A polypeptide PET molecular probe targeting breast cancer and a preparation method and application thereof

Abstract

The application discloses a polypeptide PET molecular probe for targeting breast cancer and a preparation method and application thereof. The polypeptide PET molecular probe for targeting breast cancer is obtained by labeling an AR polypeptide modified by a bifunctional chelating agent with a positron emitting radionuclide, and has the advantages of simple synthesis, small molecular weight, high specificity, no immunogenicity and good biological safety. In addition, the polypeptide PET molecular probe has high radiochemical performance and in-vivo and in-vitro stability, can better realize breast cancer targeted PET imaging compared with current commonly used imaging agents, can be used for preparing an imaging agent for breast cancer diagnosis, and is helpful for clinical diagnosis of breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a polypeptide PET molecular probe targeting breast cancer, its preparation method, and its application. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, and its accurate diagnosis is of great significance for treatment. PET / CT plays an important role in the diagnosis, clinical staging, efficacy evaluation, follow-up, prognosis prediction, and treatment guidance of breast cancer. PET imaging agents are the soul of PET / CT imaging, a powerful tool that enables precise in vivo detection of molecular-level information in the body. Developing novel and efficient PET tracers has always been a continuous pursuit for professionals.

[0003] at present, 18 F-FDG is the most widely used imaging agent in clinical practice, but its sensitivity and specificity are limited by small lesions or breast cancer subtypes, making it unable to accurately diagnose breast cancer. Furthermore, in inflamed tissues... 18 The accumulation of fibrocystic oxidative stress (F-FDG) also complicates the identification of breast tumors. Targeted PET imaging has shown significant importance in the precise detection of breast cancer. For example, tumor-targeted PET imaging based on HER2, EGFR, ER, and RGD has demonstrated value in the precision diagnosis and treatment of breast cancer. However, the application of these molecular components is limited due to insufficient expression and lack of specificity in breast cancer. Therefore, the development of novel PET molecular probes targeting breast cancer remains of significant clinical value and demand.

[0004] In 2017, Professor Mao Chuanbin screened a novel breast cancer-targeting peptide using in vivo phage technology. Its molecular sequence is AREYGTRFSLIGGYR (AR for short), and in vitro and in vivo experiments showed good targeting ability for breast cancer. However, during the preparation of PET molecular probes targeting breast cancer based on the AR peptide, it was found that the targeting and stability of PET molecular probes prepared using conventional molecular modification methods, such as modification with bifunctional chelating agents followed by labeling with positron-emitting radionuclides, did not meet the needs of breast cancer diagnosis. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the shortcomings and deficiencies of peptide PET molecular probes prepared using conventional molecular modification methods based on peptide AR, such as insufficient targeting and stability, which cannot meet the needs of breast cancer diagnosis. This invention provides a peptide PET molecular probe targeting breast cancer, its preparation method, and its application.

[0006] The first objective of this invention is to provide a polypeptide PET molecular probe that targets breast cancer.

[0007] A second objective of this invention is to provide the application of the peptide PET molecular probe in the preparation of imaging products targeting breast cancer.

[0008] A third objective of this invention is to provide the application of the aforementioned polypeptide PET molecular probe in the preparation of diagnostic reagents for breast cancer.

[0009] A fourth objective of this invention is to provide a method for preparing the polypeptide PET molecular probe.

[0010] A fifth objective of this invention is to provide an imaging agent for the diagnosis of breast cancer.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] This invention's experiments have shown that, based on the peptide AR (AREYGTRFSLIGGYR), modification with NOA or DOTA, followed by labeling with positron-emitting radionuclides, can be achieved. 68 The peptide PET molecular probes for targeting breast cancer prepared with Ga cannot adequately meet the needs of breast cancer diagnosis. To address this issue, this invention incorporates a linker block between a bifunctional chelating agent and the peptide AR. However, different linker blocks result in significant variations in the stability, hydrophilicity, and other properties of the obtained peptide PET molecular probes. Through continuous adjustments and optimizations, this invention finally yields a peptide PET molecular probe with high stability and hydrophilicity, good in vivo safety, and the ability to specifically target breast cancer cells.

[0013] This invention provides a peptide PET molecular probe for targeting breast cancer. The peptide PET molecular probe is obtained by labeling an AR peptide modified with a bifunctional chelating agent with a positron-emitting radionuclide. The AR peptide is AREYGTRFSLIGGYR. The bifunctional chelating agent and the AR peptide also contain a linker. The linker is GGGKKKK-PEG3 or K-PEG3.

[0014] Specifically, the bifunctional chelating agent is DOTA or NOA.

[0015] Specifically, the positron-emitting radionuclide is 68 Ga or 18 F.

[0016] Specifically, the bifunctional chelating agent used in this invention is NOTA.

[0017] As an optional implementation, the polypeptide PET molecular probe of the present invention is AREYGTRFSLIGGYR-GGGKKKK-PEG3-NOTA, abbreviated as Nota-P-AR.

[0018] As an optional implementation, the polypeptide PET molecular probe of the present invention is AREYGTRFSLIGGYR-K-PEG3-NOTA, abbreviated as Nota-PK-AR.

[0019] Specifically, this invention labels the NOA end of the peptide PET molecular probe NOA-P-AR. 68 Ga, named 68 Ga-NOTA-P-AR; or labeled at the NOA end of the peptide PET molecular probe NOA-PK-AR. 18 F, named Al 18 F-NOTA-PK-AR.

[0020] Specifically, the preparation method of the peptide PET molecular probe targeting breast cancer according to the present invention includes the following steps:

[0021] S1. Artificially synthesize the AR polypeptide modified with the bifunctional chelating agent described in this invention;

[0022] S2. The AR polypeptide modified with the bifunctional chelating agent described in step S1 is labeled with a positron emission tomography (PET) radionuclide.

[0023] Specifically, the positron-emitting radionuclide is 68 Ga or 18 F.

[0024] Specifically, the positron-emitting radionuclide 68 The Ga labeling method is as follows: The synthesized bifunctional chelating agent modified AR peptide is dissolved in water to obtain a precursor solution. The germanium gallium generator is rinsed into the container with 0.8-1.2 mL of 0.1 mol / L hydrochloric acid solution. 80-100 μg of the precursor solution is added and mixed well. Then, 80-120 μL of 1 mol / L sodium acetate solution is added and mixed well. The pH of the mixture is adjusted to 3.5-4.5, and the mixture is heated at 98-100℃ for 8-12 min. After the reaction is completed, the reaction solution is cooled to room temperature.

[0025] More specifically, the NOTA-P-AR synthesized using a solid-phase chemical method was dissolved in deionized water to obtain a precursor solution with a concentration of 1 μg / μL, and germanium-gallium was rinsed with a 0.1 mol / L high-purity hydrochloric acid solution. 68 Ge / 68 The Ga) generator was connected to an EP tube, and the 1 mL with the highest radioactivity content was collected. 100 μg of precursor solution (1 μg / μL, 100 μL) was added and mixed well. Then, 100 μL of sodium acetate (1 mol / L) solution was added and mixed well. The pH of the mixture was adjusted to 3.5–4.5, and the mixture was heated at 100 °C for 10 min. After the reaction was complete, the reaction solution was cooled to room temperature to obtain… 68 Ga-NOTA-P-AR.

[0026] Specifically, the positron-emitting radionuclide 18 The labeling method for F is as follows: the synthesized bifunctional chelating agent-modified AR peptide is dissolved in water to obtain a precursor solution, which is then eluted and enriched on a QMA column with 0.4–0.6 mL of physiological saline. 18 F ions were collected, and the eluent was mixed with 24–26 μL of AlCl3 sodium acetate buffer and 80–100 μg of precursor solution. The pH of the mixture was adjusted to 4 with dilute hydrochloric acid, and the mixture was heated at 110 °C for 12–15 min. After the reaction was completed, the reaction solution was cooled to room temperature.

[0027] More specifically, the NOTA-PK-AR synthesized using solid-phase chemistry was dissolved in deionized water to obtain a precursor solution with a concentration of 1 μg / μL, which was then eluted and enriched on a QMA column using 0.5 mL of physiological saline. 18 F ions were collected, and the eluent was mixed with 25 μL of AlCl3 sodium acetate buffer and 100 μg of precursor solution (1 μg / μL, 100 μL). Then, 0.05 mol / L dilute hydrochloric acid was added to adjust the pH of the mixture to 4. The mixture was heated at 110 °C for 15 min. After the reaction was complete, the reaction solution was cooled to room temperature to obtain Al. 18 F-NOTA-PK-AR.

[0028] The present invention also claims protection for the use of the peptide PET molecular probe in the preparation of imaging products targeting breast cancer.

[0029] This invention also claims protection for the use of the peptide PET molecular probe in the preparation of breast cancer diagnostic reagents.

[0030] The present invention also provides an imaging agent for breast cancer diagnosis, wherein the imaging agent contains the polypeptide PET molecular probe described in the present invention.

[0031] The present invention has the following beneficial effects:

[0032] This invention provides a peptide-based PET molecular probe targeting breast cancer and its preparation method. The peptide-based PET molecular probe of this invention is obtained by labeling an AR peptide modified with a bifunctional chelating agent using a positron-emitting radionuclide. It has advantages such as simple synthesis, small molecular weight, high specificity, no immunogenicity, and good biosafety. Furthermore, the peptide-based PET molecular probe of this invention exhibits high radiochemical performance and in vitro and in vivo stability. Compared with commonly used imaging agents, this probe can better achieve targeted PET imaging of breast cancer and can be used to prepare imaging agents for breast cancer diagnosis, thus contributing to the clinical diagnosis of breast cancer. Attached Figure Description

[0033] Figure 1 The present invention provides the chemical structural formula of the NOA-P-AR and the polypeptide PET molecular probe prepared therefrom. 68 The radiochemical purity test results of Ga-NOTA-P-AR; where Figure A is the chemical structural formula of NOTA-P-AR, and Figures B and C are respectively... 68 HPLC chromatograms of Ga-NOTA-P-AR in PBS and FBS.

[0034] Figure 2 The chemical structural formula of the NOTA-PK-AR described in this invention and the polypeptide PET molecular probe Al prepared therefrom are described in this invention. 18 The radiochemical purity test results of F-NOTA-PK-AR; Figure A shows the chemical structural formula of NOTA-PK-AR, and Figures B and C show the chemical structural formulas of Al, respectively. 18 HPLC chromatograms of F-NOTA-PK-AR in PBS and FBS.

[0035] Figure 3 The uptake of AR-FITC by MCF-7 and MCF-10A cells.

[0036] Figure 4 For MCF-7 cells and 4T1 cells against 68 The internalization status of Ga-NOTA-P-AR.

[0037] Figure 5 For MCF-7 cells to respond to Al 18 The internalization status of F-NOTA-PK-AR.

[0038] Figure 6 for 68 PET imaging of Ga-NOTA-P-AR in MCF-7 and 4T1 tumor-bearing mice and comparison of tumor radioactive uptake and tumor / muscle ratio at different time points; where Figures A and B are respectively 68 Representative PET images of MCF-7(A) and 4T1(B) tumor-bearing mice at 10, 30, 60, and 120 min after Ga-NOTA-P-AR injection; Figures C and D show quantitative analysis based on PET images. 68 Tumor uptake of Ga-NOTA-P-AR and tumor / muscle (T / M) ratio.

[0039] Figure 7 for 68 Dynamic imaging results of Ga-NOTA-P-AR in MCF-7 tumor-bearing mice; Figure A shows the intravenous injection... 68A representative coronal Micro-PET image of the MCF-7 tumor model after dynamic scanning 120 min following Ga-NOTA-P-AR; Figure B shows the quantitative time-activity curves of MCF-7 tumor and muscle.

[0040] Figure 8 for 68 Ga-NOTA-P-AR, Blocking Group and 18 Comparison of F-FDG imaging in MCF-7 tumor-bearing mice and 68 Imaging of Ga-NOTA-P-AR and the blocking group in MCF-7 mice; Figure A shows... 68 30 minutes after Ga-NOTA-P-AR injection and in the blocking group and 18 Representative PET images of F-FDG in MCF-7 tumor-bearing mice; Figure B shows the effect of MCF-7 tumors on the tumor in the blocking and non-blocking groups. 68 Ga-NOTA-P-AR uptake, ***p<0.001; Figure C shows... 68 Ga-NOTA-P-AR and 18 Tumor / muscle (T / M) ratio at different time points using F-FDG.

[0041] Figure 9 For Al 18 Dynamic imaging results of F-NOTA-PK-AR in MCF-7 tumor-bearing mice; where Figure A shows Al 18 PET images of F-NOTA-PK-AR in MCF-7 tumor-bearing mice, with the tumor tissue indicated by white arrows; Figure B shows the time-activity curves of tumor and muscle over 120 min; Figure C shows the quantitative calculation of the tumor-to-muscle uptake ratio (T / M).

[0042] Figure 10 For Al 18 Static imaging of the F-NOTA-PK-AR / blockade group in MCF-7 tumor-bearing mice at 0.5 h; Figure A shows the in vivo Al in MCF-7 tumor-bearing mice. 18 Normal and blocked imaging of F-NOTA-PK-AR and 30 min after co-injection of NOTA-AR; Figure B shows the tumor uptake value of the blocked experiment for quantitative analysis; **P<0.01.

[0043] Figure 11 for 68 Biodistribution and blood half-life of Ga-NOTA-P-AR in key tissues and organs of normal Balb / c mice; Figure A shows the time-activity curve of blood samples obtained by fitting a classic two-compartment model; Figure B shows... 68Biodistribution of Ga-NOTA-P-AR in important tissues and organs of normal balb / c mice.

[0044] Figure 12 For Al 18 F-NOTA-PK-AR represents the biodistribution and blood half-life of F-NOTA-PK-AR in key tissues and organs of normal Balb / c mice; Figure A shows the time-activity curve of the blood sample obtained by fitting the classic two-compartment model; Figure B shows the biodistribution and blood half-life of F-NOTA-PK-AR in normal Balb / c mice. 18 Biodistribution of F-NOTA-PK-AR in important tissues and organs of normal balb / c mice.

[0045] Figure 13 The results show the safety of NOTA-P-AR in mice; Figure A shows the histopathological H&E staining results, and Figure B shows the blood routine and blood biochemistry results.

[0046] Figure 14 The results show the safety of NOTA-PK-AR in mice; Figure A shows the histopathological H&E staining results, and Figure B shows the complete blood count and blood biochemistry results of mice. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] Example 1 Molecular probe 68 Ga-NOTA-P-AR and Al 18 Preparation of F-NOTA-PK-AR

[0050] 1. Peptide PET molecular probe 68 Preparation of Ga-NOTA-P-AR

[0051] The polypeptide PET molecular probe of the present invention 68 The preparation of Ga-NOTA-P-AR is as follows:

[0052] S1. A novela-modified AR polypeptide (AREYGTRFSLIGG YR-GGGKKKK-PEG3-NOTA, abbreviated as Nota-P-AR) was synthesized using a solid-phase chemical method; the Nota-P-AR was synthesized by Hefei Guotai Biotechnology Co., Ltd.

[0053] S2. Perform on Nota-P-AR68 Ga-labeled; NOA-P-AR synthesized using solid-phase chemical methods was dissolved in deionized water to obtain a precursor solution with a concentration of 1 μg / μL, which was then rinsed with 0.1 mol / L high-purity hydrochloric acid solution. 68 Ge / 68 The Ga) generator was connected to an EP tube, and the 1 mL with the highest radioactivity content was collected. 100 μg of precursor solution (1 μg / μL, 100 μL) was added and mixed well. Then, 100 μL of sodium acetate (1 mol / L) solution was added and mixed well. The pH of the mixture was adjusted to 3.5–4.5, and the mixture was heated at 100 °C for 10 min. After the reaction was complete, the reaction solution was cooled to room temperature to obtain… 68 Ga-NOTA-P-AR.

[0054] 2. Peptide PET molecular probe Al 18 Preparation of F-NOTA-PK-AR

[0055] The polypeptide PET molecular probe Al of the present invention 18 The preparation of F-NOTA-PK-AR is as follows:

[0056] S1. A novela-modified AR polypeptide (AREYGTRFSLIGG YR-K-PEG3-NOTA, abbreviated as Nota-PK-AR) was synthesized using a solid-phase chemical method; the Nota-PK-AR was synthesized by Hefei Guotai Biotechnology Co., Ltd.

[0057] S2. Perform a test on NOTA-PK-AR 18 F-labeled; NOA-PK-AR synthesized using solid-phase chemistry was dissolved in deionized water to obtain a precursor solution with a concentration of 1 μg / μL, which was then eluted and enriched on a QMA column with 0.5 mL of physiological saline. 18 F ions were collected, and the eluent was mixed with 25 μL of AlCl3 sodium acetate buffer and 100 μg of precursor solution (1 μg / μL, 100 μL). Then, 0.05 mol / L dilute hydrochloric acid was added to adjust the pH of the mixture to 4. The mixture was heated at 110 °C for 15 min. After the reaction was complete, the reaction solution was cooled to room temperature to obtain Al. 18 F-NOTA-PK-AR.

[0058] Example 2 Molecular probe 68 Ga-NOTA-P-AR and Al 18 The characterization of F-NOTA-PK-AR

[0059] 1. High-performance liquid chromatography (HPLC) for labeling rate determination

[0060] (1) High-performance liquid chromatography (Shimadzu Corporation, Japan) was used to analyze...68 The radioactivity of Ga-NOTA-P-AR was detected using the following method: A Waters HPLC system equipped with a Waters C18 analytical column (4.6 mm × 250 mm) was used. Mobile phase A was water (containing 0.1% trifluoroacetic acid (TFA)), and mobile phase B was acetonitrile (containing 0.1% TFA). The flow rate was 1 mL / min. Gradient elution conditions were used: 0–20 min, 15% B–90% B; 20–30 min, 90% B–15% B. The eluent contained 0.1% TFA, and the flow rate was 1 mL / min.

[0061] (2) High-performance liquid chromatography (Agilent) was used to analyze Al 18 The radioactivity of F-NOTA-PK-AR was detected using the following method: An Agilent HPLC system equipped with an Agilent C18 analytical column (ZORBAX 300SB-C18 5μm, 4.6mm × 250mm) was used. Mobile phase A was water (containing 0.1% trifluoroacetic acid), and mobile phase B was acetonitrile. The flow rate was 1 mL / min. Gradient elution conditions were: 0–10 min, 0% B–100% B; 10–15 min, 100% B, flow rate 1 mL / min. 4. Determination of radiochemical purity and stability.

[0062] Take 50 μL (approximately 7.4 MBq) of... 68 Ga-NOTA-P-AR reaction solution was added to 500 μL of PBS and 500 μL of fetal bovine serum (FBS), respectively. After incubation at 37°C for 1 h and 2 h, small samples were taken for HPLC analysis to determine their radiochemical purity and observe their in vitro stability. For FBS, plasma proteins were first diluted with an equal volume of acetonitrile, then centrifuged at 12000 r / min for 5 min, and a small amount of supernatant was taken for HPLC analysis. Similarly, Al 18 The in vitro stability of F-NOTA-PK-AR was observed after incubation with PBS and FBS for 1 h and 2 h, respectively.

[0063] 2. Determination of the lipid-water partition coefficient

[0064] Take 10 μL (approximately 1.85 MBq) of... 68 In the Ga-NOTA-P-AR reaction solution, 0.5 mL of PBS and 0.5 mL of n-octanol were added sequentially. After sealing and thorough mixing, the mixture was centrifuged at 1000 rpm for 5 minutes. 100 μL of each organic and aqueous phase were collected for gamma counter counting. The average LogP value of the labeled product was calculated using the formula LogP = Log(counted n-octanol / counted PBS). This process was repeated three times. The same method was used for Al... 18 F-NOTA-PK-AR was used to calculate its lipid-water partition coefficient.

[0065] 3. Results

[0066] The chemical structural formula of NOA-P-AR and the polypeptide PET molecular probe prepared therefrom are described in this invention. 68 The radiochemical purity test results of Ga-NOTA-P-AR are as follows: Figure 1 As shown, Figure 1 In this context, A represents the chemical structural formula of NOTA-P-AR. Figure 1 B and C in the middle are respectively 68 HPLC chromatograms of Ga-NOTA-P-AR in PBS and FBS. Figure 1 From B and C, we can see that 68 The retention time of Ga-NOTA-P-AR was 11.098 min, and its radiochemical purity was >97%. Its radioactivity purity after incubation in PBS and FBS for 1 h and 2 h was both higher than 95%, indicating... 68 Ga-NOTA-P-AR exhibits good stability.

[0067] The chemical structural formula of NOTA-PK-AR and the polypeptide PET molecular probe Al prepared therefrom described in this invention 18 The radiochemical purity test results of F-NOTA-PK-AR are as follows: Figure 2 As shown; Figure 2 In this context, A represents the chemical structural formula of NOTA-PK-AR. Figure 2 B and C in the equation are respectively Al 18 HPLC chromatograms of F-NOTA-PK-AR in PBS and FBS. Figure 2 From B and C in the diagram, we can see that Al 18 The retention time of F-NOTA-PK-AR was 9.004 min, and its radiochemical purity was >97%. Its radioactivity purity after incubation in PBS and FBS for 1 h and 2 h was both higher than 95%, further indicating that Al... 18 The F-NOTA-PK-AR probe exhibits good stability.

[0068] Furthermore, the results of the lipid-water partition coefficient determination showed that... 68 The lipid-water partition coefficient of the Ga-NOTA-P-AR probe is -2.98±0.12, and Al 18 The lipid-water partition coefficient of the F-NOTA-PK-AR probe is -2.73±0.09, indicating that the polypeptide PET molecular probe of the present invention has good hydrophilicity and is conducive to in vivo metabolic clearance.

[0069] The results above show that the polypeptide PET molecular probe prepared by this invention is effective. 68 Ga-NOTA-P-AR and Al 18F-NOTA-PK-AR has high radiochemical purity, good hydrophilicity and stability, which lays a good foundation for its application in breast cancer diagnosis.

[0070] Example 3: Imaging application of molecular probes in a mouse model of breast cancer

[0071] This implementation uses a polypeptide PET molecular probe. 68 Ga-NOTA-P-AR and Al 18 The in vivo subcutaneous tumor scintigraphy efficacy of F-NOTA-PK-AR was evaluated. First, the AR peptide (AREYGTRFSLIGGYR) was modified with fluorescein isothiocyanate (FITC) to obtain the probe AR-FITC (synthesized by Hefei Guotai Biotechnology Co., Ltd.). Then, the specific targeting ability of the AR-FITC probe to MCF-7 (human breast cancer cell line) and MCF-10A (human breast epithelial cells) was qualitatively assessed by cellular fluorescence staining. Additionally, an internalization assay was used to evaluate… 68 Ga-NOTA-P-AR and Al 18 The affinity of F-NOTA-PK-AR for MCF-7 and / or 4T1 cells. Furthermore, animal models, including MCF-7 and 4T1 tumor-bearing mouse models, were constructed, and static and dynamic PET imaging was performed. The region of interest (ROI) and tumor / background ratio were analyzed to determine the optimal imaging time and evaluate the imaging effect. The experimental group consisted of: 68 Ga-NOTA-P-AR or Al 18 F-NOTA-PK-AR, the blocking group is: 68 Ga-NOTA-P-AR+NOTA-AR (AREYGTRFSLIGGYR-NOTA, without linker blocks and radioactive modifications) or Al 18 F-NOTA-PK-AR+NOTA-AR. Finally, biodistribution experiments were conducted and the blood half-life of the probe was determined. The organ and time-dependent metabolic characteristics of radioactive concentration distribution were analyzed based on the percentage of radioactive uptake per unit mass (%ID / g) in each organ and tissue. 68 The imaging time of the Ga-NOTA-P-AR probe was set to 10 min, 0.5, 1, 2 h, and 2 h dynamically; Al 18 The imaging time for the F-NOTA-PK-AR probe is set to 0.5 hours for static imaging and 2 hours for dynamic imaging. 68 The biodistribution time of the Ga-NOTA-P-AR probe was set to 0.5, 1, and 2 hours; Al 18The biodistribution time of the F-NOTA-PK-AR probe was set to 10 min, 0.5, 1 and 2 h; 68 The blood half-life of the Ga-NOTA-P-AR probe was set to 0, 3, 5, 10, 20, 30, 60, 120, 240, and 360 min; Al 18 The blood half-life of the F-NOTA-PK-AR probe was set to 1, 3, 5, 10, 20, 30, 45, 60, 120, and 240 min.

[0072] Specifically as follows:

[0073] 1. Cell confocal microscopy experiment

[0074] MCF-7 tumor cells and MCF-10A cells were placed at an appropriate density (5×10⁶). 5 Cells were seeded in confocal culture dishes (number per well) and incubated overnight. Then, 0.5 mL of AR-FITC (30 μg / mL) was added to each well, and the cells were incubated at 37°C for 1 h. After washing, fixing, and staining the nuclei with DAPI, the cells were fluorescently imaged under a confocal microscope.

[0075] 2. Cell internalization experiment

[0076] MCF-7 and 4T1 cells were respectively placed at an appropriate density (5×10⁻⁶). 5 Seeds were planted in 12-well plates (number per well), and after overnight incubation, 7.4 kBq of [unspecified substance] was added to each well. 68 Serum-free Ga-NOTA-P-AR culture medium was incubated at 37°C for 30 min, washed three times with frozen PBS (pH 7.4), and then treated with glycine hydrochloric acid solution (1M, pH 2.2) for 10 min. The supernatant (membrane-bound probe) and cell pellet were collected separately. Cells were then lysed with sodium hydroxide solution (1N), and the internalization probe was measured. The cell suspension was collected and radioactivity was measured using a gamma counter. Al2O3 was then performed using MCF-7 cells. 18 The cell internalization experiment of F-NOTA-PK-AR was the same as the cell internalization experiment described above, except that the incubation time was 10, 30, 60 and 120 min.

[0077] 3. Animal Model Construction

[0078] The animal models were constructed using NOG mice and BALB / c nude mice, female, 4–6 weeks old, SPF grade. Each NOG mouse received a subcutaneous injection of 1.0 × 10⁻⁶ mg / L of vaccine in the right axilla. 7 5.0 × 10⁸ MCF-7 cells (cell suspension in 100 μL of matrix gel and PBS) were subcutaneously seeded in the right axilla of BALB / c nude mice. 6 Animal experiments were conducted using 4 T1 cells when the tumor volume reached 0.8–1 cm.

[0079] 4. PET Imaging

[0080] 3.7–5.55 MBq (for multiple imaging, the injection volume should fall within this range) were injected intravenously into the tail vein of MCF-7 or 4T1 tumor-bearing mice. 68 Ga-NOTA-P-AR and 18F-FDG (n=3 per group), the blocking group mice were simultaneously injected with the same activity 68 Ga-NOTA-P-AR and an excess of non-radiolabeled peptide (NOTA-AR); static PET images were acquired at 10 min, 0.5, 1, and 2 h post-injection; dynamic images were acquired in MCF-7 tumor-bearing mice at 2 h. The same method was used to inject Al into MCF-7 tumor-bearing mice. 18 F-NOTA-PK-AR performs static and dynamic imaging.

[0081] 5. Biodistribution and pharmacokinetics

[0082] Each female BALB / c mouse (n=3 / time point) was injected with 3.7 MBq (100 μCi) via the tail vein. 68 Ga-NOTA-P-AR; Animals were euthanized by cervical dislocation 0.5h, 1h, and 2h after injection, and their blood, heart, liver, spleen, lungs, kidneys, stomach, large intestine, small intestine, muscles, bones, and brain were collected, weighed, and their radioactivity counts were determined using a gamma counter; similarly, after injection of Al... 18 Radioactivity counts were measured 10 min, 0.5 h, 1 h, and 2 h after F-NOTA-PK-AR probe administration, and the results were expressed as a percentage of the injected dose per gram of tissue (%ID / g). For evaluation... 68 Blood retention time of Ga-NOTA-P-AR was measured using a gamma counter at 0, 0.05, 0.08, 0.17, 0.33, 0.5, 1, 2, 4, and 6 hours post-injection; similarly, to evaluate Al... 18 The blood retention time of F-NOTA-PK-AR was determined by counting blood samples collected at 0.02, 0.05, 0.08, 0.17, 0.33, 0.5, 0.75, 1, 2, and 4 hours after injection using a gamma counter; statistical analysis was performed using Origin 8.5.

[0083] 6. In vivo safety experiments

[0084] Mice (n=3 / group) were intravenously injected with 100 μL PBS and either NOA-P-AR or NOA-PK-AR (5 mg / kg). Blood samples were collected at 24 h and 4 weeks post-injection to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine, and to perform a complete blood count. The mice were then sacrificed, and the major organs (spleen, liver, lung, heart, and kidney) were collected for histopathological analysis.

[0085] 7. Results

[0086] The uptake of AR-FITC by MCF-7 cells and MCF-10A cells is as follows: Figure 3 As shown, by Figure 3 It can be seen that the uptake of AR-FITC by MCF-7 cells and MCF-10A cells can be detected by cell immunofluorescence, and the fluorescence intensity of MCF-7 cells is significantly higher than that of MCF-10A cells.

[0087] MCF-7 cells and 4T1 cells against 68 The internalization of Ga-NOTA-P-AR is as follows: Figure 4 As shown, by Figure 4 It can be seen that, 68 After co-incubation of Ga-NOTA-P-AR with MCF-7 cells for 30 min, the internalized and membrane-bound uptake values ​​were 0.69±0.06%AD and 1.28±0.08%AD, respectively, with a total internalization rate of 34.8±0.6%. For 4T1 cells, the internalized and membrane-bound uptake values ​​were 0.56±0.02%AD and 1.04±0.05%AD, respectively, with a total internalization rate of 35.2±0.3%. MCF-7 cells showed a higher tolerance to Al... 18 The internalization of F-NOTA-PK-AR is as follows: Figure 5 As shown, by Figure 5 It can be seen that as the incubation time increases, Al 18 The internalization and membrane-bound uptake values ​​of F-NOTA-PK-AR in MCF-7 cells gradually increased, reaching their highest values ​​at 120 min, at 4.14 ± 0.06% AD and 47.58 ± 1.46% AD, respectively. The total internalization rate at 120 min was 8.0 ± 0.17% AD. From the above results, it can be concluded that the present invention... 68 Ga-NOTA-P-AR and Al 18 The internalization rates of F-NOTA-PK-AR were 35.2±0.3% and 37.41±8.70%, respectively.

[0088] 68PET imaging of Ga-NOTA-P-AR in MCF-7 and 4T1 tumor-bearing mice and comparison of tumor radiouptake and tumor / muscle ratio at different time points, as follows: Figure 6 As shown, 68 Dynamic imaging of Ga-NOTA-P-AR in MCF-7 tumor-bearing mice, such as Figure 7 As shown, 68 Ga-NOTA-P-AR and 18 Comparison of F-FDG imaging in MCF-7 tumor-bearing mice and 68 Imaging results of the Ga-NOTA-P-AR / blockade group in MCF-7 mice are as follows: Figure 8 As shown. By Figures 6-8 It can be seen that in PET static and dynamic imaging, 68 Ga-NOTA-P-AR successfully targeted and visualized MCF-7 subcutaneous tumors in an MCF-7 tumor-bearing mouse model. Ten minutes after injection... 68 Ga-NOTA-P-AR was rapidly and highly uptaken by MCF-7 tumors (3.60±0.14% ID / g), with the highest tumor / muscle ratio of 9.1±1.02 1 hour post-injection. In MCF-7 tumor-bearing mice pretreated with an excess of unlabeled peptide-NOTA-AR, tumor uptake was significantly reduced to 0.31±0.06% ID / g, indicating that... 68 Ga-NOTA-P-AR can be specifically taken up and retained by MCF-7 subcutaneous tumors, and good contrast-resolution images can be obtained 60 minutes after injection. 18 Compared to F-FDG, MCF-7 tumors 68 Ga-NOTA-P-AR has weak radioactive uptake, but due to its low background uptake and high lesion uptake, it can be effective in tumors. 68 It is easier to identify on Ga-NOTA-P-AR images.

[0089] Al 18 The F-NOTA-PK-AR probe was observed in dynamic PET imaging of MCF-7 tumor-bearing mice as follows: Figure 9 As shown, Al 18 Static imaging of the F-NOTA-PK-AR / blockade group in MCF-7 tumor-bearing mice at 0.5 h is as follows: Figure 10 As shown. By Figure 9 and Figure 10 It can be seen that in PET static and dynamic imaging, Al 18 F-NOTA-PK-AR successfully targeted and visualized MCF-7 subcutaneous tumors in a mouse model bearing MCF-7. In dynamic imaging, Al was observed 10 minutes after injection. 18F-NOTA-PK-AR was rapidly and highly uptaken by MCF-7 tumors (1.59±0.42% ID / g), with the highest tumor / muscle ratio of 3.09±0.19 1 hour post-injection. Good contrast-resolution images were obtained in 0.5-hour static imaging, with tumor uptake of 3.4±0.53% ID / g. In contrast, tumor uptake was significantly reduced in MCF-7 tumor-bearing mice pretreated with excessive amounts of unlabeled peptide NOTA-AR, at 1.4±0.21% ID / g, indicating that F-NOTA-PK-AR... 18 F-NOTA-PK-AR can be specifically taken up and retained by MCF-7 subcutaneous tumors. Significant radioactive uptake was observed in both the kidneys and bladder in images at different time points, indicating that the drug is primarily excreted via the kidneys.

[0090] 68 Biodistribution and blood half-life of Ga-NOTA-P-AR in important tissues and organs of normal Balb / c mice, such as Figure 11 As shown, Al 18 F-NOTA-PK-AR was used to determine the biodistribution and blood half-life of F-NOTA-PK-AR in important tissues and organs of normal Balb / C mice. Figure 12 As shown. By Figure 11 and 12 It can be seen that the biodistribution and blood half-life of the peptide PET molecular probe in important tissues and organs are basically consistent with the imaging results. In the biodistribution experiment in normal mice, the molecular probe... 68 Ga-NOTA-P-AR and Al 18 F-NOTA-PK-AR has a high renal uptake rate, among which, 68 The uptake (%ID / g) of Ga-NOTA-P-AR at 0.5h, 1h, and 2h were 84.02±4.08, 89.17±3.94, and 61.72±6.88 (n=3), respectively; Al 18 The uptake (%ID / g) of F-NOTA-PK-AR at 10 min, 0.5 h, 1 h, and 2 h were 14.10±1.91, 4.48±0.65, 2.44±0.36, and 2.03±0.18 (n=3), respectively. Both probes showed low uptake in the liver and gastrointestinal tract and rapid elimination from the bloodstream, indicating... 68 Ga-NOTA-P-AR and Al 18 F-NOTA-PK-AR is excreted via the kidneys, not the liver and gallbladder. A classic two-compartment model was used to fit the time-activity curves of blood samples. 68 The half-life of Ga-NOTA-P-AR is 12 minutes. Figure 11 A, r 2 =0.99), Al 18The half-life of F-NOTA-PK-AR is 18 minutes. Figure 12 A, r 2 =0.99), indicating that both probes were rapidly cleared from the blood.

[0091] The safety results of NOTA-P-AR and NOTA-PK-AR in mice are as follows: Figure 13 and Figure 14 As shown, Figure 13 and Figure 14 In the text, A represents the results of HE staining of pathological tissues. Figure 13 and 14 In the figures, B represents the results of complete blood count and blood biochemistry. Figure 13 and Figure 14 It was found that no structural or histopathological changes were observed in the organ tissue sections. Compared with the control group, the experimental group showed slight transient changes in ALT, BUN and creatinine levels 24 hours after injection, while the levels almost returned to baseline levels 2 weeks after injection, but the AST level increased slightly. The whole blood test results showed that except for a slight change in the number of white blood cells, there were no significant differences in other cells, indicating that the peptide PET molecular probe described in this invention has good safety.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide PET molecular probe targeting breast cancer, characterized in that, The polypeptide PET molecular probe is obtained by labeling the AR polypeptide modified by a bifunctional chelator with a positron emitting radionuclide, the bifunctional chelator and the polypeptide AR further contain a linker; the AR polypeptide is AREYGTRFSLIGGYR, the bifunctional chelator is NOTA, the linker is GGGKKKK-PEG3, and the positron emitting radionuclide is 68 Ga; or the AR polypeptide is AREYGTRFSLIGGYR, the bifunctional chelator is NOTA, the linker is K-PEG3, the positron-emitting radionuclide is 18 F.

2. Use of the polypeptide PET molecular probe of claim 1 in the preparation of an imaging product targeting breast cancer.

3. Use of the polypeptide PET molecular probe of claim 1 in the preparation of a diagnostic reagent for breast cancer.

4. A method of preparing the polypeptide PET molecular probe of claim 1, wherein, comprising the following steps: S1. Synthesis of the bifunctional chelator-modified AR polypeptide as described in claim 1; S2. Positron emitting radionuclide labeling of the bifunctional chelator-modified AR polypeptide obtained in step S1.

5. The method of claim 4, wherein, When the positron emitting radionuclide is 68 Ga, the labeling method is as follows: dissolving the synthesized bifunctional chelator modified AR polypeptide in water to obtain a precursor solution, eluting a germanium gallium generator into a container with 0.8-1.2 mL of 0.1 mol / L hydrochloric acid solution, adding 80-100 μg of the precursor solution and mixing, then adding 80-120 μL of 1 mol / L sodium acetate solution and mixing, adjusting the pH value of the mixture to 3.5-4.5, heating at 98-100°C for 8-12 min, and cooling the reaction solution to room temperature after the reaction is completed.

6. The method of claim 4, wherein When the positron-emitting radionuclide is 18 At F, the labeling method is as follows: the synthesized bifunctional chelating agent-modified AR peptide is dissolved in water to obtain a precursor solution, and the QMA column is eluted with 0.4–0.6 mL of physiological saline. 18 F ions were collected, and the eluent was mixed with 24–26 μL of AlCl3 sodium acetate buffer and 80–100 μg of precursor solution. The pH of the mixture was adjusted to 4 with dilute hydrochloric acid, and the mixture was heated at 110 °C for 12–15 min. After the reaction was completed, the reaction solution was cooled to room temperature.

7. An imaging agent for breast cancer diagnosis, characterized by, containing the polypeptide PET molecular probe of claim 1.

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