Preparation and application of polypeptide ligand and radioactive molecular probe for brain glioma diagnosis

Abstract

The application discloses a polypeptide ligand for brain glioma diagnosis and preparation and application of a radioactive molecular probe. The polypeptide targeting a TREM2 protein has the amino acid sequence as shown in the following: HLRKLRKR. The targeted polypeptide designed by the application has the characteristics of small molecular weight, strong blood brain barrier penetration and strong TREM2 protein binding capacity. The radioactive nuclide labeled molecular probe prepared by the application has the characteristics of strong targeting and easy observation as a brain glioma imaging drug, and is convenient for clinical application. The application improves the diagnosis effect of brain glioma and provides a new idea for early discovery and early treatment of brain glioma.

Description

Technical Field

[0001] This invention falls under the category of new biomedical technologies and relates to the preparation and application of polypeptide ligands and specific targeted radioactive molecular probes for the diagnosis of gliomas. Background Technology

[0002] Gliomas are the most common primary brain tumors, accounting for 45% of all primary brain tumors. According to the World Health Organization (WHO) classification, gliomas are divided into four pathological types (grades I, II, III, and IV), among which glioblastoma multiforme (GBM, WHO grade IV) is the most common and most aggressive malignant type. Despite significant improvements in diagnostic methods and treatment strategies for gliomas, the prognosis for high-grade GBM remains poor, with an average survival of less than one year, and only 5% of GBM patients surviving more than 15 months. The emergence of targeted therapies for gliomas in recent years has brought new hope to GBM treatment to a large extent, compensating for the shortcomings of chemotherapy drugs. However, only a few targeted drugs, such as bevacizumab, are used in clinical treatment, and the development of targeted diagnostic and therapeutic drugs for GBM remains a long-term problem for researchers. Therefore, early detection and early treatment remain the direction for achieving precision treatment of GBM.

[0003] Analysis based on the TGAC database revealed that, compared to other tumor types, the triggering receptor expressed on myeloid cells 2 (TREM2) showed the highest expression abundance in GBM. Furthermore, the higher the pathological grade of glioma, the higher the TREM2 expression level, and the lower the patient's prognosis and survival rate. Therefore, TREM2 has the potential to be developed as a diagnostic and therapeutic target for GBM. Peptides, with their small molecular weight, ease of modification and optimization, chemical stability, and ability to cross the blood-brain barrier, are important ligand molecules for the development of drugs for the diagnosis and treatment of glioma. Currently, there are no targeted peptides against TREM2 protein for the diagnosis and treatment of glioma, and there is an urgent need for molecular probes that can specifically bind to TREM2 protein for the clinical diagnosis and treatment of GBM. Summary of the Invention

[0004] Based on the above background, this invention designs a polypeptide sequence targeting the TREM2 protein, which exhibits stable binding and strong affinity to the TREM2 protein. A radioactive molecular probe with specific targeting for gliomas was designed and synthesized, and its application in cellular experiments and with radionuclides was investigated. 68Ga-labeled HLR was used for targeted imaging of U87-MG glioma cell xenografts. The results indicate that HLR has the potential to be used as a molecular imaging probe for the clinical diagnosis of malignant gliomas and has potential clinical application value.

[0005] One of the objectives of this invention is to provide a polypeptide that targets the TREM2 protein, the amino acid sequence of which is shown below: HLRKLRKR (histidine-leucine-arginine-lysine-leucine-arginine-lysine-arginine, His-Leu-Arg-Lys-Leu-Arg-Lys-Arg), abbreviated as HLR.

[0006] A second objective of this invention is to provide the application of the aforementioned peptide targeting the TREM2 protein in the preparation of diagnostic reagents for glioma.

[0007] The glioma mentioned is specifically glioblastoma multiforme.

[0008] This invention designs a polypeptide sequence that targets the TREM2 protein, which has the characteristics of stable binding and strong affinity to the TREM2 protein. The affinity of the polypeptide to the TREM2 protein is identified, and its ability to cross the blood-brain barrier is verified.

[0009] In the embodiments of this invention, the amino acid sequence of the designed polypeptide is HLRKLRKR, abbreviated as HLR. Molecular docking technology was employed, taking into account the limitation on polypeptide length for crossing the blood-brain barrier, using an octapeptide as the length basis, to analyze the binding mode of the polypeptide with the TREM2 protein.

[0010] In an embodiment of the present invention, the affinity determination method used to identify the peptide and TREM2 protein is biomembrane interferometry, that is, 20 μg / mL TREM2 protein is immobilized on an NTA chip, 200 μL of peptide with a concentration of 100 μM is added, and the affinity determination result between HLR and TREM2 is obtained by biomembrane interferometry analysis.

[0011] In an embodiment of the present invention, when verifying the ability of the polypeptide to cross the blood-brain barrier, the imaging effect of Cy5.5-HLR in the brain of nude mice with U87-MG cell orthotopic xenografts was observed using small animal fluorescent in vivo. 100 μL (1 μg / μL) of Cy5.5-HLR (dissolved in PBS) was injected into the tail vein of nude mice with U87-MG cell orthotopic xenografts.

[0012] The third objective of this invention is to provide a radioactive molecular probe.

[0013] The radioactive molecular probe provided by the present invention comprises: a polypeptide targeting the TREM2 protein, a chelating agent, and a radionuclide, wherein the polypeptide targeting the TREM2 protein is covalently coupled to the chelating agent, and the radionuclide is chelated to the chelating agent.

[0014] The polypeptide targeting the TREM2 protein has the following amino acid sequence: HLRKLRKR, abbreviated as HLR.

[0015] The chelating agent is one of DTPA, EDTA, and DOTA:

[0016]

[0017]

[0018] The radionuclides are selected from 99m TcO, 18 F, 89 Zr、 68 Any one of Ga;

[0019] Among them, radioactive nuclides 99m TcO can chelate with DTPA;

[0020] 99m TcO can chelate with DTPA;

[0021] 89 Zr can chelate with EDTA;

[0022] 18 F, 68 Ga can chelate with DOTA.

[0023] Specifically, the radioactive molecular probe is represented as follows: 68 Ga-DOTA-HLR, where... 68 Ga is a radioactive isotope, DOTA is a chelating agent, and HLR is a polypeptide that targets the TREM2 protein.

[0024] This invention designs a peptide ligand with optimal binding mode and affinity to the TREM2 protein and prepares a radioactive molecular probe with specific targeting activity. This molecular probe couples the targeting peptide to the chelating agent DOTA via a coupling bond, and then labels the DOTA with a radionuclide. 68 Ga, using PET / CT to achieve accurate diagnosis of GBM, shows potential application prospects in the efficient diagnosis and treatment of glioma.

[0025] Another object of the present invention is to provide a method for preparing the above-mentioned radioactive molecular probe.

[0026] The above-mentioned radioactive molecular probes are prepared by a method including the following steps:

[0027] 1) Preparation of chelating agents-peptides

[0028] In the presence of a catalyst and a condensing agent, a chelating agent is coupled with a polypeptide to obtain a chelating agent-polypeptide.

[0029] 2) Preparation of radionuclide-labeled chelating agents-peptides

[0030] A radionuclide is added to a mixed solution of chelating agent-peptide, water, and hydrogen ion buffer, and the reaction is carried out to obtain a radionuclide-labeled chelating agent-peptide, i.e., a radioactive molecular probe.

[0031] In step 1) of the above method, the catalyst may specifically be NMM;

[0032] The condensing agent may specifically be HBTU.

[0033] The molar ratio of polypeptide to chelating agent, catalyst and condensing agent can be 1:1-5:1-5:1-5, specifically 1:3:3:3;

[0034] The coupling reaction is carried out in an inert atmosphere, which is provided by nitrogen.

[0035] The coupling reaction is carried out in an organic solvent, specifically DCM.

[0036] The reaction time can be 5-15 minutes.

[0037] In step 2) of the above method, the hydrogen ion buffer can specifically be hydroxyethylpiperazine ethanethiolic acid (HEPES).

[0038] The pH value of the reaction is 4 to 4.5.

[0039] The reaction temperature can be 80-100℃, specifically 95℃, and the time can be 10-20min, specifically 15min.

[0040] The chelating agent is DOTA, and it is made with a radioactive nuclide. 68 For Ga labeling, step 2) involves rinsing with 0.1M HCl. 68 Ge- 68 Ga generator obtains 68 GaCl3 was added to a mixture of DOTA-peptide, water, and HEPES. 68 The Ga eluent was mixed thoroughly, sealed, and reacted at 95°C for 15 min. The mixture was then purified by column chromatography to obtain... 68 Ga-DOTA-HLR radioactive molecular probe.

[0041] The application of the aforementioned radioactive molecular probes in the preparation of diagnostic reagents for gliomas also falls within the scope of protection of this invention.

[0042] In this application, the glioma is specifically glioblastoma multiforme.

[0043] The diagnostic reagent is used for imaging human glioma cells and in vivo glioma xenografts.

[0044] According to an embodiment of the present invention, the selected human glioma cells are U87-MG cells, and the selected in vivo glioma xenografts are BABL / c nude mouse subcutaneous and orthotopic U87-MG cell-bearing xenografts.

[0045] The radioactive molecular probe described in this invention (specifically, 68 Compared with existing technologies, Ga-DOTA-HLR has the following advantages:

[0046] 1) This invention utilizes molecular docking technology to screen and design targeting peptides with high affinity for TREM2. The design method is simple and rapid, filling the gap in high affinity peptide ligands for TREM2.

[0047] 2) Since the presence of the blood-brain barrier limits the efficiency of molecular probes crossing the blood-brain barrier, the targeting peptide designed in this invention has a small molecular weight and its sequence is HLRKLRKR, which has the characteristics of crossing the blood-brain barrier and having a strong ability to bind to TREM2 protein.

[0048] 3) The radionuclide-labeled molecular probe prepared in this invention has the characteristics of strong targeting and easy observation as an imaging drug for glioma, which is convenient for clinical application.

[0049] 4) This invention improves the diagnostic effect of glioma and provides a new approach for the early detection and treatment of glioma. Attached Figure Description

[0050] Figure 1 This is the structural formula of the polypeptide HLR in this invention.

[0051] Figure 2 This is a three-dimensional docking model of peptide HLR and TREM2 protein in Example 1 of the present invention.

[0052] Figure 3 This is a synthetic route diagram of the polypeptide HLR in Example 2 of the present invention.

[0053] Figure 4 The results are HPLC and MS identification of the polypeptide HLR obtained in Example 2 of this invention.

[0054] Figure 5The results of the affinity test between HLR and TREM2 protein in Example 2 of this invention are shown.

[0055] Figure 6 The results show the effect of HLR on the survival rate of U87-MG cells in Example 3 of this invention. *p<0.05, **p<0.01.

[0056] Figure 7 This is a synthesis route diagram of FITC-HLR in Embodiment 4 of the present invention.

[0057] Figure 8 This is the structural formula of FITC-HLR in Embodiment 4 of the present invention.

[0058] Figure 9 The results are HPLC and MS identification of the FITC-HLR prepared in Example 4 of this invention.

[0059] Figure 10 This is an example of immunofluorescence analysis of the binding of TREM2 protein and HLR in U87-MG cells in Example 4 of the present invention.

[0060] Figure 11 This is a flow cytometry comparison of HLR uptake in U87-MG cells in Example 4 of the present invention.

[0061] Figure 12 This is a synthesis route diagram of Cy5.5-HLR in Embodiment 5 of the present invention.

[0062] Figure 13 The structure of Cy5.5-HLR obtained in Example 5 of this invention is shown.

[0063] Figure 14 The HPLC and MS identification results are for Cy5.5-HLR prepared in Example 5 of this invention.

[0064] Figure 15 This describes the distribution and brain uptake of Cy5.5-HLR in nude mice with U87-MG cell orthotopic xenografts in Example 5 of this invention. # p<0.001, A. Fluorescence distribution at different time points after peptide uptake in nude mouse brain; B. Fluorescence intensity at different time points after peptide uptake in nude mouse brain.

[0065] Figure 16 This figure shows the uptake of Cy5.5-HLR in the organs and brain of nude mice with U87-MG cell orthotopic xenografts 24 hours after administration in Example 5 of the present invention. # p<0.001, A. Fluorescence distribution in the organs and brain of nude mice after taking up the peptide; B. Fluorescence intensity in the brain of nude mice after taking up the peptide.

[0066] Figure 17This is the synthesis route diagram of DOTA-HLR in Embodiment 6 of the present invention.

[0067] Figure 18 The structure of DOTA-HLR prepared in Example 6 of this invention is shown below.

[0068] Figure 19 The HPLC and MS identification results are for the DOTA-HLR prepared in Example 6 of this invention.

[0069] Figure 20 Prepared as described in Example 6 of this invention 68 HPLC detection results of Ga-DOTA-HLR.

[0070] Figure 21 The U87-MG cell pair in Example 6 of this invention 68 Intake results of Ga-DOTA-HLR. **p<0.01.

[0071] Figure 22 In Example 7 of this invention, BABL / c nude mice that had not been inoculated with U87-MG cells were injected via the tail vein. 68 Micro-PET / CT imaging results of Ga-DOTA-HLR.

[0072] Figure 23 The U87-MG cell subcutaneous xenograft model of BABL / c nude mice in Example 7 of this invention was established by tail vein injection. 68 Micro-PET / CT imaging results of Ga-DOTA-HLR.

[0073] Figure 24 The U87-MG cell orthotopic xenograft model of BABL / c nude mice in Example 7 of this invention was established by tail vein injection. 68 Micro-PET / CT imaging results of Ga-DOTA-HLR. Detailed Implementation

[0074] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0075] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0076] Example 1: Molecular docking of HLRKLRKR with TREM2 protein

[0077] 1) Determination of the TREM2 protein structure

[0078] The human TREM2 protein, with a total length of 230 amino acids, uses the A chain of a 5UD7 crystal structure and is scored for docking using the Cluspro algorithm.

[0079] 2) Molecular binding mode analysis

[0080] The binding mode of the highest-scoring peptide to the TREM2 protein was analyzed. The peptide sequence is histidine-leu-leucine-arginine-lysine-leucine-arginine-lysine-arginine (His-Leu-Arg-Lys-Leu-Arg-Lys-Arg, abbreviated HLRKLRKR, structural formula see [link to structural formula]). Figure 1 The results of the interaction positions of the HLRKLRKR and TREM2 docking are shown in [the table below]. Figure 2 The results showed that HLRKLRKR can effectively bind to the TREM2 protein.

[0081] Example 2: Affinity determination of HLR and TREM2 proteins

[0082] 1) Artificial synthesis of HLR

[0083] See the synthesis route diagram of HLR. Figure 3Synthesis sequence: from C-terminus to N-terminus. ① Resin swelling: Place 2-ChlorotritylChloride Resin into a reaction tube, add DCM (dichloromethane) (15 mL / g), and shake for 30 min; ② Introduce the first amino acid: Filter the solvent through a stencil, add 3 times the molar excess of Fmoc-His(Trt)-OH amino acid, add DMF (dimethylformamide) to dissolve, then add 10 times the molar excess of DIEA (diisopropylethylamine), and shake for 60 min. ③ Block with methanol; ④ Deprotection: Remove DMF, add 20% piperidine DMF solution (15 mL / g), react for 5 min, remove and add another 20% piperidine DMF solution (15 mL / g), react for 15 min; ⑤ Detection: Remove piperidine solution, take a dozen resin grains, wash three times with ethanol, add test reagent, heat at 105℃~110℃ for 5 min, a deep blue color indicates a positive reaction; ⑥ Washing: DMF (10 mL / g) twice, DCM (10 mL / g) twice, DMF (10 mL / g) twice; ⑦ Condensation: Protect amino acid in three-fold excess, HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate) in three-fold excess, dissolve both in as little DMF as possible, add to the reaction tube, immediately add DIEA in ten-fold excess, react for 30 min; ⑧ Detection: Take a dozen resin grains, wash three times with ethanol, add test reagent, heat at 105℃~110℃ for 5 min, a colorless color indicates a negative reaction. ⑧ Wash twice with DMF (10 mL / g), twice with DCM (10 mL / g), twice with DMF (10 mL / g); ⑨ Repeat steps three to six, linking amino acids in the sequence from right to left; ⑩ Dry, washing steps: twice with DMF (10 mL / g), twice with methanol (10 mL / g), twice with DMF (10 mL / g), twice with DCM (10 mL / g), dry for 10 min. 11) Cut the peptide from the resin, prepare the cutting solution (10 mL / g) TFA (trifluoroacetic acid) 95%; water 1%; EDT (ninhydrin) 2%; TIS (triisopropylsilane) 2%, cutting time: 120 min; blow dry and wash, blow the lysate as dry as possible with nitrogen, wash with ether six times, and then evaporate to room temperature; analyze and purify, purify the crude product by high performance liquid chromatography; freeze dry, collect the target peptide solution and concentrate it in a freeze dryer, freeze dry into powder. The HPLC and MS identification results of HLR are shown in the figure. Figure 4 .

[0084] 2) Affinity analysis of HLR and TREM2 proteins

[0085] TREM2 protein was diluted to 20 μg / mL with PBS for chip curing, and the peptide was diluted to 100 μM with PBST (pH 7.4). The TREM2 protein solution was added dropwise to the NTA chip. 200 μL of PBST (pH 7.4) was added to different wells of the cured NTA chip as a control, and 200 μL of the 100 μM peptide was added to the other wells. 250 μL of PBS buffer (pH 7.4) was injected into the sensor, and the buffer was run at the maximum flow rate (150 μL / min) until the signal baseline was reached. The flow rate was then reduced to 20 μL / min to obtain a more stable baseline. The signal of HLR binding to TREM2 protein is shown in [the figure]. Figure 5 The results showed that HLR and TREM2 protein have a strong binding interaction, and the equilibrium dissociation constant KD between them is 8.17 μM.

[0086] Example 3: Effect of HLR on U87-MG cell survival

[0087] Cell viability was determined using a CCK-8 assay kit. Human glioblastoma U87-MG cells were seeded in 96-well plates. When the cell density reached 70%, different concentrations of HLR were added, and the plates were cultured at 37°C and 5% CO2 for 24 hours. Then, 10 μL of CCK-8 reaction solution was added to each well, and the plates were cultured at 37°C and 5% CO2 for another 4 hours. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: [A(sample well) – A(blank)] / [A(PBS) – A(blank)] × 100%. Results showed no significant difference in toxicity to U87-MG cells at concentrations of 5, 10, and 20 μg / mL. Therefore, 20 μg / mL was selected for the cell uptake experiment. Figure 6 ).

[0088] Example 4: Analysis of HLR uptake by U87-MG cells

[0089] 1) Preparation of FITC-HLR

[0090] The FITC-HLR synthesis route is as follows: Figure 7 As shown. The polypeptide synthesis is the same as steps ① to ⑧ in Example 2, with amino acids linked sequentially from right to left, followed by 5-FTIC linkage. Subsequent steps are the same as step ⑩ in Example 2. The structural formula of FITC-HLR is shown below. Figure 8 The HPLC and MS identification results are shown in the figure. Figure 9 .

[0091] 2) Qualitative analysis of HLR binding to TERM2 in U87-MG cells

[0092] U87-MG cells were seeded onto glass slides (placed in Φ35 dishes). After reaching a cell density of 60%, the cells were washed three times with pre-cooled PBS, fixed with 4% paraformaldehyde for 15 min, and allowed to air dry. Then, 1 μg / mL of FITC-HLR was added, and the cells were incubated overnight at 4°C. The nuclei were stained with 5 μg / mL of DAPI. Finally, the slides were mounted with an anti-quenching agent and observed under a fluorescence microscope. The results showed that HLR could bind to U87-MG cells (…). Figure 10 ).

[0093] 3) Quantitative analysis of HLR uptake in U87-MG cells

[0094] Human umbilical vein endothelial cells (HUVECs) and U87-MG cells were seeded in the upper and lower chambers of a Transwell at a 1:5 ratio and cultured for 48 hours to simulate a tumor microvascular environment. The HUVEC cell culture medium in the upper chamber was aspirated and FITC-HLR (containing 10% FBS to a final concentration of 20 μg / mL) was added. The cells were incubated at 37°C and digested with 0.25% trypsin at 2 and 4 hours, washed three times with cold PBS, and then resuspended in 300 μl of cold PBS for flow cytometry analysis of cell fluorescence intensity. The results showed that the FITC fluorescence intensity of U87-MG cells at 4 hours was greater than that at 2 hours and in the PBS group, indicating that HLR could be taken up by U87-MG cells. Figure 11 ).

[0095] Example 5: HLR crossing the blood-brain barrier experiment

[0096] 1) Preparation of Cy5.5-HLR

[0097] The synthetic route of Cy5.5-HLR is as follows: Figure 12 As shown. The polypeptide synthesis follows steps ① to ⑧ of Example 2, with amino acids linked sequentially from right to left, followed by linking Cy5.5. Subsequent steps are the same as step ⑩ of Example 2. The structural formula of Cy5.5-HLR is shown below. Figure 13 The HPLC and MS identification results are shown in the figure. Figure 14 .

[0098] 2) Establishment of U87-MG cell orthotopic tumor animal model

[0099] A mouse orthotopic tumor model of U87-MG cells was established using a stereotaxic apparatus. U87-MG-luc cells in logarithmic growth phase were harvested, digested with 0.25% trypsin, and then the cell density was adjusted to 1×10⁻⁶ cells / cells. 5 / mL, using a stereotaxic instrument, 5μL of cell suspension was aspirated and injected into the posterior ventricle of the cranium (striatum, 1.8mm to the right of the anterior fontanelle, depth 3mm) of female BABL / c nude mice. Tumor formation was observed 14 days after inoculation using in vivo imaging.

[0100] 3) Observation of intravenous administration of medication to tumor-bearing rats and HLR imaging

[0101] Tumor-bearing nude mice were injected intravenously with 100 μL (1 μg / μL) of Cy5.5-HLR saline solution as described in Example 5. Images were acquired using a small animal fluorescence in vivo imaging system at 1, 3, 6, 12, and 24 hours post-injection. Figure 15 Control group tumor-bearing mice were injected with the same volume of PBS as Cy5.5-HLR. Results showed that the brains of nude mice in the treatment group continuously displayed fluorescent signals. Twenty-four hours after injection, the mouse organs and brains were separated, and images were acquired using a small animal fluorescence in vivo imaging system. The results showed that, compared with the PBS group, the fluorescent signal in the brains of nude mice in the treatment group was significantly increased. Figure 16 This indicates that HLR can cross the blood-brain barrier and has a sustained targeted binding effect.

[0102] Example 6 68 Preparation of Ga-DOTA-HLR molecular probes

[0103] 1) Preparation of DOTA-HLR

[0104] DOTA-HLR crafting route as follows: Figure 17 As shown. The polypeptide synthesis is the same as steps ① to ⑧ in Example 2, with the amino acids in the sequence linked sequentially from right to left. Weigh three molar amounts of DOTA (tetraazacyclododecanetetraacetic acid-diphenylcyclooctylene), three molar amounts of HBTU, and three molar amounts of NMM (N-methylmorpholine), dissolve in DMF, add DCM, stir under nitrogen, and react for 5–15 min. Subsequent steps are the same as step ⑩ in Example 2. The structural formula of DOTA-HLR is shown below. Figure 18 The HPLC and MS identification results are shown in the figure. Figure 19 .

[0105] 2) 68 Ga-marked DOTA-HLR

[0106] DOTA-HLR was dissolved in ultrapure water to a concentration of 1 mg / mL and stored at -20°C; HEPES (hydroxyethylpiperazine ethanethioic acid) was prepared with ultrapure water to a concentration of 2M for later use; 100 μg peptide solution + 350 μL H2O + 450 μL HEPES (HEPES concentration of 1M); rinsed with 0.1M HCl. 68 Ge- 68 Ga generator obtains 68 Add 380 μL of GaCl3 to the mixture of peptide and HEPES. 68Ga eluent (0.1M HCl) was mixed thoroughly, and the pH was determined to be 4–4.5 using pH test paper. After sealing, the mixture was reacted at 95°C for 15 min. Purification was performed using a Zobax C-18 column with a mobile phase of 40% ACN (acetonitrile) and 60% 0.05% TFA (flow rate 1 mL / min). The results were confirmed by Radio-HPLC quality control. See attached table. Figure 20 .

[0107] 3) 68 Cellular uptake assay of Ga-DOTA-HLR

[0108] 100 μg of HLR was used with 2.6 mCi 68 Ga radiolabeling for 15 min, total volume 1.3 mL. Before use, U87-MG cells were seeded into 6-well plates and incubated for 24 hours. 10 μL of Ga radiolabeling was then added. 68 Ga-DOTA-HLR (approximately 2 μCi) was added to each well of a 6-well plate. For the competitive inhibition group, 30× unlabeled peptide DOTA-HLR (~25 μg) was added to wells containing... 68 Cells were cultured in Ga-DOTA-HLR wells for 2 hours, washed three times with cold PBS, and then lysed with 0.5 mL of 1 M NaOH. Cells were then counted using a Wizard 2470 gamma counter. Uptake rate = cell gamma count / 2 μCi 68 Ga gamma count × 100%. Results showed that the addition of 30× unlabeled peptide DOTA-HLR significantly reduced the gamma response of U87-MG cells to... 68 Ga-DOTA-HLR uptake indicates 68 Ga-DOTA-HLR exhibits a strong targeted binding effect on U87-MG. Figure 21 ).

[0109] Example 7: In vivo imaging of U87-MG cell transplanted tumors

[0110] 1) Establishment of U87-MG cell subcutaneous tumor animal model

[0111] U87-MG-luc cells in the logarithmic growth phase were digested with 0.25% trypsin and the cell density was adjusted to 1×10⁻⁶. 5 / mL, 5μL of cell suspension was injected into the axilla of the right forelimb of mice. Thirty days after inoculation, in vivo imaging was used to observe tumor formation.

[0112] 2) Establishment of U87-MG cell orthotopic tumor animal model

[0113] The steps are the same as in Example 5.

[0114] 3) PET / CT imaging of nude mice

[0115] Nude mice without U87-MG cells and nude mice with subcutaneous U87-MG cell xenografts were injected via tail vein with 3.7 MBq of the solution prepared in Example 6. 68 Ga-DOTA-HLR (physiological saline injection) was administered, and PET / CT images were acquired 0.5, 1, and 2 hours post-injection. The results showed that in nude mice that had not been inoculated with U87-MG cells, no imaging was observed in the axilla of the right forelimb. Figure 22 The subcutaneous transplanted tumor in the right forelimb axilla of a nude mouse can be visualized (indicated by the arrow), and the tumor is most clearly visualized after 1 hour. Figure 23 This indicates that U87-MG subcutaneous xenografts are effective against... 68 Ga-DOTA-HLR has excellent uptake properties.

[0116] Nude mice without U87-MG cells and nude mice with U87-MG cell orthotopic xenografts were injected via tail vein with 3.7 MBq of the solution prepared in Example 6. 68 Ga-DOTA-HLR (physiological saline injection), images were acquired using PET / CT one hour after injection. Figure 24 The results showed that no brain imaging was observed in nude mice that had not been inoculated with U87-MG cells after drug administration; however, brain imaging was possible in nude mice with orthotopic xenograft tumors (arrows indicate the dotted area), indicating that... 68 Ga-DOTA-HLR can cross the blood-brain barrier to reach the brain and be taken up by U87-MG cells in situ xenografts, thereby imaging the tumor.

[0117] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A polypeptide targeting the TREM2 protein, with the following amino acid sequence: HLRKLRKR.

2. The use of the polypeptide according to claim 1 in the preparation of diagnostic reagents for glioma.

3. Radioactive molecular probes, including: The peptide targeting TREM2 protein, the chelating agent, and the radionuclide according to claim 1, wherein the peptide targeting TREM2 protein is covalently coupled to the chelating agent, and the radionuclide is chelated to the chelating agent. The radioactive molecular probe is represented as follows: 68 Ga-DOTA-HLR, where... 68 Ga is a radioactive nuclide, DOTA is a chelating agent, and HLR is a polypeptide that targets the TREM2 protein (HLRKLRKR). 。 4. A method for preparing the radioactive molecular probe of claim 3, comprising the following steps: 1) Preparation of chelating agents-peptides In the presence of a catalyst and a condensing agent, a chelating agent is coupled with a polypeptide to obtain a chelating agent-polypeptide. 2) Preparation of radionuclide-labeled chelating agents-peptides A radionuclide was added to a mixed solution of chelating agent-peptide, water and hydrogen ion buffer, and the reaction was carried out to obtain a radioactive molecular probe.

5. The method according to claim 4, characterized in that: In step 1), the catalyst is NMM; The condensing agent is HBTU. The molar ratio of the polypeptide to the chelating agent, catalyst, and condensing agent is 1:1-5:1-5:1-5, respectively. The coupling reaction was carried out in a nitrogen atmosphere; The coupling reaction is carried out in an organic solvent; The reaction time is 5-15 minutes; In step 2), the hydrogen ion buffer is hydroxyethylpiperazine ethanethiolic acid; The pH value of the reaction is 4~4.

5. The reaction temperature can be 80-100℃, and the time can be 10-20 min.

6. The application of the radioactive molecular probe according to claim 3 in the preparation of diagnostic reagents for glioma.

7. The application according to claim 6, characterized in that: The glioma in question is glioblastoma multiforme.

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