Preparation for osteosarcoma diagnosis and / or treatment and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a preparation for osteosarcoma diagnosis and / or treatment and a preparation method and application thereof, and the preparation is obtained by coupling CS polypeptide and a bifunctional chelating agent and then chelating gadolinium; the amino acid sequence of the CS polypeptide is as shown in SEQ ID NO. 1. The preparation provided by the invention can specifically target osteosarcoma, can also realize visual MR imaging of osteosarcoma focuses, and realizes neutron capture therapy and common photon sensitization therapy of osteosarcoma, namely diagnosis and treatment integration. The preparation has the advantages of being high in treatment specificity, remarkable in effect and small in damaged normal tissue, is a new treatment mode with good clinical application prospects, and is expected to finally improve prognosis of osteosarcoma patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a preparation for the diagnosis and / or treatment of osteosarcoma, its preparation method, and its application. Background Technology

[0002] Osteosarcoma, or OS for short, is the most common primary malignant bone tumor in children and adolescents, with an average age of 20 years. The standard treatment for osteosarcoma, namely surgery and chemotherapy, was established in the 1980s, but the survival rate for osteosarcoma patients remains low, with a 5-year survival rate of approximately 50%–60% and a 10-year survival rate of only about 30%. In fact, local surgical control is a crucial prognostic factor for all osteosarcomas; surgery is often challenging depending on the tumor's location and extent of invasion into peripheral tissues. Although surgery and chemotherapy are the preferred treatment options, radiotherapy plays a vital role in the treatment of osteosarcoma. Over the past few decades, many advanced radiotherapy techniques have been developed to ensure better local control of osteosarcoma; the development of new technologies has made radiotherapy an effective method for treating incomplete or unresectable tumors, tumors located near critical organs, and metastases.

[0003] Neutron capture therapy (NCT) is a precision medicine technique. This method involves introducing a nuclide with a large thermal neutron capture cross-section into a targeted drug and injecting it into the cancer patient. After the drug accumulates in the tumor tissue, the tumor site is irradiated with an ultrathermal neutron beam or a thermal neutron beam to induce a neutron capture reaction. Because it not only provides high-linear-energy transferred radiation to kill tumors, but its targeting properties also reduce radiation damage to adjacent healthy tissues, it is considered one of the most promising treatment methods in radiotherapy. Boron neutron capture therapy (BNCT) involves introducing a sufficient amount of... 10 Selective delivery of beta-lactone (B) to tumor cells is crucial for successful treatment. Currently, approved drugs for BNCT clinical trials have evolved from the first-generation sodium boropropionate (BSH) to the second-generation l-boronylphenylalanine (BPA). However, the second-generation BPA still suffers from issues such as high uptake doses and low tumor selectivity in the blood, meaning its targeting is not yet ideal. Furthermore, its distribution in various tissues and tumor tissues after introduction into the body is difficult to visualize. Therefore, next-generation drugs targeting improved tumor specificity are key to neutron capture therapy. Summary of the Invention

[0004] This invention provides a formulation for integrated diagnosis and treatment of osteosarcoma. The formulation not only specifically targets osteosarcoma, but also enables visualized MR imaging of osteosarcoma lesions, thus achieving integrated diagnosis and treatment of osteosarcoma.

[0005] The technical solution adopted in this invention is: The present invention provides a formulation for the diagnosis and / or treatment of osteosarcoma, the formulation being obtained by conjugating a CS peptide with a bifunctional chelating agent and then chelating gadolinium; the amino acid sequence of the CS peptide is shown in SEQ ID NO.1.

[0006] Preferably, the bifunctional chelating agent is tetraazacyclododecanetetraacetic acid and / or a tetraazacyclododecanetetraacetic acid derivative.

[0007] Preferably, the structural formula of the formulation is as follows: .

[0008] The second invention provides a method for preparing the formulation, comprising the following steps: Based on the amino acid sequence shown in SEQ ID NO.1, a CS polypeptide loaded on resin was synthesized in a solid phase. The CS peptide loaded on the resin was coupled with a bifunctional chelating agent to obtain a bifunctional chelating agent-resin peptide. The bifunctional chelating agent and CS peptide are cleaved from the bifunctional chelating agent-resin peptide to obtain the bifunctional chelating agent-CS peptide. Gadolinium was added to the bifunctional chelating agent-CS peptide for chelation to obtain the formulation.

[0009] Preferably, the coupling process is as follows: After dissolving the bifunctional chelating agent and activator, they were added to the CS peptide loaded on the resin, followed by the addition of a condensing agent. After mixing, a nitrogen bubbling reaction was carried out.

[0010] Preferably, the activator is 1-hydroxy-1H-pyrrole-2,3-dione-4-carboxylate; The condensing agent is dimethylmethylene carbon anhydride.

[0011] Preferably, the molar ratio of the CS polypeptide loaded on the resin to the bifunctional chelating agent is 1:1 to 5.

[0012] Preferably, a cleavage reagent is used to cleave the bifunctional chelating agent and CS peptide from the bifunctional chelating agent-resin peptide, wherein the volume ratios of each substance in the cleavage reagent are as follows: Trifluoroacetic acid:water:1,2-ethanedithiol:Tis = 95:1:2:2.

[0013] Preferably, the mass-to-volume ratio of the bifunctional chelating agent-resin peptide cleavage agent to the cleavage reagent is 1g:10mL.

[0014] Preferably, the mass ratio of the bifunctional chelating agent-CS polypeptide to gadolinium is 20:7~9.

[0015] The third invention provides an application of the formulation, the formulation being used to prepare a formulation for the diagnosis and / or treatment of osteosarcoma, comprising at least one of the following: 1) Contrast agents used in the preparation of magnetic resonance imaging; 2) Drugs used in the preparation of photon radiotherapy sensitizers; 3) Used to prepare drugs for neutron capture therapy.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a formulation for the diagnosis and / or treatment of osteosarcoma, wherein the formulation is obtained by coupling a CS peptide with a bifunctional chelating agent and then chelating gadolinium; the amino acid sequence of the CS peptide is shown in SEQ ID NO.1. This invention utilizes Gd, which is highly concentrated in tumor tissue and has a large thermal neutron capture cross-section, with the specifically targeting CS peptide to prepare a formulation for integrated diagnosis and treatment of osteosarcoma. This formulation can specifically target osteosarcoma and also achieve visualized MRI imaging of osteosarcoma lesions, realizing neutron capture therapy and conventional photon sensitization therapy for osteosarcoma, i.e., integrated diagnosis and treatment. The formulation of this invention has the advantages of high therapeutic specificity, significant efficacy, and minimal damage to normal tissue, representing a new treatment modality with good clinical application prospects and the potential to ultimately improve the prognosis of osteosarcoma patients.

[0017] The chelating agent DOTA used in this invention has a closed-ring structure. The compound formed by DOTA and the lanthanide trivalent ion Gd exhibits superior thermodynamic stability and kinetic inertness, significantly better than the open-ring chelating agent DTPA currently used in magnetic resonance imaging (MRI) contrast agents. Therefore, the Gd-DOTA-CS compound can specifically deliver Gd to the tumor site in an osteosarcoma animal model. This invention utilizes this compound to explore the efficacy of neutron capture therapy / photon therapy in an osteosarcoma animal model and the potential clinical value of this technology in the treatment of other malignant tumors; and utilizes the neutron capture and photon sensitization properties of gadolinium to explore the radiosensitization effect of photons or neutrons in the presence of Gd. Attached Figure Description

[0018] Figure 1 This is the process of coupling amino acids to resin peptides.

[0019] Figure 2 This is a resin peptide structure with all amino acids linked together.

[0020] Figure 3 The structure of resin peptide coupled with DOTA (3-TBU) is shown.

[0021] Figure 4 This refers to the process of cleaving polypeptides from a solid support.

[0022] Figure 5For mass spectrometry detection of DOTA-CS.

[0023] Figure 6 The 3D structure of Gd-DOTA-CS.

[0024] Figure 7 For mass spectrometry detection of Gd-DOTA-CS.

[0025] Figure 8 To validate the cytotoxicity detection of different concentrations of Gd-DOTA-CS in the CCK-8 assay.

[0026] Figure 9 For in vivo hepatotoxicity and nephrotoxicity testing of Gd-DOTA-CS, A: serum AST level; B: serum ALT level; C: serum BUN level; D: serum TBIL level; E: serum CRE level.

[0027] Figure 10 For in vivo MRI imaging and distribution of Gd-DOTA-CS, A: representative MRI images; B: T / N and T / B ratios calculated based on the tissue distribution of Gd-DOTA-CS, where T / N: tumor tissue / normal tissue; T / B: tumor blood / normal blood.

[0028] Figure 11 The radiation damage effect of Gd-DOTA-CS on cells: A: Western blot results; B: Cell cloning experiment results; C: Immunofluorescence experiment results.

[0029] Figure 12 The changes in tumor volume in tumor-bearing mice after NCT treatment are shown in Figure A: Statistical results of tumor size in different groups; Figure B: Representative images of tumors in different groups.

[0030] Figure 13 To show the survival and weight changes of tumor-bearing mice after NCT treatment, A: Survival curves for different groups; B: Weight statistics for different groups.

[0031] Figure 14 The structure is Gd-DOTA-CS. Detailed Implementation

[0032] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0033] The inventive concept of this invention is as follows: IL-11 receptor α, or IL-11Rα for short, is highly expressed in primary osteosarcoma lesions and lung metastases, and its expression is closely related to tumor progression, metastasis, and immune infiltration. Interleukin-11 activates downstream pathways, such as the JAK / STAT3 and MAPK pathways, by transmitting signals through its receptor IL-11Rα and the shared gp130 subunit. This invention utilizes identified mimic motifs—short peptide sequences that can mimic the binding of IL-11 to IL-11Rα and the shared gp130 subunit and perform signal transduction—to conduct targeted therapy research for osteosarcoma.

[0034] Compared to boron drugs, gadolinium drugs have unique advantages: ① elemental gadolinium is highly concentrated in tumor tissues; ② its natural abundance is relatively high, at 15.68%; ③ gadolinium has a large thermal neutron capture cross section; ④ clinically, gadolinium is used in magnetic resonance MRI, allowing for the observation of gadolinium aggregation in tumor cells through MRI imaging, thus achieving integrated diagnosis and treatment. Existing technologies have conducted extensive research on novel gadolinium drugs, but these new molecules have almost entirely remained at the level of structural design and basic research, encountering developmental bottlenecks. Therefore, this invention provides a gadolinium-based integrated diagnostic and therapeutic formulation for osteosarcoma. The formulation is obtained by coupling a CS peptide with a bifunctional chelating agent, followed by chelation of Gd; the amino acid sequence of the CS peptide is shown in SEQ ID NO.1; the bifunctional chelating agent is tetraazacyclododecanetetraacetic acid and / or a tetraazacyclododecanetetraacetic acid derivative.

[0035] SEQ ID NO.1: CGRRAGGSC.

[0036] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0037] The list of abbreviations for this invention is shown in Table 1.

[0038] Table 1 Experimental equipment: 0.01% electronic balance, 20mm×250mm vertical reactor with No. 1 sand core, 50mL centrifuge tubes, centrifuge, HPLC semi-preparative unit, 2cm C18 column, disposable pipettes, nitrogen, circulating water vacuum pump, wash bottle, test tubes, long-necked pipettes, and constant temperature heater.

[0039] Experimental materials and reagents: 2-Cl resin, anhydrous DCM, DIC, DIEA, methanol, industrial grade DMF, analytical grade DMF, 20% piperidine / DMF, detection reagent A, detection reagent B, HOBT, acetonitrile, diethyl ether, TFA, EDT, TIS, DOTA (3-TBU), Gd solution.

[0040] 20% piperidine / DMF: 20% piperidine + 80% DMF, v / v.

[0041] Test reagent A: 5g ninhydrin + 100mL anhydrous ethanol.

[0042] Test reagent B: Analytical grade pyridine.

[0043] The required amino acids are: FMOC-Cys(TRT)-OH, FMOC-Ser(TBU)-OH, FMOC-Gly-OH, FMOC-Ala-OH, and FMOC-Arg(PBF)-OH. The entire polypeptide consists of 9 amino acids, namely Cys-Gly-Arg-Arg-Ala-Gly-Gly-Ser-Cys, with several amino acids repeated. The linkage begins with the first Cys at the C-terminus, followed by Ser and Gly, and then the remaining amino acids.

[0044] Example 1 A method for preparing an agent for the diagnosis and / or treatment of osteosarcoma, as detailed below: 1. Based on the amino acid sequence shown in SEQ ID NO.1, a CS polypeptide loaded on resin was synthesized in a solid-phase manner, as follows: 1.1 Resin activation.

[0045] A vertical reactor was selected, and the peptide name was labeled on the reactor. 1.5 g of 2-Cl resin was weighed using an electronic balance and placed in the reactor. DCM was added and the mixture was soaked for 30 min. The resin was then washed five times with DMF to activate the chlorine groups and enhance its reactivity with the carboxyl groups of amino acids.

[0046] 1.2 C-terminal amino acid loading: The first amino acid Cys at the C-terminus is covalently linked to the solid support.

[0047] Weigh 0.15 mmol of FMOC-Cys(TRT)-OH and 0.15 mmol of HOBt into a centrifuge tube to activate the Cys carboxyl group. Add 10 mL of anhydrous DCM to maintain resin stability, and add 0.5 mmol of DIEA to neutralize the H released during HOBt activation of the carboxyl group. + Shake well. Use a disposable pipette to add the solution to the vertical reactor in step 1.1. Bubble the reaction with nitrogen for 90 minutes to ensure that the carboxyl groups are fully activated and react completely with the resin. After the reaction is complete, add 2 mL of methanol and 6 mL of DCM, and react for 20 minutes to seal any unreacted groups on the resin.

[0048] 1.3 Washing: After the liquid in the vertical reactor is drained by a circulating water vacuum pump, industrial-grade DMF is added to the reactor through a wash bottle. The volume of the reagent should be about 3 times the volume of the resin to completely immerse the resin in the solution. Wash for 30 seconds, and then drain the liquid in the reactor by a circulating water vacuum pump for about 30 seconds. Repeat this operation 4 times.

[0049] 1.4 De-FMOC: Add v / v 20% piperidine / DMF to the vertical reactor using a wash bottle. The reagent volume should be approximately three times the resin volume, ensuring the resin is completely immersed in the solution. Bubble the reaction under nitrogen for 20 minutes. The purpose of this step is to remove the FMOC group, exposing the α-amino group for coupling with the carboxyl group of the next amino acid.

[0050] 1.5 Washing: Refer to 1.3, and replace the industrial-grade DMF with analytical-grade DMF during the final washing process.

[0051] 1.6 Resin Detection: Use a long-necked pipette to take 20 resin beads from the vertical reactor and place them at the bottom of the test tube. Then, use a dropper to add two drops each of test reagent A and test reagent B to the test tube, ensuring that the resin and test reagents are in full contact. Then, place the test tube in a constant temperature of 100℃ for 2 minutes and observe the resin color. If the resin shows color, it indicates that the FMOC removal is successful. If no color develops, repeat the steps 1.4 to 1.6.

[0052] 1.7 Assemble the second Ser: Couple FMOC-Ser(tBu)-OH.

[0053] Weigh 0.5 mmol FMOC-Ser(tBu)-OH and 0.5 mmol HOBt into a centrifuge tube, dissolve them thoroughly in 5 mL DMF, then add 1.5 mmol DIC, mix for 1 min, and add to the vertical reactor from step 1.4. React under nitrogen bubbling for 1 h. The purpose of this step is to couple the second amino acid, FMOC-Ser(tBu)-OH, to the N-terminus of the first amino acid already loaded on the resin during peptide solid-phase synthesis, thereby extending the peptide chain. See the flowchart below. Figure 1 .

[0054] 1.8 Resin test: Refer to 1.6 and observe the color of the resin. If there is no color, it indicates that the connection is complete and proceed to step 1.9. If there is color, repeat step 1.7.

[0055] 1.9 Washing: Same as 1.3.

[0056] 1.10 Assemble the third Gly: Couple FMOC-Gly-OH.

[0057] Repeat steps 1.4 to 1.8, replacing FMOC-Ser(tBu)-OH in step 1.7 with FMOC-Gly-OH, while keeping the other steps the same.

[0058] 1.11 Assemble the fourth Gly: Couple FMOC-Gly-OH.

[0059] Repeat steps 1.4 to 1.8, replacing FMOC-Ser(tBu)-OH in step 1.7 with FMOC-Gly-OH, while keeping the other steps the same.

[0060] 1.12. Assemble the fifth Ala: Couple FMOC-Ala-OH.

[0061] Repeat steps 1.4 to 1.8, replacing FMOC-Ser(tBu)-OH in step 1.7 with FMOC-Ala-OH, while keeping the other steps the same.

[0062] 1.13 Assemble the sixth Arg: Couple FMOC-Arg(PBF)-OH.

[0063] Repeat steps 1.4 to 1.8, replacing FMOC-Ser(tBu)-OH in step 1.7 with FMOC-Arg(PBF)-OH, while keeping the other steps the same.

[0064] 1.14. Assemble the seventh Arg: Couple FMOC-Arg(PBF)-OH.

[0065] Repeat steps 1.4 to 1.8, replacing FMOC-Ser(tBu)-OH in step 1.7 with FMOC-Arg(PBF)-OH, keeping all other steps the same.

[0066] 1.15. Assemble the eighth Gly: Couple FMOC-Gly-OH.

[0067] Repeat steps 1.4 to 1.8, replacing FMOC-Ser(tBu)-OH in step 1.7 with FMOC-Gly-OH, while keeping the other steps the same.

[0068] 1.16. Assemble the ninth Cys: Couple FMOC-Cys(TRT)-OH.

[0069] Repeat steps 1.4 to 1.8, replacing FMOC-Ser(tBu)-OH in step 1.7 with FMOC-Cys(TRT)-OH, while keeping the other steps the same.

[0070] Continue until the last amino acid is linked, then remove FMOC and wash. See [link to complete resin peptide structure]. Figure 2 .

[0071] 2. The CS peptide loaded on the resin is coupled with a bifunctional chelating agent to obtain a bifunctional chelating agent-resin peptide. The steps are as follows: 2.1 DOTA Coupling: Weigh 0.5 mmol DOTA(3-TBU) and 0.5 mmol HOBt into a centrifuge tube, dissolve them thoroughly with 5 mL LDMF, then add 1.5 mmol DIC, mix for 1 min, and add to the dried resin (1.16 g). React under nitrogen bubbling for 1 h. See [link to documentation]. Figure 3 .

[0072] 2.2 Resin testing: Observe the color of the resin. If there is no color, it indicates that the connection is complete; if there is repeated color, then 2.1.

[0073] 2.3 Resin drying: Wash the resin three times with methanol, filter and dry to obtain the bifunctional chelating agent - resin peptide.

[0074] 3. The bifunctional chelating agent and CS peptide are cleaved from the bifunctional chelating agent-resin peptide to obtain the bifunctional chelating agent-CS peptide.

[0075] 3.1 Preparation of the cutting reagent, taking a 100mL system as an example, the formula is as follows: Prepare 95mL TFA + 1mL water + 2mL EDT + 2mL Tis for later use.

[0076] The amount of cleavage reagent used is: 1g of bifunctional chelating agent-resin peptide plus 10mL of cleavage reagent.

[0077] 3.2 Preparation for sedimentation: Pre-cool anhydrous diethyl ether at -20℃ for at least 2 hours. Use 1 mL of diethyl ether to precipitate the polypeptide with 1 mL of cleavage reagent.

[0078] 3.3 Peptide Cleavage: The dried bifunctional chelating agent-resin peptide was weighed, and cleavage reagent was added for 2 hours to cleave the peptide from the resin and remove the side-chain protecting groups. Then, the peptide was precipitated with diethyl ether to form a crude peptide. (See flowchart). Figure 4 .

[0079] 4. Purification of the bifunctional chelating agent-CS peptide, the steps are as follows: 4.1 Parameter settings.

[0080] Chromatographic column: 20mm×250mm diagesol 8μm.

[0081] Mobile phase: A: 0.1% v / v TFA + water; B: 0.1% v / v TFA + acetonitrile.

[0082] Flow rate: 10 mL / min.

[0083] 4.2 Load the sample onto pump A, then run 10% acetonitrile solution for 5 minutes to start the gradient test. See Table 2 for the conditions.

[0084] Table 2 Operating Conditions 4.3. Prepare and collect the sample peaks for detection, ensuring an analytical purity greater than 95%. Identify the products using mass spectrometry, see [link to relevant documentation]. Figure 5 It was confirmed as the target product, denoted as DOTA-CS, with a molecular weight of 1250.4; the purity was 99.1% as determined by HPLC analysis.

[0085] 4.4. Sample freeze-drying: Transfer the sample into a freeze-drying dish and freeze-dry in a freeze dryer for 24 hours to obtain the pure product before chelation.

[0086] 5. After the pure product forms disulfide bonds before chelation, gadolinium is added for chelation.

[0087] Dissolve 20 mg of the pure product before chelation in 30% acetonitrile and water (v / v), adjust the pH to around 8, and stir overnight to form disulfide bonds. Then add 7.5 mg of Gd, adjust the pH to around 7.2 with ammonium bicarbonate solution, and stir overnight to chelate.

[0088] 6. Purification and identification after chelation.

[0089] Sample preparation after chelation: The chelated peptide solution was diluted with pure water to a concentration of 10% acetonitrile, then filtered. The sample was loaded via pump A, and the gradient was started after running a 10% acetonitrile water equilibrium for 5 minutes. The sample peaks were collected and analyzed, and the purity was greater than 95%.

[0090] See 3D structure diagram Figure 6 Results identification: The above products were identified by mass spectrometry, see [see results]. Figure 7 The compound was confirmed to be the target product, Gd-DOTA-CS, with a theoretical molecular weight of 1404.68 and an actual molecular weight of 1404.40 determined by MS. HPLC analysis showed a purity of 98.1%. Therefore, the prepared compound is indeed the target product, and its structural formula is shown below. Figure 14 .

[0091] Example 2 For the application of the diagnostic and / or therapeutic agents for osteosarcoma, the Gd-DOTA-CS prepared in Example 1 was validated as follows: 1. The effect of different concentrations of Gd-DOTA-CS on cell viability was verified using CCK-8 assay. The experimental method is as follows:

[0092] The cytotoxicity of Gd-DOTA-CS was detected using a CCK-8 assay kit. The specific steps are as follows:

[0093] 1) When rat osteosarcoma cells UMR-106 are in the logarithmic growth phase, in good condition and with a confluence of 80%, enzymatic digestion is performed to count the cells.

[0094] 2) 3000 cells were seeded in one well of a 96-well plate at a volume of 100 μL. After the cells adhered to the plate, 0 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL of Gd-DOTA-CS were added respectively, and the cells were treated for 24 h and 48 h respectively.

[0095] 3) After the drug treatment time is over, add freshly prepared CCK-8 solution containing 10 μL of detection solution to each well. Be careful to avoid generating air bubbles during the liquid addition process, otherwise it will affect the measurement value.

[0096] 4) Place the 96-well plate containing the CCK-8 detection solution in the incubator and incubate for 2 hours.

[0097] 5) After incubation, use an ELISA reader to measure the absorbance of each well of the 96-well plate at 450 nm, i.e., the OD value.

[0098] 6) The data obtained from the microplate reader is processed by Excel and then plotted using Graphpad.

[0099] The results are as follows Figure 8 As shown. By Figure 8 It can be seen that when the concentration of Gd-DOTA-CS is below 25 μg / mL, the viability of UMR-106 cells does not decrease significantly.

[0100] 2. The experimental method for detecting the hepatotoxicity and nephrotoxicity of Gd-DOTA-CS in rats is as follows: Five rats were injected with Gd-DOTA-CS via the tail vein at a dose of 350 μg per rat. Blood samples were collected one day before, three days after, and ten days after the injection. The results are as follows: Figure 9 As shown. By Figure 9 It was found that no obvious toxicity was observed in the liver and kidneys of tumor-bearing mice three days and ten days later.

[0101] 3. In vivo MRI imaging of Gd-DOTA-CS, the experimental method is as follows: UMR-106 was cultured using standard methods. Under aseptic conditions, a concentration of 1×10⁻⁶ was prepared. 7A subcutaneous tumor animal model was established by subcutaneously injecting a cell suspension of 100 μL cells into the leg of a rat. When the tumor diameter was 1 cm, the tumor-bearing rat was injected with 350 μg of Gd-DOTA-CS via the tail vein, fixed in a scaffold, and subjected to MRI imaging in a 3.0 T strong magnetic field.

[0102] MRI results are shown Figure 10 Four hours after tail vein injection of the drug in rats, the tumor site showed significant imaging, which was due to increased blood supply. The signal weakened after 4 hours, proving that Gd-DOTA-CS is targeted at tumors and can be visualized by MRI.

[0103] 4. Verification of the neutron radiation damage effect of Gd-DOTA-CS on osteosarcoma cells. The experimental method is as follows: UMR-106 osteosarcoma cells were incubated with 10 µg / ml Gd-DOTA-CS for 4 h, then the peptide-containing medium was removed, followed immediately by neutron irradiation. SDS-PAGE gel electrophoresis was then performed on the total proteins. Western blotting results showed that the expression level of the DNA-damaging protein γ-H2AX was increased in the Gd-DOTA-CS group compared to the Control and Neutron groups, demonstrating that Gd-DOTA-CS has a radiation-damaging effect on osteosarcoma cells under neutron irradiation. Furthermore, cell cloning experiments showed similar results; the Gd-DOTA-CS group had the lowest colony formation rate compared to the control and Neutron groups, further demonstrating the inhibitory effect of Gd-DOTA-CS on osteosarcoma cells in vitro. Finally, immunofluorescence experiments showed that the Gd-DOTA-CS group had the strongest fluorescence intensity, and the co-localization of the DNA-damaging protein γ-H2AX with DAPI indicated that Gd-DOTA-CS has a significant inhibitory effect on osteosarcoma cells. These results are shown in […]. Figure 11 .

[0104] 5. The neutron capture therapy effect of Gd-DOTA-CS was investigated using the following experimental method: UMR-106 was cultured using standard methods. Under aseptic conditions, a concentration of 1×10⁻⁶ was prepared. 7 A subcutaneous tumor animal model was established by subcutaneously injecting a cell suspension of 1 cell per 100 μL into the leg of rats. When the tumor diameter reached 1 cm, the tumor-bearing rats were divided into groups and irradiated with moderated thermal neutrons (DT neutrons with an energy of 2.5 MeV) at a specific site for 1 hour. The rats were then fed continuously, and changes in body weight and tumor growth were continuously monitored. Results are as follows: Figure 13 and Figure 14 As shown in the figure, rats that received Gd-DOTA-CS via tail vein injection after thermal neutron irradiation exhibited slow tumor growth and significantly smaller tumor volume. Figure 12The rats showed almost no change in body weight, indicating the safety of Gd-DOTA-CS. Survival curves of the rats revealed that the rats in the group successfully treated with NCT had the longest survival time, suggesting the effectiveness of neutron capture therapy in rats following Gd-DOTA-CS injection.

[0105] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0106] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A formulation for the diagnosis and / or treatment of osteosarcoma, characterized in that, The formulation is obtained by coupling CS peptide with a bifunctional chelating agent and then chelating gadolinium. The amino acid sequence of the CS polypeptide is shown in SEQ ID NO.

1.

2. The formulation according to claim 1, characterized in that, The bifunctional chelating agent is tetraazacyclododecanetetraacetic acid and / or a tetraazacyclododecanetetraacetic acid derivative.

3. The formulation according to claim 1, characterized in that, The structural formula of the formulation is as follows: 。 4. The method for preparing the formulation according to claim 1, characterized in that, Includes the following steps: Based on the amino acid sequence shown in SEQ ID NO.1, a CS polypeptide loaded on resin was synthesized in a solid phase. The CS peptide loaded on the resin was coupled with a bifunctional chelating agent to obtain a bifunctional chelating agent-resin peptide. The bifunctional chelating agent and CS peptide are cleaved from the bifunctional chelating agent-resin peptide to obtain the bifunctional chelating agent-CS peptide. Gadolinium was added to the bifunctional chelating agent-CS peptide for chelation to obtain the formulation.

5. The preparation method according to claim 4, characterized in that, The coupling process is as follows: After dissolving the bifunctional chelating agent and activator, they were added to the CS peptide loaded on the resin, followed by the addition of a condensing agent. After mixing, a nitrogen bubbling reaction was carried out.

6. The preparation method according to claim 5, characterized in that, The activator is 1-hydroxy-1H-pyrrole-2,3-dione-4-carboxylate; The condensing agent is dimethylmethylene carbon anhydride.

7. The preparation method according to claim 4, characterized in that, The bifunctional chelating agent and CS peptide are cleaved from the bifunctional chelating agent-resin peptide using a cleaving reagent, wherein the volume ratios of each substance in the cleaving reagent are as follows: Trifluoroacetic acid:water:1,2-ethanedithiol:Tis = 95:1:2:

2.

8. The preparation method according to claim 7, characterized in that, The mass-to-volume ratio of the bifunctional chelating agent-resin peptide cleavage agent to the cleavage reagent is 1 g: 10 mL.

9. The preparation method according to claim 4, characterized in that, The mass ratio of the bifunctional chelating agent-CS peptide to gadolinium is 20:7~9.

10. The application of the formulation as described in claim 1, characterized in that, The formulation is used to prepare a formulation for the diagnosis and / or treatment of osteosarcoma, comprising at least one of the following: 1) Contrast agents used in the preparation of magnetic resonance imaging; 2) Drugs used in the preparation of photon radiotherapy sensitizers; 3) Used to prepare drugs for neutron capture therapy.

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