A molecular imaging probe for diagnosing lung cancer and its preparation method

Abstract

The present invention belongs to the field of molecular imaging probes for cancer diagnosis, and discloses a nucleic acid aptamer targeting tumor-related calcium channel sensor protein 2 and a molecular imaging probe for diagnosing lung cancer and a preparation method thereof. The nucleic acid aptamer contains 46 nucleotides, can specifically target Trop‑2 on the surface of lung cancer tumor cells, and has a specific targeting recognition effect on lung cancer cells overexpressing Trop‑2, such as non-small cell lung cancer cells H1650, small cell lung cancer cells SBC‑2, etc., and can be used for molecular imaging of Trop‑2 positive lung cancer patients. Molecular imaging probes can be used for non-invasive visual molecular imaging of lung cancer, and have the advantages of high specificity, high sensitivity, good imaging effect, etc. The preparation method of the molecular imaging probe has simple reaction conditions, low cost, and easy clinical transformation.

Description

Technical Field

[0001] The invention relates to a molecular imaging probe for cancer diagnosis, in particular to a nucleic acid aptamer and a molecular imaging probe for lung cancer diagnosis and a preparation method thereof. Background Art

[0002] Lung cancer is the leading cause of cancer-related death. Based on the microscopic histological characteristics of cancer cells, lung cancer can be divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with the former accounting for 85%. Since the early symptoms of lung cancer are not obvious, most patients are already in the late stage when diagnosed, resulting in poor treatment outcomes and poor prognosis. Therefore, early detection, accurate diagnosis, precise staging and effective treatment are crucial to improving the survival rate and quality of life of lung cancer patients.

[0003] Imaging examination of tumor tissue is of great significance for the prevention and treatment of cancer. At present, fluorescence, ultrasound, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography-computed tomography (PET-CT) and other methods are commonly used in clinical practice to track and image tumors. Among them, PET / CT, as an imaging technology for nuclear medicine clinical examination, is currently the only technology that uses anatomical morphology to perform functional, metabolic and receptor imaging. It is non-invasive and can provide three-dimensional and functional images of the whole body. Therefore, PET / CT technology has a large number of applications in the research of clinical medical imaging and cancer spread in oncology.

[0004] Positron emission tomography / computed tomography (PET / CT) is widely used in nuclear medicine for the diagnosis, staging, treatment evaluation and monitoring of lung cancer. It uses radionuclide-labeled biomolecules as imaging agents to display the functional or metabolic state of tissues or organs at the molecular level and provide anatomical details through CT. Molecular imaging diagnosis based on PET / CT technology can image and molecularly type primary and metastatic tumors at an early stage and non-invasively, providing rich and reliable diagnostic basis for the implementation of corresponding treatment plans. The most commonly used PET imaging tracer in clinical applications is 18 F-FDG. It takes advantage of the physiological characteristics of tumor tissues, such as their vigorous metabolism and large uptake of sugar molecules, and can efficiently enrich in the tumor area to achieve targeted imaging of tumor tissues. In recent years, with the introduction of the concept of molecular imaging and the rapid development of related technologies, researchers have constructed radionuclide molecular probes based on targeting molecules such as antibodies, small molecules, and nucleic acid aptamers. This type of probe can specifically bind to tumor markers, and through PET / CT imaging, it can achieve molecular imaging of biomarkers that are highly expressed in tumor tissues, and provide molecular diagnostic information of tumor tissues. Imaging molecular diagnosis can provide complete molecular physiological information of tumors, avoiding problems such as incomplete diagnostic information that may exist in immunohistochemistry of pathological sections. However, at present,18 F-FDG PET / CT imaging lacks specificity in tumor diagnosis and monitoring. Several factors affect its clinical application: (1) 18 F-FDG is absorbed not only by tumor cells but also by normal tissues or non-tumor lesions, which may lead to false positive results. (2) In some poorly differentiated or hypoxic tumor cells, F-FDG is absorbed by normal tissues or non-tumor lesions, which may lead to false positive results. 18 Low F-FDG uptake may lead to false negatives; (3) 18 F-FDG shows high background signals in important metabolically active organs such as the brain, heart, liver, and kidneys, which affects tumor detection in these areas; (4) 18 Preparation for F-FDG PET / CT requires patients to fast and regulate their blood sugar levels before the scan to reduce 18 Distribution and uptake of F-FDG in healthy tissues.

[0005] Aptamers are highly structured DNA or RNA fragments that can specifically bind to cellular target molecules. Compared with traditional biological targeting molecules such as antibodies and enzymes, aptamers have good chemical stability and can be widely used in a variety of radionuclide molecular labeling methods. The labeled radionuclide of molecular probes can be customized according to actual needs to provide suitable probes for tumor metabolism and immunogenomic analysis. With the development of immuno-PET, precision targeting probes have been gradually developed. Among them, although aptamers have shown great potential for clinical application, the clinical application of aptamer-based PET / CT molecular probes has not yet been clinically applied in lung cancer. There are still many challenges, such as aptamers are easily degraded by nucleases, and the in vivo stability and targeting are insufficient. Therefore, it is urgent to develop new aptamer PET / CT molecular probes with high specificity and stability to achieve accurate diagnosis and treatment of lung cancer. Summary of the invention

[0006] The purpose of the present invention is to provide a nucleic acid aptamer that specifically targets tumor-associated calcium channel sensor protein 2 on the surface of tumor cell membrane, and a molecular imaging probe for diagnosing lung cancer prepared by using the nucleic acid aptamer and having high stability, high specificity and high sensitivity. At the same time, the present invention also provides a preparation method of the molecular imaging probe.

[0007] To achieve the above object, on the one hand, the present invention provides a nucleic acid aptamer, the nucleotide sequence of the nucleic acid aptamer is as shown in SEQ ID NO: 1, specifically 5'-CTC GAC ACC CTC GTC CCC GGC CGC TCC TGC TCAATT TCA TAG CAG G-3'.

[0008] The nucleic acid aptamer described in the present invention is a nucleic acid aptamer targeting tumor-associated calcium channel sensor protein 2 (Trop-2). The present invention screened a single-stranded nucleic acid aptamer DNA targeting Trop-2 through exponential enrichment system evolution (SELEX) technology, and after in vitro affinity detection and targeting detection at the level of lung cancer tumor cells, and after a series of experiments, it was confirmed that the nucleic acid aptamer specifically targeted and recognized lung cancer cells overexpressing Trop-2, such as non-small cell lung cancer cells H1650 and small cell lung cancer cells SBC-2, and could distinguish Trop-2 positive expression and negative expression cells at the cellular level and in tumor-bearing mouse models. Furthermore, by constructing a nucleic acid aptamer-nuclide targeted contrast agent targeting Trop-2, it is expected to achieve molecular imaging of Trop-2 positive lung cancer patients and avoid the invasiveness of puncture biopsy. While ensuring high specificity in identifying targeted molecules, it reduces the pain of patients.

[0009] Preferably, the nucleic acid aptamer is a freeze-dried powder. The nucleic acid aptamer of the present invention is synthesized by solid phase synthesis method, and is preferably in the form of a freeze-dried powder.

[0010] In the second aspect, the present invention provides the use of the above-mentioned nucleic acid aptamer in the preparation of a molecular imaging probe for diagnosing lung cancer. The nucleic acid aptamer designed and screened by the present invention obtains a specific nucleotide sequence, and the specific targeted recognition of lung cancer cells overexpressing Trop-2, such as non-small cell lung cancer cells H1650, small cell lung cancer cells SBC-2, etc., has been confirmed by experiments, and can distinguish Trop-2 positive expression and negative expression cells at the cellular level and in tumor-bearing mouse models, and can be used to prepare a molecular imaging probe for diagnosing lung cancer, and has the advantages of high stability, high specificity, etc.

[0011] In the third aspect, the present invention provides a molecular imaging probe for diagnosing lung cancer. To achieve this purpose, the technical solution adopted by the present invention is: a molecular imaging probe for diagnosing lung cancer, the molecular imaging probe comprising the nucleic acid aptamer, radionuclide chelating ligand and labeling radionuclide as described above; the nucleic acid aptamer is coupled to the radionuclide chelating ligand and labeled with the labeling radionuclide.

[0012] The molecular imaging probe for diagnosing lung cancer described in the present invention is formed by coupling the nucleic acid aptamer of the specific sequence with the radionuclide chelating ligand and labeling it with the labeling radionuclide. The obtained molecular imaging probe can be used for non-invasive visualized molecular imaging of lung cancer and has the advantages of high specificity, high sensitivity, good imaging effect, etc.

[0013] Preferably, the nuclide chelating ligand includes at least one of deferoxamine, 1,4,7-triazacyclononane-1,4,7-triacetic acid, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. The nuclide chelating ligand includes but is not limited to deferoxamine, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). More preferably, the nuclide chelating ligand is 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0014] Preferably, the labeling nuclides are Tc-99m, Ga-68, F-18, I-123, I-125, I-131, I-124, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, Y-86, Mn-52, Sc-44, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-1 Preferably, the labeling nuclide is at least one of Ga-68, Ga-68, and Ga-69.

[0015] Preferably, the nuclide chelating ligand is 1,4,7-triazacyclononane-1,4,7-triacetic acid, and the labeling nuclide is Ga-68. When the nuclide chelating ligand is 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and the labeling nuclide is Ga-68, the resulting molecular imaging probe is represented by [ 68 Ga]Ga-NOTA-TRP, verified by the inventors of the present application, realizes non-invasive visual detection of Trop-2 receptor protein expression, and further realizes non-invasive molecular imaging of lung cancer.

[0016] In a fourth aspect, the present invention provides a method for preparing a molecular imaging probe with simple reaction conditions and low cost. To achieve this purpose, the technical solution adopted by the present invention is: a method for preparing a molecular imaging probe for diagnosing lung cancer, the preparation method comprising the following steps:

[0017] Step (1): synthesizing nucleic acid aptamers by solid phase synthesis method;

[0018] Step (2): coupling the synthesized nucleic acid aptamer with the nuclide chelating ligand to obtain a nucleic acid aptamer-nuclide molecular probe precursor;

[0019] Step (3): The nucleic acid aptamer-nuclide molecular probe precursor obtained in step (2) is mixed with the labeling nuclide solution to obtain a molecular imaging probe.

[0020] The preparation method of the molecular imaging probe of the present invention firstly synthesizes the nucleic acid aptamer of the nucleotide sequence as described above by a solid phase synthesis method, the synthesis time of the solid phase synthesis method is generally 1 to 2 hours, and then the nucleic acid aptamer is coupled with a nuclide chelating ligand to obtain a nucleic acid aptamer-nuclide molecular probe precursor, and finally the precursor is mixed with a labeling nuclide solution to obtain a molecular imaging probe. The preparation method has simple reaction conditions and low cost.

[0021] Preferably, the step (3) is specifically as follows: dissolving the labeling nuclide with hydrochloric acid to obtain a nuclide hydrochloric acid solution, then adding a sodium acetate buffer solution to obtain a labeling nuclide solution; mixing the labeling nuclide solution with the nucleic acid aptamer-nuclide molecular probe precursor, heating the reaction, and then cooling to room temperature to obtain a molecular imaging probe. When the labeling nuclide is used to label the nucleic acid aptamer-nuclide molecular probe precursor, the labeling nuclide is first prepared into a labeling nuclide solution with a specific pH. Specifically, the labeling nuclide is first dissolved with a hydrochloric acid solution, and the hydrochloric acid solution is preferably a 0.1 M hydrochloric acid solution. After dissolving, the nuclide hydrochloric acid solution is obtained, and then sodium acetate buffer is added to adjust the pH value to a specific range. The concentration of the sodium acetate buffer is preferably 1 M, and the labeling nuclide solution is obtained.

[0022] Preferably, in step (3), the radioactive dose of the labeling nuclide in the nuclide hydrochloric acid solution is 7 mCi to 10 mCi. The radioactive dose of the labeling nuclide in the nuclide hydrochloric acid solution is preferably, but not limited to, 7 mCi to 10 mCi, for example, 7 mCi, 8 mCi, 9 mCi, 10 mCi, and any intermediate value therebetween.

[0023] Preferably, the volume ratio of the radionuclide hydrochloric acid solution to the sodium acetate buffer is 1:(5-6); more preferably, the pH of the labeling radionuclide solution is 4.0.

[0024] Preferably, in step (3), the reaction temperature is 100° C. and the reaction time is 20 minutes.

[0025] Preferably, the product of the mixed reaction of the nucleic acid aptamer-nuclide molecular probe precursor and the labeling nuclide solution is purified to obtain a molecular imaging probe, and the purification is preferably but not limited to purification using a Cytiva PD MiniTrap G-25 desalting purification column.

[0026] The nucleic acid aptamer described in the present invention is a nucleic acid aptamer composed of 46 nucleotides that specifically targets Trop-2 on the surface of lung cancer tumor cells. It specifically targets and recognizes lung cancer cells overexpressing Trop-2, such as non-small cell lung cancer cells H1650 and small cell lung cancer cells SBC-2, and can distinguish Trop-2 positive expression and negative expression cells at the cellular level and in tumor-bearing mouse models.

[0027] The molecular imaging probe for diagnosing lung cancer described in the present invention uses the specific nucleic acid aptamer for nuclide chelate ligand coupling and further performs radionuclide labeling to construct a PET molecular probe for a specific molecular marker Trop-2, and uses a lung cancer tumor model tumor-bearing mouse to verify the molecular imaging efficacy of the probe, thereby achieving non-invasive visual detection of Trop-2 receptor protein expression, further achieving non-invasive molecular imaging of lung cancer, and can be used as an imaging agent in PET / CT molecular imaging, which is of great significance for the diagnosis of lung cancer.

[0028] The method for preparing a molecular imaging probe for diagnosing lung cancer described in the present invention first uses a DNA synthesizer to perform solid-phase synthesis of a nucleic acid aptamer of a specific sequence. The sequence can be customized and the difference between batches is small. Compared with the preparation of traditional antibody probes, the reaction conditions are simple, and the probe synthesis precursor and the probe itself have good stability and are not affected by temperature and pH. Moreover, the method can be generally applied to the preparation of molecular probes for other molecular targets and different nuclides. It has broad application prospects and very high clinical transformation value in the diagnosis and precision treatment of tumor tissues in the future. It has the advantages of simple preparation process, low cost, high specificity, high stability, high imaging signal-to-noise ratio, and easy clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the preparation process of the nucleic acid aptamer of the present invention;

[0030] Figure 2 This is a flow cytometry image of the in vitro specific targeted recognition of lung cancer tumor cells by the nucleic acid aptamer of the present invention;

[0031] Figure 3 This is a schematic diagram of the process of coupling the nucleic acid aptamer and the nuclide chelating ligand of the present invention;

[0032] Figure 4This is a high performance liquid chromatogram after the nucleic acid aptamer of the present invention is coupled with the nuclide chelating ligand NOTA;

[0033] Figure 5 It is a high-resolution mass spectrum after the nucleic acid aptamer of the present invention is coupled with the nuclide chelating ligand NOTA;

[0034] Figure 6 This is a PET / CT molecular imaging diagram of a non-small cell lung cancer cell H1650 tumor-bearing mouse 30 minutes after the molecular imaging probe of one embodiment of the present invention was injected into the tail vein;

[0035] Figure 7 This is a PET / CT molecular imaging diagram of a mouse bearing RP21 small cell lung cancer cell 30 minutes after the molecular imaging probe of one embodiment of the present invention was injected into the tail vein;

[0036] Figure 8 This is a PET / CT molecular imaging diagram of a mouse bearing a human small cell lung cancer cell SBC-2 30 minutes after the molecular imaging probe of one embodiment of the present invention was injected into the tail vein;

[0037] Fig. 9 The data of biodistribution of various organs of mice bearing human non-small cell lung cancer cells H1650 30 minutes and 60 minutes after tail vein injection of the molecular imaging probe of one embodiment of the present invention;

[0038] Fig.10 The data of biodistribution of various organs of mice bearing RP21 lung cancer cells of a molecular imaging probe according to an embodiment of the present invention 30 minutes and 60 minutes after tail vein injection;

[0039] Fig.11 The data of biodistribution of various organs of mice bearing human small cell lung cancer cells SBC-2 30 minutes and 60 minutes after tail vein injection of the molecular imaging probe of one embodiment of the present invention;

[0040] Fig.12 The signal intensity ratio of the tumor site and muscle tissue of a molecular imaging probe of an embodiment of the present invention in a non-small cell lung cancer cell H1650 tumor-bearing mouse 30 minutes and 60 minutes after tail vein injection;

[0041] Fig.13 The molecular imaging probe of one embodiment of the present invention is injected into the tail vein 30 minutes and 60 minutes after the signal intensity ratio of the tumor site and muscle tissue of mice bearing mouse small cell lung cancer cells RP21;

[0042] Fig.14This is the signal intensity ratio of the tumor site to the muscle tissue of human small cell lung cancer cell SBC-2 lung cancer bearing mice 30 minutes and 60 minutes after tail vein injection of the molecular imaging probe of one embodiment of the present invention. DETAILED DESCRIPTION

[0043] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Embodiment 1

[0044] An embodiment of the nucleic acid aptamer of the present invention, the nucleic acid aptamer of this embodiment is synthesized by solid phase synthesis method, through multi-step solid phase synthesis of DNA containing solid phase carrier and protective group, and finally amino deprotection and purification to obtain the final product. The schematic diagram of the preparation process is shown in the attached Figure 1 As shown. Oligonucleotide sequences were synthesized using the phosphoramidite technique in solid phase synthesis.

[0045] Specifically, the solid phase synthesis method described in this embodiment includes:

[0046] First, according to the scale, a nucleic acid synthesizer is used to synthesize on a solid support made of controlled pore glass (CPG carrier); all phosphoramidite monomers can be removed under alkaline conditions; all phosphoramidite monomers are dissolved in anhydrous acetonitrile (100 mM) and molecular sieves are added for drying; 5-ethylthiotetrazolyl (0.6 M) acetonitrile solution is used as an activator solution; the coupling time is 200 seconds; for unreacted active groups, acetic anhydride, N-methylimidazole, pyridine and acetonitrile mixed reagents (N-methylimidazole / acetonitrile is 1:4; acetic anhydride / pyridine / acetonitrile is 2:3:5) are used as capping reagents, and the oligomers bound to the solid support are cleaved and deprotected;

[0047] Secondly, after the solid phase synthesis is completed, the dried solid support is treated with an aqueous ammonia solution at 55°C for 16 hours, the solution is evaporated and the solid residue is redissolved in water, and the target nucleic acid chain is obtained by HPLC purification.

[0048] The nucleotide sequence of the synthesized nucleic acid aptamer TRP is shown in SEQ ID NO: 1, specifically:

[0049] TPR: 5'-CTC GAC ACC CTC GTC CCC GGC CGC TCC TGC TCA ATT TCA TAG CAG G-3'. Embodiment 2

[0050] Specificity experiment of nucleic acid aptamer of the present invention

[0051] The dissolved Cy5 activated ester (DMSO solution, equivalent to 10) was added to the amino-modified nucleic acid aptamer described in Example 1, and an appropriate amount of carbonate solution was added. Ultrasonic mixing was performed until completely dissolved, and the reaction was carried out at 25°C for 1 h. After the reaction was completed, the nucleic acid aptamer labeled with Cy5 was purified by HPLC to obtain the nucleic acid aptamer. The nucleic acid aptamer described in Example 1 was labeled with Cy5, and then flow cytometry was performed on human and mouse lung cancer cell lines H1650, SBC-2 and RP21, respectively. The cell lines (5 10 5 cells / tube), washed twice with wash buffer (the wash buffer was Dulbecco's phosphate buffered saline (DPBS) supplemented with 4.5 mg / mL glucose and 5 mg MgCl2), and then incubated with DNA aptamer (250 nM) in binding buffer at 4°C for 30 minutes (the binding buffer was prepared by adding 0.1 mg / mL tRNA and 1 mg / mL BSA to the wash buffer). After the incubation, the samples were washed three times with wash buffer and analyzed by the BD FACSVerse™ system. The results are shown in the attached figure. Figure 2 shown.

[0052] By the attached Figure 2 It can be seen that the nucleic acid aptamer can specifically target human and mouse lung cancer cell lines H1650, SBC-2 and RP21. Embodiment 3

[0053] An embodiment of the molecular imaging probe of the present invention is prepared by the following method:

[0054] Preparation of nucleic acid aptamer-nuclide molecular probe precursor: as shown in the attached Figure 3 As shown, the nucleic acid aptamer prepared in Example 1 is coupled with the nuclide chelating ligand 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), specifically: 3 mg of the nucleic acid aptamer-NH2 synthesized in Example 1 is dissolved in 80 μl PBS buffer solution, 2 mg NOTA-NHS (10 times equivalent, 240 ul DMSO) is dissolved in 240 μl DMSO solution, and then the two solutions are evenly mixed, reacted at 25°C overnight to obtain a nucleic acid aptamer-nuclide molecular probe precursor, which is then purified by high performance liquid chromatography HPLC, and the product molecular weight is confirmed by high resolution mass spectrometry;

[0055] The HPLC chromatogram of the nucleic acid aptamer-nuclide molecular probe precursor NOTA-TRP is shown in the attached figure. Figure 4 As shown in the attached Figure 4It can be seen that the main peak with a retention time of 13.267 min is the main product nucleic acid aptamer-nuclide molecular probe precursor NOTA-TRP; the high-resolution mass spectrum of the nucleic acid aptamer-nuclide molecular probe precursor is shown in the attached figure. Figure 5 As shown in the attached Figure 5 It can be seen that the molecular weight of 16210.6 is the main product, nucleic acid aptamer-nuclide molecular probe precursor NOTA-TRP;

[0056] (2) Preparation of labeling nuclide solution: Dissolve the labeling nuclide in 0.1M hydrochloric acid. 68 Ga, obtain a radionuclide hydrochloric acid solution, wherein the radionuclide hydrochloric acid solution 68 The radioactive dose of Ga is 3 mCi; take 1 ml of radionuclide hydrochloric acid solution, add 156uL of 1 M sodium acetate solution, adjust the pH value to about 4, and mix well to form a labeling radionuclide solution;

[0057] (3) Preparation of molecular imaging probe: The nucleic acid aptamer-nuclide molecular probe precursor NOTA-TRP obtained in step (1) was mixed with the labeling nuclide solution prepared in step (2), and the mixture was shaken at 100°C for 20 minutes, cooled to room temperature, and then DPBS was used as the mobile phase. The free ions in the product were purified and separated using a pre-equilibrated PD-10 desalting column (Cytiva). 68 Ga ions were removed and finally the radiochemical purity (RCP) of the probe was determined using a radio-thin layer chromatography (Radio-TLC, Eckert&Ziegler Radiopharma Inc) to obtain a molecular imaging probe.

[0058] The molecular imaging probe prepared in this example is represented by [ 68 Ga]Ga-NOTA-TRP.

[0059] Effect Example 1

[0060] The molecular imaging probe of the present invention is used to image subcutaneous lung cancer

[0061] This effect example requires the construction of a Trop-2-positive mouse and human lung cancer tumor-bearing mouse model. The model construction method includes the following steps:

[0062] (1) Use mouse and human lung cancer tumor cell lines H1650, RP21, and SBC-2 that were confirmed to be Trop-2 positive by Western blot for pre-culture;

[0063] (2) Digest the cultured tumor cells and resuspend them in DPBS, then count the cells and mix them with Matrigel (Corning) at a ratio of 1:1. Place the mixed cell suspension on ice and perform tumor implantation as soon as possible.

[0064] (3) Take 100 μl of the cell suspension from step (2) (containing 5×10 6 The subcutaneous tumor model was established by injecting 5-week-old Balb / c nude mice (Viton Liva) into the right flank.

[0065] The imaging experiment was conducted using the Trop-2 expression-positive mouse and human lung cancer tumor-bearing mouse models constructed above, specifically including the following steps:

[0066] (1) Three mice bearing H1650 non-small cell lung cancer cells were selected as group 1; three mice bearing RP21 murine small cell lung cancer cells were selected as group 2; three mice bearing SBC-2 human small cell lung cancer cells were selected as group 3; 3.7-7.4 MBq of the molecular imaging probe prepared in Example 3 was injected into each tumor-bearing nude mouse in each group through the tail vein;

[0067] (2) At a specific time point of 30 min after injection, the tumor-bearing nude mice were anesthetized with isoflurane (concentration of 3%) mixed with oxygen and placed in a prone position on the Siemens Inveon PET / CT scanner bed in a deeply anesthetized state. PET and CT images were acquired and image reconstruction was completed using Inveon Research Workplace 4.2. The results are shown in the attached figure. Figures 6-8 shown.

[0068] Attached Figure 6 The PET / CT molecular imaging images of human non-small cell lung cancer H1650 tumor-bearing mice in Group 1 were obtained 30 minutes after tail vein injection of the molecular imaging probe. It can be seen that the molecular imaging probe described in Example 3 can accurately molecularly image subcutaneous mouse lung cancer tumors, namely H1650 tumors.

[0069] Attached Figure 7 The PET / CT molecular imaging images of the mouse RP21 tumor-bearing mice in Group 2 were taken 30 minutes after the tail vein injection of the molecular imaging probe. It can be seen that the molecular imaging probe described in Example 3 can accurately molecularly image subcutaneous mouse small cell lung cancer tumors, namely RP21 tumors.

[0070] Attached Figure 8The PET / CT molecular imaging images of human SBC-2 tumor-bearing mice in Group 3 were obtained 30 minutes after tail vein injection of the molecular imaging probe. It can be seen that the molecular imaging probe described in Example 3 can accurately molecularly image subcutaneous human small cell lung cancer tumors, namely SBC-2 tumors.

[0071] Use Inveon Research Workplace 4.2 to outline the regions of interest such as tumor, heart, and major tissue organs (liver, spleen, lung, kidney, muscle) on the reconstructed PET images, as shown in the attached figure. Figures 6-8 As shown in the figure, the gray dotted line indicates the tumor area. Figures 6-8 It can be seen that 30 minutes after injection, the molecular imaging probe has obvious high uptake in the tumor tissue.

[0072] Effect Example 2

[0073] Biodistribution experiment of the molecular imaging probe of the present invention in a tumor-bearing mouse model

[0074] This effect example requires the construction of Trop-2 expression-positive mouse and human lung cancer tumor-bearing mouse models, and the construction method is the same as that of effect example 1.

[0075] The molecular probe biodistribution experiment was conducted using the constructed Trop-2 expression positive mouse and human lung cancer tumor-bearing mouse models, which specifically included the following steps:

[0076] (1) Three mice bearing H1650 non-small cell lung cancer cells were selected as group 1; three mice bearing RP21 mouse small cell lung cancer cells were selected as group 2; three mice bearing SBC-2 human small cell lung cancer cells were selected as group 3; each nude mouse bearing tumor in each group was injected with 0.37-0.74 MBq of the molecular imaging probe described in Example 3 through the tail vein;

[0077] (2) At specific time points after injection (30 minutes and 60 minutes), mice were euthanized and dissected; tumors, hearts, and major tissues and organs (liver, spleen, lungs, kidneys, bladder, brain, large intestine, small intestine, bones, muscles, etc.) were collected and weighed, and then the radioactivity of each tissue was measured using a γ-counter. The radioactivity uptake values ​​of the tumor and each tissue were calculated in %ID / g. The results are shown in the attached figure. Figures 9-11 shown.

[0078] Figures 9-11 The experimental results of mice in groups 1 to 3 are shown in Figure 2. Figures 9-11The organ biodistribution data of the molecular imaging probe prepared in Example 3 30 minutes and 60 minutes after the tail vein injection of human and mouse tumor-bearing mice showed that the molecular imaging probe prepared in Example 3 had a higher uptake in tumor tissues and also had non-specific uptake in major excretory and metabolic organs (liver, kidneys).

[0079] Figures 12-14 The experimental results of mice in groups 1 to 3 are shown in the attached figure. Figures 12-14 The signal intensity ratio of the tumor site to the muscle tissue 30 minutes and 60 minutes after the tail vein injection of the molecular imaging probe prepared in Example 3 is as follows. 30 minutes and 60 minutes after the injection, the signal of the tumor area of ​​the non-small cell lung cancer tumor-bearing mice is more than 30 times higher than that of the muscle tissue, and the signal of the tumor area of ​​the small cell lung cancer mouse-derived and human-derived cell tumor-bearing mice is more than 20 times higher than that of the muscle tissue, which can achieve high-contrast PET / CT imaging results.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A nucleic acid aptamer, characterized in that: The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO:

1.

2. The nucleic acid aptamer according to claim 1, characterized in that The nucleic acid aptamer is a freeze-dried powder.

3. Use of the nucleic acid aptamer according to any one of claims 1 to 2 in the preparation of molecular imaging probes for diagnosing lung cancer.

4. A molecular imaging probe for diagnosing lung cancer, characterized in that: The molecular imaging probe comprises the nucleic acid aptamer, the radionuclide chelating ligand and the labeling radionuclide as described in any one of claims 1 to 2; the nucleic acid aptamer is coupled to the radionuclide chelating ligand and is labeled with the labeling radionuclide.

5. The molecular imaging probe for diagnosing lung cancer according to claim 4, characterized in that: The nuclide chelating ligand comprises at least one of deferoxamine, 1,4,7-triazacyclononane-1,4,7-triacetic acid, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

6. The molecular imaging probe for diagnosing lung cancer according to claim 4, characterized in that: The labeling nuclides are Tc-99m, Ga-68, F-18, I-123, I-125, I-131, I-124, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, Y-86, Mn-52, Sc-44, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165 , Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra-223, Ru-106, Na-24, Sr-89, Tb149, Th-227, Xe-133, Yb-169, and Yb-177.

7. The molecular imaging probe for diagnosing lung cancer according to claim 4, characterized in that: The nuclide chelating ligand is 1,4,7-triazacyclononane-1,4,7-triacetic acid, and the labeling nuclide is Ga-68.

8. A method for preparing a molecular imaging probe for diagnosing lung cancer according to any one of claims 4 to 7, characterized in that: The following steps are involved: Step (1): synthesizing nucleic acid aptamers by solid phase synthesis method; Step (2): coupling the synthesized nucleic acid aptamer with the nuclide chelating ligand to obtain a nucleic acid aptamer-nuclide molecular probe precursor; Step (3): The nucleic acid aptamer-nuclide molecular probe precursor obtained in step (2) is mixed with the labeling nuclide solution to obtain a molecular imaging probe.

9. The preparation method according to claim 8, characterized in that: The step (3) is specifically as follows: Dissolving the labeling nuclide with hydrochloric acid to obtain a nuclide hydrochloric acid solution, and then adding a sodium acetate buffer solution to obtain a labeling nuclide solution; The labeling nuclide solution is mixed with the nucleic acid aptamer-nuclide molecular probe precursor, heated for reaction and then cooled to room temperature to obtain a molecular imaging probe.

10. The preparation method according to claim 9, characterized in that In the step (3), the radioactive dose of the labeling nuclide in the nuclide hydrochloric acid solution is 7 mCi to 10 mCi; and / or, the volume ratio of the radionuclide hydrochloric acid solution to the sodium acetate buffer solution is 1:(5-6); And / or, in step (3), the reaction temperature is 100° C. and the reaction time is 20 minutes.

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