form a PEG-Ca-PEG linker. The PEG-Ca-PEG linker further reacts with (NH4)2HPO4 to generate nano-hydroxyapatite. Then, the first reaction vessel is sealed and placed at room temperature to allow the nano-hydroxyapatite to fully incubate and obtain the label. 223 Ra nano-hydroxyapatite;

[0020] S05: Centrifuge the product incubated in the first reaction vessel, wash it with deionized water, and then freeze-dry the product;

[0021] S06: Then... 89 ZrCl4 solution was added to the freeze-dried product, 223 RaCl2 solution and the 89 The ZrCl4 solution was used in a 1:1 ratio, shaken, and the free nuclides were removed by centrifugation and washing with distilled water to obtain the dual-nuctopic labeled nano-hydroxyapatite.

[0022] Furthermore, the volume of the second reaction vessel is 1 / 4 of the volume of the first reaction vessel.

[0023] As a preferred embodiment, the PEG6000 aqueous solution has a mass percentage concentration of 6%.

[0024] Furthermore, the pH range of the PEG6000 aqueous solution is 10-12.

[0025] As a preferred embodiment, the pH value of the PEG6000 aqueous solution is 11.

[0026] Furthermore, the freeze-drying conditions in S05 are set as follows: temperature range of -50℃ to -90℃; pressure range of 0.01-0.1MPa; and freezing time of 48-72 h.

[0027] Furthermore, the centrifugation conditions in S05 and S06 are 1000-2000 rpm for 3-10 min; the shaking conditions in S06 are 30℃-37℃, 100-500 rpm for 0.5-1 h.

[0028] Beneficial technical effects:

[0029] (1) This invention provides a method for preparing radionuclide-labeled nano-hydroxyapatite using a radionuclide "homogeneous" in-situ substitution (doping) method. Specifically, based on nanomaterials... 223 Ra can partially replace Ca ions in hydroxyapatite, becoming the basis for the composition of nano-hydroxyapatite. In terms of implementation, the method of this invention uses PEG as a template, firstly mixing the PEG solution with... 223 The RaCl2 solution is mixed, and then the calcium ions in the reaction system react fully with PEG to form a PEG-Ca-PEG linker. This PEG-Ca-PEG linker further reacts with (NH4)2HPO4 to generate nano-hydroxyapatite. This process is used to achieve the transfer of radioactive elements... 223 Ra is doped into the molecular structure of hydroxyapatite. The labeling of nuclides on nano-hydroxyapatite using this method exhibits extremely high stability, with almost no delabeling, and can withstand the challenges of complex physiological environments; nuclides... 223 Ra is slowly released and exerts its therapeutic effect, providing a long-lasting radiotherapy to target cells in the body. Then it is labeled. 89 Zr, 89 Zr is used for in vivo tracking. Therefore, the present invention prepares this... 223 Ra / 89 Zr@nHA has strong security, and any problems can be intervened in a timely manner.

[0030] (2) The present invention prepares this 223 Ra / 89 Zr@nHA is suitable for the preparation of drugs for the treatment of malignant bone tumors. Bone tumors are often accompanied by a decrease in bone biomechanical strength, and bone filling with biomaterials can enhance the biomechanical properties of bone.

[0031] (3) The preparation method of the present invention uses low reagent amounts and low costs, and is suitable for large-scale industrial production. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0033] Figure 1 This is a schematic diagram of the reaction system structure of the present invention;

[0034] Figure 2 The images shown are verification diagrams of the dual-nucleolabeled nano-hydroxyapatite prepared in this invention (A is an electron microscope image, scale bar 100 nm; B is a particle size measurement result; C is an X-ray diffraction pattern; D is a Fourier transform infrared spectrum).

[0035] Figure 3 The results of the stability study are as follows: (A represents the stability of nHA labeled with nuclides obtained by different preparation methods, where C represents the "homogeneous" in-situ substitution (doping) method and S represents the adsorption method; B represents the stability of nHA labeled with nuclides prepared by the "homogeneous" in-situ substitution (doping) method with and without PEG; C represents the pH stability of nHA labeled with nuclides prepared by the method of this invention).

[0036] Figure 4 The results of cytotoxicity and apoptosis detection are shown in Figure A (where A represents the dual-nucleoside-labeled nHA prepared by the method of this invention). 223 A) Cytotoxicity assay of RaCl2 solution; B) Flow cytometry assay of dual-nucleoside-labeled nHA prepared by the method of this invention; C) 223 (Flow cytometry diagram of RaCl2 solution). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0039] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0040] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0041] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges 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 the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0042] Example 1

[0043] This embodiment provides an example of a method for preparing dual-nucleolabeled nano-hydroxyapatite.

[0044] S01: Place Ca(NO3)2·4H2O at the bottom of the 100ml first reaction vessel, place (NH4)2HPO4 at the bottom of the 25ml second reaction vessel, and then place the second reaction vessel into the first reaction vessel.

[0045] S02: Mix the PEG solution with... 223 The RaCl2 solution mixture was added dropwise to the second reaction vessel until it was full. The PEG solution was a 6.00% (w / w) aqueous solution of PEG6000 (the pH of the PEG solution was adjusted to 11 by adding ammonia).

[0046] S03: Mix the PEG solution from S02 with... 223 The RaCl2 solution mixture was added dropwise to the first reaction vessel until the liquid levels in both the first and second reaction vessels were equal. The entire system was allowed to stand for 5 minutes. Then, the PEG solution was continued to be added dropwise to the first reaction vessel. 223The RaCl2 solution mixture is prepared until the liquid level in the first reaction vessel is 5 mm higher than the liquid level in the second reaction vessel. At this 5 mm liquid level difference, calcium ions react fully with PEG to form a PEG-Ca-PEG linker. The PEG-Ca-PEG linker then reacts with (NH4)2HPO4 to generate nano-hydroxyapatite.

[0047] S04: Seal the first reaction vessel and place it at room temperature for 4 days to allow the compounds in the reaction system to fully incubate and obtain the label. 223 Ra nano-hydroxyapatite ( 223 Ra@nHA).

[0048] S05: Centrifuge the product incubated in the first reaction vessel, wash with deionized water, and then freeze-dry the product. The centrifugation parameters are: 1500 rpm for 5 min. The freeze-drying parameters are: temperature range of -50℃ to -90℃, pressure range of 0.01-0.1 MPa, and freezing time of 48-72 h.

[0049] S06: Then... 89 ZrCl4 solution was added to the freeze-dried product, shaken, and the free nuclides were removed by centrifugation and washing with distilled water to obtain dual-nucleic acid labeled nano-hydroxyapatite. The centrifugation parameters were 1500 rpm for 5 min, and the shaking parameters were 37℃, 200 rpm, and 1 h.

[0050] In the above preparation method, the calcium-to-phosphorus ratio of Ca(NO3)2·4H2O and (NH4)2HPO4 is 0.6.

[0051] In the above preparation method, 223 The radioactivity of the RaCl2 solution is 0.37 MBq; 89 The radioactivity of the ZrCl4 solution is 150 MBq, and the volume used is 1 mL.

[0052] Example 2

[0053] This embodiment provides a dual-nucleolabeled nano-hydroxyapatite prepared by the method of Example 1.

[0054] The dual-nucleolabeled nano-hydroxyapatite prepared by the method in Example 1 is referred to as... 223 Ra / 89 Zr nano-hydroxyapatite ( 223 Ra / 89 Zr@nHA). Among them, radionuclides 223Ra was introduced into the interior of nano-hydroxyapatite molecules through a "homogeneous" in-situ substitution (doping) method, and then through positron-emitting nuclides. 89 Zr was used for in vivo tracing. Results are shown below. Figure 2 , Figure 2 This proves that PEG was successfully synthesized using PEG as a template. 223 Ra / 89 Zr nano-hydroxyapatite.

[0055] Example 3

[0056] This embodiment provides an effect verification of dual-nucleoside-labeled nano-hydroxyapatite.

[0057] 3.1 Comparative Experiment of Different Synthesis Methods

[0058] A method for labeling radionuclides using adsorption is provided.

[0059] The specific operation is the same as in Example 1, except that no additives are used during the synthesis process. 223 RaCl2. The labeling and purification steps are as follows:

[0060] The reaction products and 223 After mixing with RaCl2, the mixture was incubated in a dry constant temperature metal bath (30 min, 200 rpm, 40 °C), centrifuged three times (5 min, 1500 rpm), and the free nuclides were removed by centrifugation and washing with distilled water.

[0061] 3.2 Stability test investigation

[0062] The label prepared in Example 1 223 Ra nano-hydroxyapatite (“homogeneous” in-situ substitution (doping) method and 3.1 prepared label 223 Ra nano-hydroxyapatite (adsorption method): 3 μL (1.11 MBq) of the test product was added to 27 μL of phosphate buffer (PBS) and newborn calf serum (FBS), respectively, and mixed thoroughly before being stored at room temperature. The radioactive mixture was spotted and developed at different time points (depending on the half-life of the different radionuclides) using a glass capillary tube. Free radionuclides were removed by centrifugation and washing with distilled water. The stability of the obtained product is defined by formula (1):

[0063] (1);

[0064] Where LR is the labeling rate of the radionuclide. C0 is the cpm value of the introduced radionuclide, measured by a γ-counter. C1 is the cpm value of the supernatant and the washed distilled water, measured by a γ-counter.

[0065] The results are as follows Figure 3 As shown in Figure A. Compared to the control experiment which uses adsorption to label nuclides, the method of this invention differs in that it utilizes a strategy of "homogeneous" in-situ substitution (doping) of radionuclides. 223 The synthesis of Ra@nHA significantly improved the effect achieved. 223 Stability of Ra.

[0066] 3.3 Control experiment without using PEG template in chemical synthesis method

[0067] Methods: Calcium and phosphorus were obtained from calcium nitrate and disodium hydrogen phosphate, respectively. The pH was adjusted to 9 using ammonium hydroxide. 0.25 M calcium nitrate solution was added to a 0.15 M disodium hydrogen phosphate solution and stirred until the Ca / P molar ratio reached 1.67. Then, 0.015 M sodium tripolyphosphate (STPP) was added. 223 RaCl2. The temperature was adjusted to 40-60℃ to form the precursor of nHA. The precipitate was transferred to a stirred hydrothermal reactor and incubated continuously at 160℃ for 8 h. The cooled nHA precipitate was washed successively with deionized water and ethanol and filtered to remove free radionuclides. Finally, the precipitate was dried in a laboratory oven at 50℃ for 24 h.

[0068] 3.4 Stability Test

[0069] The label prepared in Example 1 223 Ra nano-hydroxyapatite (“homogeneous” in-situ substitution (doping) method with PEG) and 3.3 prepared label 223 Ra nano-hydroxyapatite (“homogeneous” in-situ substitution (doping) method no PEG) was tested according to the stability test in section 3.2.

[0070] The results are as follows Figure 3 As shown in B. Compared to the control experiment which did not use a PEG template, the difference in the method of this invention lies in the strategy of "homogeneous" in-situ substitution (doping) of radionuclides based on a PEG template. 223 The synthesis of Ra@nHA significantly improved the effect achieved. 223 Stability of Ra.

[0071] Furthermore, the marker prepared in Example 1 of this invention 223 Ra nano-hydroxyapatite also exhibits good pH stability, such as Figure 3 As shown in C, it can maintain stability in an environment with pH 7.4 ~ pH 10.

[0072] Example 4

[0073] The cytotoxicity and apoptosis detection of the dual-nucleoside-labeled nano-hydroxyapatite prepared in this invention.

[0074] 4.1 Cytotoxicity

[0075] Method steps: (1) Prepare a solution with a concentration of 2.5×10 4 Human osteosarcoma cells (MG-63 cells) and human embryonic lung fibroblasts (MRC-5 cells) resuspended per mL. Three 96-well plates were taken from each group. The outermost ring of wells was blocked with 200 µL of PBS, and 100 µL of the aforementioned cell resuspended solution was added to each of the 6×10 wells in the middle. The plates were incubated for 24 h. After the incubation period, the supernatant in the wells was removed, and 190 µL of the corresponding cell culture medium was added. (2) The cells were then placed in the wells. 223 Ra / 89 Zr@nHA diluted to 7.4 KBq / µL (as shown in the original text) 223 (The dosage of Ra is determined), and 10 gradients are prepared by 2-fold concentration serial dilution, 10 µL per well, and added to cells in 96-well plates, with three controls per gradient. 223 Ra / 89 After Zr@nHA, the 96-well plate was placed in a constant temperature cell incubator. After 96 h, it was removed, the supernatant was aspirated, and the plate was washed three times with 200 µL PBS. (3) Using the same dose 223 RaCl2 solution was used as a control group. (4) Take the MTS cytotoxicity assay kit and prepare the MTS solution using the following method: 20% MTS + 80% cell culture medium. Add 100 µL of the prepared MTS solution to each well of a 96-well plate, mix well, and place in an incubator. Observe the color change every half hour. After the color is suitable, use a spectrophotometer for detection (wavelength 490 nm). Calculate the data and fit the images using Origin software.

[0076] 4.2 Apoptosis Detection

[0077] Method steps: (1) Prepare a solution with a concentration of 2.5×10 4 MG-63 and MRC-5 cell resuspension at 1 / mL. Take two 12-well plates, add 2 mL of the above cell resuspension to each well, and incubate in a cell incubator. (2) After the cells have completely adhered to the plate the next day, divide them into groups of 4, and add 0 KBq, 18.5 KBq, 37 KBq, and 74 KBq of the above resuspension to each group respectively. 223 Ra / 89 Zr@nHA (with 223(The dosage of Ra is determined). After mixing thoroughly by pipetting, place in an incubator for culture. (3) After 96 h, remove the above 12-well plate, transfer the supernatant to a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and remove the supernatant. Add 1 mL of PBS to wash, transfer the washed PBS to the same 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, remove the supernatant, and 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 over, add 1 mL of the corresponding culture medium to stop the digestion. After mixing thoroughly by pipetting, transfer to a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, remove the supernatant, add 1 mL of PBS to wash, transfer the washed PBS to the same 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, remove the supernatant, and repeat three times to completely collect the adherent cells. (5) Use the same dosage 223 RaCl2 solution was used as a control group. (6) Finally, Annexin V-FITC apoptosis detection kit was used, and 500 µL of 1×Binding Buffer solution was added to the centrifuge tube to resuspend the cells. Under light-protected conditions, 5 µL of Annexin V-FITC staining solution and 5 µL of propidium iodide (PI) staining solution were added to the tube respectively, and the mixture was mixed by pipetting and incubated at room temperature in the dark for 20 min. After incubation, flow cytometry was used to detect the cells immediately (if immediate detection is not possible, the cells should be placed on ice in the dark and the detection should be completed within 1 h). The data were calculated and images were fitted using Origin software.

[0078] The results are as follows Figure 4 As shown. Figure 4 As shown in A, 223 Ra / 89 Zr@nHA exhibited dose-dependent cytotoxicity against both cell lines, and the toxicity was greater than [a certain value] in both cell lines. 223 RaCl2. Secondly, compared to human embryonic lung cells MRC-5, 223 Ra / 89 Zr@nHA exhibits specific dose-dependent cytotoxicity against human osteosarcoma cells MG-63. After treatment with the highest dose (37 KBq) for 72 h, MG-63 cells showed [results in] cytotoxicity [inhibiting cytotoxicity]. 223 Ra / 89 The survival rate of Zr@nHA treatment was approximately 32.72%, significantly lower than that of treatments without Zr@nHA. 223 65.22% after RaCl2 treatment. (Explanation) 223 Ra / 89 Zr@nHA can effectively kill target cells.

[0079] Flow cytometry was used to analyze the cells. 223 Ra / 89 Zr@nHA and 223 Whether the killing effect of RaCl2 on MG-63 cells is related to its triggering of programmed cell death is as follows: Figure 4 As shown in B and C. Analysis of the apoptotic cell population (Annexin V-fitc / PI) revealed that at high doses, 223 Ra / 89 Zr@nHA showed the strongest apoptosis-inducing ability, and both drugs exhibited dose-dependent apoptosis induction. After treatment with the highest dose of 37 KBq for 72 hours, [the following results were observed]. 223 Ra / 89 The total apoptosis rate of Zr@nHA-treated MG-63 cells was approximately 30.86%, with early apoptosis (Annexin V+ / PI-) at approximately 7.85% and late apoptosis (Annexin V+ / PI+) at approximately 23.01%. The same dose... 223 After RaCl2 treatment of MG-63 cells for the same duration, the total apoptosis rate was approximately 19.41%, with early apoptosis (Annexin V+ / PI-) at approximately 3.27% and late apoptosis (Annexin V+ / PI+) at approximately 15.75%. MG-63 cells at higher doses... 223 Ra / 89 Under the action of Zr@nHA, apoptosis was more pronounced. Finally, analysis of cell necrosis (Annexin V- / PI+) showed that both drugs induced cell necrosis in MG-63 cells, but the proportion was small and there was no dose-dependent or specific induction of cell necrosis. This indicates that... 223 Ra / 89 Zr@nHA and 223 RaCl2-induced cell death primarily occurs by activating programmed cell death and inducing apoptosis.

[0080] in conclusion: 223 Ra / 89 Zr@nHA exhibits dose-dependent and specific cytotoxicity against MG-63 bone tumor cells, and at the same dosage, its cell-killing effect is superior to that of other treatments. 223 RaCl2 solution.

[0081] Overall, using PEG doping as a template improved 223 Ra@nHA exhibits specific activity and a more effective killing effect on bone tumor cells compared to the same dose. 223 Ra is higher and stronger. It can kill tumors while... 89 Zr labeling can enable real-time tracking of radiopharmaceuticals within living organisms.

[0082] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0083] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A dual-nucleolabeled nano-hydroxyapatite, characterized in that, The nano-hydroxyapatite was synthesized from Ca(NO3)2·4H2O and (NH4)2HPO4; the two nuclides were respectively... 223 Ra and 89 Zr; the 223 Ra is incorporated into the nano-hydroxyapatite molecules through homogeneous in-situ substitution of Ca ions. The method involves using PEG as a template, first mixing a PEG solution with a 223RaCl2 solution, then reacting the calcium ions in the reaction system with PEG to form a PEG-Ca-PEG linker. This PEG-Ca-PEG linker further reacts with (NH4)2HPO4 to generate nano-hydroxyapatite. 89 Zr is adsorbed onto the nano-hydroxyapatite; the particle size range of the dual-nucleolabeled nano-hydroxyapatite is 200-300 nm.

2. The dual-nucleolabeled nano-hydroxyapatite as described in claim 1, characterized in that, The calcium-to-phosphorus ratio of Ca(NO3)2·4H2O and (NH4)2HPO4 is 0.

6.

3. The dual-nucleolabeled nano-hydroxyapatite as described in claim 1, characterized in that, The 223 Ra by 223 RaCl2 solution is provided, the 223 The radioactivity of the RaCl2 solution is 0.3-0.5 MBq; 89 Zr by 89 ZrCl4 solution is provided, the 89 The radioactivity of ZrCl4 solution is 120-150 MBq.

4. The dual-nucleolabeled nano-hydroxyapatite as described in claim 3, characterized in that, The 223 RaCl2 solution and the 89 The ratio of ZrCl4 solution used is 1:

1.

5. The method for preparing dual-nucleolabeled nano-hydroxyapatite according to claim 1, characterized in that, Includes the following steps: S01: Place Ca(NO3)2·4H2O in the first reaction vessel, place (NH4)2HPO4 in the second reaction vessel, the volume of the second reaction vessel is smaller than the volume of the first reaction vessel, and then place the second reaction vessel into the first reaction vessel; S02: Mix the PEG solution with... 223 The RaCl2 solution mixture was added dropwise to the second reaction vessel until it was full; the PEG solution was an aqueous solution of PEG6000 with a mass percentage concentration of 5-10% and a pH range of 10-12. S03: Mix the PEG solution with... 223 The RaCl2 solution mixture was added dropwise to the first reaction vessel until the liquid levels in both the first and second reaction vessels were equal. The entire reaction system was then allowed to stand. Further dropwise addition of the PEG solution and... 223 The RaCl2 solution mixture is prepared until the liquid level in the first reaction vessel is higher than the liquid level in the second reaction vessel, creating a liquid level difference. S04: At the liquid level difference position, calcium ions react fully with PEG to form a PEG-Ca-PEG linker. The PEG-Ca-PEG linker further reacts with (NH4)2HPO4 to generate nano-hydroxyapatite. Then, the first reaction vessel is sealed and placed at room temperature to allow the nano-hydroxyapatite to fully incubate and obtain the label. 223 Ra nano-hydroxyapatite; S05: Centrifuge the product incubated in the first reaction vessel, wash it with deionized water, and then freeze-dry the product; S06: Then... 89 ZrCl4 solution was added to the freeze-dried product, 223 RaCl2 solution and the 89 The ZrCl4 solution was used in a 1:1 ratio, shaken, and the free nuclides were removed by centrifugation and washing with distilled water to obtain the dual-nuctopic labeled nano-hydroxyapatite.

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

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

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

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

Images