A targeting peptide and radioactive tracer for brain glioma

Abstract

The present invention belongs to the field of new biomedical technology applications, and specifically relates to a targeted polypeptide and a radioactive tracer for brain glioma. The amino acid sequence of the polypeptide is: KINWKFRLRY, Lysine-Isoleucine-Asparagine-Tryptophan-Lysine-Phenylalanine-Arginine-Leucine-Arginine-Tyr, Lys-Ile-Asn-Trp-Lys-Phe-Arg-Leu-Arg-Tyr. The radioactive tracer is represented by: 68 Ga‑DOTA‑KINW, where, 68 Ga is a radionuclide, DOTA is a chelating agent, and KINW is a polypeptide targeting the NPY1R receptor protein. The present invention uses mutation scanning and molecular docking technology to screen and modify a targeting polypeptide that specifically binds to NPY1R. Compared with the sequences reported previously, KINW has more binding sites to NPY1R and less cytotoxicity. KINW can cross the blood-brain barrier and 68 Ga chelation can image in situ brain glioma; the radioactive tracer prepared by the invention is used as an imaging drug for brain glioma, and has the characteristics of long imaging time, easy preparation and the like.

Description

Technical Field

[0001] The present invention belongs to the field of new biomedical technology applications, and in particular relates to a targeting polypeptide and a radioactive tracer for brain glioma. Background Art

[0002] According to the guidelines of the World Health Organization (WHO), brain tumors are divided into four grades. Glioblastoma multiforme (GBM) is the highest grade astrocytic tumor, with a median survival of less than 15 months and a 5-year survival rate of only 5%. Due to the strong invasiveness of GBM, it is difficult to completely remove it by surgery, and radiotherapy and chemotherapy remain the main treatments. However, due to the existence of the blood-brain barrier (BBB) ​​and the blood-brain tumor barrier (BBTB), most chemotherapy drugs find it difficult to reach the tumor area. Therefore, the development of targeted diagnostic and therapeutic drugs that can cross the BBB and BBTB is of great significance to improving the survival rate of GMB patients, and it is also a hot spot and difficulty in current research in this field.

[0003] Discovering GBM-specific expression targets and screening high-affinity ligands are prerequisites for developing GBM-targeted drugs. Therefore, it is urgent to find new high-affinity ligands based on the molecular characteristics of GBM-specific expression targets and prepare targeted molecular probes that can cross the BBB and BBTB. Neuropeptide Y (NPY) is the most abundant neuropeptide expressed in the mammalian brain and is responsible for regulating multiple biological mechanisms such as appetite, anxiety, circadian rhythm, memory, and blood pressure. The action of NPY is mediated by NPY receptor subtypes Y1R-6R. Y1R and Y2R are upregulated in many types of human tumors, including breast cancer, adrenal tumors, renal cell carcinoma, ovarian cancer, and glioma. Therefore, Y1R and Y2R can be used as potential targets for tumor diagnosis and treatment. NPY activates Y1R to promote tumor cell proliferation, angiogenesis, and metastasis. Compared with specific ligands such as monoclonal antibodies, peptides have the advantages of small molecular weight, rapid tumor uptake, and rapid clearance from normal tissues. Currently, targeted peptides such as RGD have been used in brain glioma diagnosis. Therefore, developing targeted peptides for NPY1R and applying them to the diagnosis and treatment of brain gliomas has important clinical practical value. Summary of the invention

[0004] One of the purposes of the present invention is to provide a polypeptide ligand with a higher binding mode and affinity to the NPY1R protein, and ultimately obtain a polypeptide sequence targeting the NPY1R protein.

[0005] The polypeptide targeting NPY1R protein provided by the present invention has an amino acid sequence as follows: KINWKFRLRY, lysine-isoleucine-asparagine-tryptophan-lysine-phenylalanine-arginine-leucine-arginine-tyrosine, Lys-Ile-Asn-Trp-Lys-Phe-Arg-Leu-Arg-Tyr, abbreviated as KINW.

[0006] The use of the above polypeptide KINW in the preparation of drugs for diagnosing and treating tumors such as brain glioma, breast cancer and lung cancer also falls within the protection scope of the present invention.

[0007] The second object of the present invention is to provide a radioactive tracer based on the above polypeptide KINW that specifically targets brain glioma.

[0008] The brain glioma may specifically be glioblastoma multiforme.

[0009] The present invention designs and synthesizes a radioactive tracer with specific targeting to human brain glioma cell transplants, and explores the cell experiments and radionuclides 68 The targeting effect of Ga-labeled KINW on human glioma U87-MG cell transplanted tumors showed that KINW has a targeting effect on gliomas and has potential application value in imaging gliomas.

[0010] The radioactive tracer provided by the present invention is composed of the following components: a polypeptide KINW targeting NPY1R protein, a chelating agent DOTA and a radioactive nuclide 68 Ga, where the peptide KINW targeting NPY1R protein was covalently coupled to the chelator DOTA, and the radionuclide 68 Ga is chelated to the chelating agent DOTA.

[0011] The structural formula of the chelating agent DOTA is as follows:

[0012]

[0013] The radioactive tracer is represented by: 68 Ga-DOTA-KINW, where, 68 Ga is a radionuclide, DOTA is a chelating agent, and KINW is a polypeptide targeting NPY1R protein, and its amino acid sequence is as follows: KINWKFRLRY, lysine-isoleucine-asparagine-tryptophan-lysine-phenylalanine-arginine-leucine-arginine-tyrosine, Lys-Ile-Asn-Trp-Lys-Phe-Arg-Leu-Arg-Tyr.

[0014] Another object of the present invention is to provide a method for preparing the above radioactive tracer.

[0015] The above radioactive tracer is prepared by a method comprising the following steps:

[0016] 1) Preparation of Chelating Agent-Peptide

[0017] In the presence of a catalyst and a condensing agent, the chelating agent DOTA and the polypeptide KINW are subjected to a coupling reaction to obtain a chelating agent-polypeptide DOTA-KINW;

[0018] 2) Preparation of radionuclide labeled chelator-peptide

[0019] Add radioactive nuclide to the mixed solution of chelating agent-peptide DOTA-KINW, water and hydrogen ion buffer 68 Ga, the reaction produces a radionuclide labeled chelator-peptide, i.e., a radioactive tracer 68 Ga-DOTA-KINW.

[0020] In step 1) of the above method, the molar ratio of the polypeptide to the chelating agent, the catalyst and the condensing agent is 1:1-5:1-5:1-5, specifically 1:3:3:3.

[0021] The catalyst is an organic base, specifically N-methylmorpholine;

[0022] The condensing agent may specifically be: HBTU;

[0023] The coupling reaction is carried out in an inert atmosphere, which is a nitrogen atmosphere;

[0024] The coupling reaction time is 5-15 min, specifically 15 min.

[0025] In step 2) of the above method, the hydrogen ion buffer is 4-hydroxyethylpiperazine ethanesulfonic acid HEPES, and the HEPES concentration in the mixed solution of water and hydrogen ion buffer is 1M, and the pH is 4-4.5;

[0026] The chelating agent-polypeptide DOTA-KINW and the radionuclide 68 The ratio of Ga can be: 0.1mg:2mCi-0.2mg:2mCi;

[0027] The reaction temperature may be 80-100° C., and the reaction time may be 15-30 min, specifically 30 min.

[0028] Specifically, the operation of step 2) of the above method is:

[0029] The chelating agent-peptide DOTA-KINW was dissolved in ultrapure water to a final concentration of 1 mg / mL. 100 μL of DOTA-KINW solution was mixed with 2 mCi68 Ga was mixed in 2 mL of HEPES buffer at pH = 4.5 and reacted at 90 °C for 30 min to obtain a radioactive tracer. 68 Ga-DOTA-KINW.

[0030] The use of the above-mentioned radioactive tracers in the preparation of tumor diagnostic reagents also falls within the protection scope of the present invention.

[0031] In the application, the tumor includes brain glioma, breast cancer and lung cancer, and more specifically may be glioblastoma multiforme.

[0032] The diagnostic reagent is used for imaging human brain glioma cells and brain glioma transplanted tumors in vivo.

[0033] According to the embodiment of the present invention, the selected human brain glioma cells are U87-MG cells, and the selected human brain glioma transplanted in vivo is a BABL / c nude mouse orthotopic transplanted tumor.

[0034] The radioactive tracer provided by the present invention (specifically 68 Compared with the prior art, Ga-DOTA-KINW) has the following beneficial effects:

[0035] 1) The present invention uses mutation scanning and molecular docking technology to screen the modified NPY1R-specific binding targeting polypeptide. Compared with the previously reported sequences, the polypeptide has more binding sites with NPY1R, less cytotoxicity, and stronger uptake of the polypeptide by human glioma cells;

[0036] 2) The radioactive tracer prepared by the present invention can cross the blood-brain barrier as an imaging drug for human brain glioma, and has the characteristics of long imaging time and easy preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The three-dimensional structure of the polypeptide KINP in Example 1 of the present invention. The yellow stick residues are the natural amino acids scanned for mutations, and the modified residues are shown as white sticks.

[0038] Figure 2 This is the structure of the polypeptide KINP-NPY1R complex in Example 1 of the present invention. The natural amino acids on the polypeptide are shown as yellow sticks, the protein residues near the natural amino acids of the polypeptide are shown as green sticks, and the protein is shown as a green stripe structure.

[0039] Figure 3 is the structural formula of the polypeptides KINP and KINW in Example 1 of the present invention.

[0040] Figure 4 This is a three-dimensional docking model of the polypeptides KINP and KINW and the NPY1R protein in Example 1 of the present invention.

[0041] Figure 5 The HPLC and MS identification results of the polypeptide KINP prepared in Example 1 of the present invention are shown.

[0042] Figure 6 The HPLC and MS identification results of the polypeptide KINW prepared in Example 1 of the present invention are shown.

[0043] Figure 7 The results are as follows: the effects of polypeptides KINP and KINW on the survival rate of U87-MG cells in Example 2 of the present invention. *p﹤0.05, **p﹤0.01.

[0044] Figure 8 The results are as follows: the effects of polypeptides KINP and KINW on the survival rate of HUVEC cells in Example 2 of the present invention. *p﹤0.05, **p﹤0.01.

[0045] Fig. 9 The results are as follows: the effects of polypeptides KINP and KINW on the survival rate of bEND.3 cells in Example 2 of the present invention. **p﹤0.01.

[0046] Fig.10 is the structural formula of FITC-KINP in Example 3 of the present invention.

[0047] Fig.11 These are the HPLC and MS identification results of FITC-KINP prepared in Example 3 of the present invention.

[0048] Fig.12 It is the structural formula of FITC-KINW in Example 3 of the present invention.

[0049] Fig.13 These are the HPLC and MS identification results of FITC-KINW prepared in Example 3 of the present invention.

[0050] Fig.14 The flow cytometry comparison of KINP and KINW uptake by U87-MG cells in Example 3 of the present invention.

[0051] Fig.15 This is the immunofluorescence comparison of the uptake of KINP and KINW by U87-MG cells in Example 3 of the present invention.

[0052] Fig.16 This is the fluorescence intensity analysis of immunofluorescence comparison of the uptake of KINP and KINW by U87-MG cells in Example 3 of the present invention. *p﹤0.05.

[0053] Fig.17This is the flow cytometry comparison of KINW uptake by U87-MG cells (co-culture) in Example 3 of the present invention, and the flow cytometry graph of co-culture of bEnd.3 and U87.

[0054] Fig.18 This is the immunofluorescence comparison of KINW uptake by U87-MG cells (co-culture) in Example 3 of the present invention, and the immunofluorescence image of co-culture of bEnd.3 and U87.

[0055] Fig.19 This is the fluorescence intensity analysis of the immunofluorescence contrast of KINW uptake by U87-MG cells in Example 3 of the present invention (co-culture), bEnd.3 and U87 co-culture immunofluorescence intensity. **p﹤0.01.

[0056] Fig. 20 This is the fluorescence intensity analysis of KINW uptake by U87-MG cells compared by flow cytometry in Example 3 of the present invention (co-culture), and the flow cytometric graph of HUVEC and U87 co-culture.

[0057] Fig.21 This is the fluorescence intensity analysis of the immunofluorescence comparison of KINW uptake by U87-MG cells in Example 3 of the present invention (co-culture), and the immunofluorescence image of HUVEC and U87 co-culture.

[0058] Fig. 22 This is the fluorescence intensity analysis of the immunofluorescence contrast of KINW uptake by U87-MG cells in Example 3 of the present invention (co-culture), and the immunofluorescence intensity of HUVEC and U87 co-culture. **p﹤0.01, ***p﹤0.001.

[0059] Fig.23 is the structural formula of Cy5.5-KINW prepared in Example 4 of the present invention.

[0060] Fig.24 These are the HPLC and MS identification results of Cy5.5-KINW prepared in Example 4 of the present invention.

[0061] Fig.25 These are fluorescent in vivo imaging images of Cy5.5-KINW in Example 4 of the present invention at different time points in nude mice with orthotopic transplanted tumors of U87-MG cells.

[0062] Fig.26 The fluorescence intensity of Cy5.5-KINW in Example 4 of the present invention at different time points in the brain of nude mice with orthotopic transplanted tumors of U87-MG cells.

[0063] Fig. 27These are in vivo imaging images of Cy5.5-KINW in the brain and different organs of nude mice with orthotopic transplanted tumors of U87-MG cells 24 hours after administration in Example 4 of the present invention.

[0064] Fig.28 This is a graph showing the fluorescence intensity of Cy5.5-KINW in the brain of nude mice with orthotopic transplanted tumors of U87-MG cells 24 hours after administration in Example 4 of the present invention. ***p﹤0.001.

[0065] Fig.29 is the structural formula of DOTA-KINW prepared in Example 5 of the present invention.

[0066] Fig.30 These are the HPLC and MS identification results of DOTA-KINW prepared in Example 5 of the present invention.

[0067] Fig.31 Prepared in Example 5 of the present invention 68 TLC detection results of Ga-DOTA-KINW.

[0068] Fig.32 Prepared in Example 5 of the present invention 68 In vitro stability experimental results of Ga-DOTA-KINW.

[0069] Fig.33 The U87-MG cell pair in Example 5 of the present invention 68 Uptake results of Ga-DOTA-KINW. *p﹤0.05.

[0070] Fig.34 The BABL / c nude mice inoculated with U87-MG cells in Example 6 of the present invention were injected via the tail vein 68 Micro-PECT / CT imaging results of Ga-DOTA-KINW and DOTA-KINW.

[0071] Fig.35 Injection of BABL / c transplanted tumor nude mice via tail vein 68 Radioactivity detection of brain tissue 2 hours after Ga-DOTA-KINW and DOTA-KINW. *p﹤0.05. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0073] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0074] Example 1. Molecular docking of KINW and NPYIR proteins

[0075] 1) Mutation scanning

[0076] The amino acid scanning function in the MOE (Molecular Operating Environment) protein design module was used to scan the natural amino acids of the ligand peptide in the complex to determine the affinity changes between the human NPY1R protein and the ligand peptide. The natural amino acids of KINPKFRLRY (KINP) were selected for mutation formation. The three-dimensional structure of KINP is shown in Figure 1 Based on the structure of the complex formed by KINP and NPY1R, the binding mode of KINP and NPY1R protein is shown in Figure 2 As shown. Each natural amino acid of the ligand polypeptide in the KINP-NPY1R complex is mutated into other amino acids, the changes in binding affinity between the ligand polypeptide and the protein are recorded, and the mutations are ranked according to the changes in binding affinity. "dAffinity" refers to the relative binding affinity of the mutation to the protein. The smaller the value, the better the affinity of the mutation. The proline (P) at the fourth position of the KINPKFRLRY (KINP) sequence is mutated into tryptophan (W), and the new sequence is KINWKFRLRY (KINW) (structural formula as shown Figure 3 This sequence has the highest affinity with NPY1R protein among the newly scanned mutant sequences, with a dAffinity value of -0.95659 kcal / mol.

[0077] 2) Molecular docking

[0078] The three-dimensional structure of the peptide KINW was constructed using the Protein Builder module in MOE (Molecular Operating Environment). The human NPY1R protein, with a total length of 384 amino acids, and the crystal structure of NPY1R was downloaded from the RCSB protein database (http: / / www.rcsb.org / ), and the PDB ID number is 5ZBQ, as the docking receptor. The HDOCK protein-protein docking software was used to dock the peptide with NPY1R, and the binding mode was predicted by a hybrid docking strategy. In the docking process, the protein was selected as the receptor and the peptide as the ligand. The binding mode of the peptide KINP with NPY1R is shown in Figure 2. Figure 4As shown in Figure 2, KINP and NPY1R have three binding sites. In the binding pocket, hydrogen bonds are formed between KINP and NPY1R. Asn at position 3 of KINP forms hydrogen bonds with Gln at positions 328 and 332 of NPY1R, respectively, and Arg at position 7 of KINP forms hydrogen bonds with Leu at position 323 of NPY1R. The binding mode of peptide KINW and NPY1R is shown in Figure 2. Figure 4 As shown in the figure, KINW has five binding sites with NPY1R and there is a π-H bond. In the binding pocket, hydrogen bonds are formed between KINW and NPY1R. Asn at position 3 of KINW forms hydrogen bonds with Arg at position 260 and Gln at position 332 of NPY1R, Trp at position 4 of KINW forms a π-H bond with Arg at position 260 of NPY1R, Arg at position 7 of KINW forms a hydrogen bond with Phe at position 322 of NPY1R, and Lys at position 5 of KINW forms a hydrogen bond with Asn at position 336 and Phe at position 337 of NPY1R. Compared with KINP, KINW has more binding sites with NPY1R, which provides the binding energy between the polypeptide and NPY1R.

[0079] Example 2: Analysis of the effects of KINP and KINW on cytotoxicity

[0080] 1) Artificial synthesis of KINP and KINW

[0081] Lysine-isoleucine-asparagine-proline-lysine-phenylalanine-arginine-leucine-arginine-tyrosine, Lys-Ile-Asn-Pro-Lys-Phe-Arg-Leu-Arg-Tyr, abbreviated as KINP.

[0082] Lysine-isoleucine-asparagine-tryptophan-lysine-phenylalanine-arginine-leucine-arginine-tyrosine, Lys-Ile-Asn-Trp-Lys-Phe-Arg-Leu-Arg-Tyr, abbreviated as KINW.

[0083] KINP and KINW were artificially synthesized from C-terminus to N-terminus. ① The resin was swollen, 2-Chlorotrityl Chloride Resin was placed in a reaction tube, DCM (15 mL / g) was added, and the mixture was shaken for 30 min; ② The first amino acid was added, the solvent was filtered out through a sand core, 3 times the molar excess of Fmoc-Tyr(tBu)-OH amino acid was added, DMF (dimethylformamide) was added to dissolve, and then 10 times the molar excess of DIEA (diisopropylethylamine) was added, and the mixture was shaken for 60 min. Block with methanol; ③ Deprotection, remove DMF, add 20% piperidine DMF solution (15mL / g), react for 5min, remove and add 20% piperidine DMF solution (15mL / g), react for 15min; ④ Detection, remove the piperidine solution, take more than a dozen resins, wash with ethanol three times, add detection reagent for detection, heat at 105℃~110℃ for 5min, turn dark blue for positive reaction; ⑤ Wash, DMF (10mL / g) twice, DCM (10mL / g) twice, DMF (10mL / g) twice; ⑥ Condensation, protect amino acid three times excess, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate) three times excess, all dissolved with as little DMF as possible, add to the reaction tube, immediately add DIEA ten times excess, react for 30min; ⑦ Detection, take more than a dozen resins, wash with ethanol three times, add detection reagent for detection, heat at 105℃~110℃ for 5min, colorless for negative reaction. ⑧ Washing, DMF (10mL / g) twice, DCM (10mL / g) twice, DMF (10mL / g) twice; ⑨ Repeat ②-⑧ operation steps, connect the amino acids in the sequence from right to left; ⑩ Drain, washing steps: DMF (10mL / g) twice, methanol (10mL / g) twice, DMF (10mL / g) twice, DCM (10mL / g) twice, drain for 10 minutes. 11) Cut the peptide from the resin, prepare the cutting solution (10mL / g) TFA (trifluoroacetic acid) 95%; water 1%; EDT (ninhydrin) 2%; TIS (triisopropylsilane) 2%, cutting time: 120min; blow dry and wash, blow the cleavage solution as dry as possible with nitrogen, wash with ether six times, and then evaporate at room temperature; analyze and purify, purify the crude product with high performance liquid chromatography; freeze dry, collect the target peptide solution and put it into a freeze dryer for concentration, and freeze dry it into powder. The results of HPLC and MS identification of KINP (molecular weight 1334.63) are shown in Figure 5 The HPLC and MS identification results of KINW (molecular weight 1423.73) are shown in Figure 6 .

[0084] 2) Effects of KINP and KINW on cell survival

[0085] The cell viability was determined using the CCK-8 kit. bEnd.3, HUVEC and U87-MG cells were seeded in 96-well plates, respectively. When the growth density reached 70%, different concentrations of KINP and KINW (0, 20, 40, 100, 200 μg / mL) were added and 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 culture was continued for 4 hours at 37°C and 5% CO2. The 96-well plate was placed in a microplate reader and the absorbance was measured at a wavelength of 450 nm. The cell viability formula is [A(sample well)–A(blank)] / [A(PBS)–A(blank)]×100%. The results showed that KINP had an inhibitory effect on the survival of human glioma cells U87-MG cells ( Figure 7 ), human umbilical vein endothelial cells HUVEC cells ( Figure 8 ) and mouse brain microvascular endothelial cells bEnd.3 cells ( Fig. 9 ) is more toxic than KINW.

[0086] Example 3: Analysis of KINW uptake by U87-MG cells

[0087] 1) Preparation of FITC-KINP and FITC-KINW

[0088] The peptide synthesis was the same as steps ① to ⑧ in Example 2, and the amino acids in the sequence were connected from right to left to link 5-FTIC. The subsequent steps were the same as step ⑩ in Example 2. The structural formula of FITC-KINP is shown in Fig.10 , HPLC and MS (molecular weight 1837.01) identification results are shown in Fig.11 The structural formula of FITC-KINW is shown in Fig.12 , HPLC and MS (molecular weight 1926.23) identification results are shown in Fig.13 .

[0089] 2) Quantitative and qualitative analysis of KINP and KINW uptake in U87-MG cells

[0090] FITC-KINP and FITC-KINW at a final concentration of 20 μg / mL containing 10% FBS were added to U87-MG cells in the logarithmic growth phase, incubated at 37°C, and digested with 0.25% trypsin at 2 and 4 hours, respectively. The cells were centrifuged at 1000 rpm / min for 5 minutes, the supernatant was removed, and the cells were washed 3 times with cold PBS, and then resuspended in 300 μL cold PBS for flow cytometry to measure the FITC fluorescence intensity of the cells. The samples for laser confocal detection were washed 3 times with pre-cooled PBS, then fixed with 4% paraformaldehyde for 15 minutes, naturally dried, and 5 μg / mL DAPI was added to stain the cell nucleus. Finally, the slides were sealed with anti-fading agent, photographed with laser confocal microscopy, and the fluorescence density was analyzed with Image J software. The results showed that 2 hours and 4 hours after drug addition, the FITC fluorescence intensity of U87-MG cells in the FITC-KINW group was greater than that in the FITC-KINP group ( Fig.14 ). 4 hours after drug addition, the FITC fluorescence intensity of U87-MG cells in the FITC-KINW group was greater than that in the FITC-KINP group ( Fig.15 , 16 ). The above results indicate that the uptake ability of brain glioma cells to KINW is stronger than that to KINP.

[0091] 3) Analysis of KINW crossing the blood-brain barrier and blood-brain tumor barrier in vitro

[0092] bEnd.3 and HUVEC were plated with U87-MG cells at a ratio of 1:5 in the upper and lower chambers of Transwell, respectively, and cultured for 48 hours to simulate the blood-brain barrier and blood-brain tumor barrier. The culture medium of bEnd.3 and HUVEC cells in the upper chamber of Transwell was aspirated, and FITC-KINW with a final concentration of 20μg / mL containing 10% FBS was added, incubated at 37℃, and the U87-MG cells in the lower chamber were digested with 0.25% trypsin at 4 and 8 hours, respectively, centrifuged at 1000rpm / min for 5min, the supernatant was removed, and washed with cold PBS three times, and then resuspended in 300μL cold PBS for flow cytometry of FITC fluorescence intensity of cells, and FITC without peptide was used as a control. Samples for laser confocal detection were washed three times with pre-cooled PBS, then fixed with 4% paraformaldehyde for 15min, naturally dried, and 5μg / mL DAPI was added to stain the cell nucleus, and finally sealed with anti-fading agent, photographed with laser confocal microscope, and analyzed by Image J software for fluorescence density. In the group of bEnd.3 cells co-cultured with U87-MG cells, flow cytometry ( Fig.17 ) and immunofluorescence ( Fig.18 , 19) showed that 4 hours and 8 hours after drug addition, the FITC fluorescence intensity of U87-MG cells in the FITC-KINW group was greater than that in the FITC group. In the HUVEC and U87-MG cell co-culture group, flow cytometry ( Fig. 20 ) and immunofluorescence ( Fig.21 , 22 ) showed that 4 hours and 8 hours after drug addition, the FITC fluorescence intensity of U87-MG cells in the FITC-KINW group was greater than that in the FITC group. The above results indicate that KINW can cross the blood-brain barrier and blood-brain tumor barrier in vitro and be taken up by U87-MG cells.

[0093] Example 4: Experiment on KINW crossing the blood-brain barrier

[0094] 1) Preparation of Cy5.5-KINW

[0095] The peptide synthesis was the same as steps ① to ⑧ in Example 2. The amino acids in the sequence were connected from right to left to link Cy5.5. The subsequent steps were the same as step ⑩ in Example 2. The structural formula of Cy5.5-KINW is shown in Fig.23 , HPLC and MS (molecular weight 1989.51) identification results are shown in Fig.24 .

[0096] 2) Establishment of U87-MG cell orthotopic tumor animal model

[0097] A U87-MG cell mouse orthotopic tumor model was established using a brain stereotaxic instrument. U87-MG-luc cells in the logarithmic growth phase were obtained and digested with 0.25% trypsin to adjust the cell density to 1×10 5 / mL, 5 μL of cell suspension was drawn using a brain stereotaxic instrument and injected into the posterior ventricle (striatum, 1.8 mm to the right of the anterior fontanelle, depth 3 mm) of the intracranial cavity of female BABL / c nude mice. 14 days after inoculation, the tumor formation was observed using in vivo imaging of small animals.

[0098] 3) Tail vein administration and KINW imaging observation in tumor-bearing mice

[0099] Tumor-bearing nude mice were injected with 100 μL (1 μg / μL) of Cy5.5-KINW saline injection of Example 4 through the tail vein, and images were collected using a small animal fluorescent in vivo imager 1, 3, 6, 12, and 24 hours after injection. The results showed that the brains of the nude mice in the drug-treated group could continue to show fluorescent signals, and the brains of the nude mice still had fluorescent signals 24 hours after administration ( Fig.25 , 26 ). 24 hours after injection, the mouse organs and brain were separated and images were collected using a small animal fluorescent in vivo imager. The results showed that compared with the Cy5.5 group, the fluorescent signal of the nude mouse brain in the Cy5.5-KINW group increased significantly ( Fig. 27 , 28 ), indicating that KINW can cross the blood-brain barrier and has a sustained targeting binding effect.

[0100] Embodiment 5, 68 Preparation of Ga-DOTA-KINW radiotracer

[0101] 1) Preparation of DOTA-KINW

[0102] The peptide synthesis was the same as steps ① to ⑧ in Example 2. The amino acids in the sequence were connected from right to left. Three times the molar amount of DOTA (tetraazacyclododecane tetraacetic acid-diphenylcyclooctyne), three times the molar amount of HBTU, and three times the molar amount of NMM (N-methylmorpholine) were weighed and dissolved in DMF. DCM was added and stirred with nitrogen. The reaction was carried out for 15 minutes. The subsequent steps were the same as step ⑩ in Example 2. The structural formula of DOTA-KINW is shown in Fig.29 , HPLC and MS (molecular weight 1810.14) identification results are shown in Fig.30 .

[0103] 2) 68 Ga marked DOTA-KINW

[0104] DOTA-KINW was dissolved in ultrapure water to a concentration of 1 mg / mL and stored at -20°C; HEPES (4-hydroxyethylpiperazineethanesulfonic acid) was prepared in ultrapure water to 2M for standby use; 100 μL of peptide solution + 2mL of HEPES (HEPES concentration is 1M); rinsed with 0.1M HCl 68 Ge- 68 Ga generator to obtain 68 GaCl3, add 2mCi to the mixture of peptide and HEPES 68 Ga eluent (0.1M HCl), mix well, test paper to determine pH = 4.5; seal and react at 90℃ for 30min to prepare 68 Ga-DOTA-KINW; identified by Radio-TLC quality control, the results are shown in Fig.31 , the radiochemical purity was 100%.

[0105] 3) 68 In vitro stability experiment of Ga-DOTA-KINW

[0106] The prepared radioactive tracer (100 μL, 100 μCi) was placed in 1 mL of 10% fetal bovine serum, PBS and saline, and incubated at 37°C for 0.5, 1, 2 and 4 hours, and the radiochemical purity of the radioactive tracer was detected by TLC. Fig.32As shown, the radiotracer was highly stable in 10% fetal bovine serum, with a radiochemical purity of 70.31% 4 hours after labeling.

[0107] 4) 68 Cellular uptake experiments of Ga-DOTA-KINW

[0108] U87-MG cells were inoculated into 6-well plates and incubated for 24 hours. 68 Ga-DOTA-KINW (about 25 μCi) was added to 6-well plates, and the control group was added with free 68 Ga was added to a 6-well plate. Cultured for 0.5 and 1 hour, the cells were washed 3 times with cold PBS, and then lysed with 0.5 mL of 1 M NaOH and counted using a gamma counter Wizard 2470. Uptake rate = gamma count of cells / 25 μCi 68 The gamma count of Ga × 100%. The results show that the free 68 The Ga control group did not significantly increase cellular uptake over time, but 68 The cellular uptake in the Ga-DOTA-KINW group gradually increased over time, indicating that 68 Ga-DOTA-KINW has a targeted binding effect on U87-MG ( Fig.33 ).

[0109] Example 6: PET / CT imaging and brain uptake analysis

[0110] 1) PET / CT imaging of orthotopic transplanted tumors of U87-MG cells

[0111] U87-MG cell orthotopic brain transplanted tumor in nude mice 68 Ga group, KINW blocking + 68 Ga-DOTA-KINW group and 68 Ga-DOTA-KINW group, 68 Ga group and 68 The nude mice in the Ga-DOTA-KINW group were injected with about 200 μCi of 68 Ga or 68 Ga-DOTA-KINW, KINW Block+ 68 The nude mice in the Ga-DOTA-KINW group were injected with 100 μg of KINW via the tail vein. 15 min later, about 200 μCi of 68 Ga-DOTA-KINW. Images were collected using micro-PET / CT at 0.5 and 1 h after administration. The results showed that 68In the Ga-DOTA-KINW group, 0.5 and 1 h after administration, the brain in situ tumors were imaged, and the SUVR value of the tumor was 1 h > 0.5 h; KINW blocked + 68 In the Ga-DOTA-KINW group, the brain in situ tumor was imaged at 1 hour after administration, but the SUVR value of the tumor was lower than that at the same time point. 68 Ga-DOTA-KINW group; 68 In the Ga group, no in situ brain tumors were visualized at 0.5 and 1 h after administration ( Fig.34 The above results indicate that due to the targeted binding effect of KINW on brain glioma, the radioactive tracer can image brain glioma. After KINW competitively binds to NPY1R, 68 The binding of Ga-DOTA-KINW to NPY1R was reduced, making the SUVR value of the tumor smaller than that at the same time point 68 Ga-DOTA-KINW group, 68 The imaging effect of Ga-DOTA-KINW on tumors was weakened.

[0112] 2) Brain uptake analysis

[0113] KINW Blocking+ 68 Ga-DOTA-KINW group and 68 The brain tissue of the nude mice in the Ga-DOTA-KINW group was isolated 2 hours after administration, and the weight was taken and counted using a gamma counter Wizard 2470. Uptake rate = gamma count of cells / 25 μCi 68 Ga's gamma count × 100%. The gamma count results show that 68 The %ID / g of Ga-DOTA-KINW group was greater than that of KINW blocking + 68 Ga-DOTA-KINW group ( Fig.35 The results showed that KINW competitively binds to NPY1R, making the tumor 68 Ga uptake is less than 68 Ga-DOTA-KINW group.

[0114] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A polypeptide targeting NPY1R protein, the amino acid sequence of which is as follows: KINWKFRLRY, lysine-isoleucine-asparagine-tryptophan-lysine-phenylalanine-arginine-leucine-arginine-tyrosine, Lys-Ile-Asn-Trp-Lys-Phe-Arg-Leu-Arg-Tyr, abbreviated as KINW.

2. A radioactive tracer consisting of the following components: Peptides, chelators DOTA and radionuclides targeting NPY1R protein 68 Ga, The peptide targeting NPY1R protein was covalently coupled to the chelator DOTA, and the radionuclide 68 Ga is chelated to the chelating agent DOTA; The radioactive tracer is represented by: 68 Ga-DOTA-KINW, where, 68 Ga is a radionuclide, DOTA is a chelating agent, and KINW is a polypeptide targeting NPY1R protein, and its amino acid sequence is as follows: KINWKFRLRY, Lys-Ile-Asn-Trp-Lys-Phe-Arg-Leu-Arg-Tyr.

3. A method for preparing the radioactive tracer according to claim 2, comprising the steps of: 1) Preparation of Chelating Agent-Peptide In the presence of a catalyst and a condensing agent, the chelating agent DOTA and the polypeptide KINW are subjected to a coupling reaction to obtain the chelating agent-polypeptide DOTA-KINW; 2) Preparation of radionuclide labeled chelator-peptide Add radioactive nuclide to the mixed solution of chelating agent-peptide DOTA-KINW, water and hydrogen ion buffer 68 Ga, the reaction produces a radionuclide labeled chelator-peptide, i.e., a radioactive tracer 68 Ga-DOTA-KINW.

4. The method according to claim 3, characterized in that The molar ratio of the polypeptide to the chelating agent, the catalyst and the condensing agent is 1:1-5: 1-5: 1-5; The catalyst is an organic base; The condensation agent is: HBTU; The coupling reaction is carried out in an inert atmosphere; The coupling reaction time is 5-15 min.

5. The method according to claim 3, characterized in that In step 2), the hydrogen ion buffer is 4-hydroxyethylpiperazine ethanesulfonic acid HEPES, and the HEPES concentration in the mixed solution of water and hydrogen ion buffer is 1 M, and the pH is 4-4.5; The chelating agent-polypeptide DOTA-KINW and the radionuclide 68 The ratio of Ga is: 0.1 mg: 2 mCi-0.2 mg: 2 mCi; The reaction temperature is 80-100°C and the reaction time is 15-30 min.

6. Use of the radioactive tracer according to claim 2 in the preparation of tumor diagnostic reagents; The tumor is a brain glioma.

7. The use according to claim 6, characterized in that: The tumor is glioblastoma multiforme.

8. The use according to claim 6, characterized in that: The diagnostic reagent is used for imaging human brain glioma cells and brain glioma transplanted tumors in vivo.

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