A sugar nucleic acid molecule imaging probe and its preparation method and use method

Abstract

The present invention relates to a molecular imaging probe that can be used for tumor diagnosis, and provides a sugar nucleic acid molecular imaging probe, which comprises a DNA nucleic acid aptamer sequence and one or more sugar nucleoside modules, and is labeled with a radioactive nuclide, wherein: the DNA nucleic acid aptamer sequence can specifically target tumor cells after functional folding; the sugar nucleoside module is a sugar-modified nucleoside derivative, which is precisely modified at the 5' end or / and 3' end of the nucleic acid aptamer through DNA solid phase synthesis, and has biological safety, high sensitivity and high resolution imaging characteristics, and has excellent diagnostic effects on some cancers that are difficult to diagnose by traditional methods. The present invention also provides a preparation method and a use method of this sugar nucleic acid molecular imaging probe, which makes the sugar modification of nucleic acid aptamers convenient, fast, precise, controllable, and modular, and has great clinical application potential and transformation value in cancer diagnosis.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a molecular imaging probe that can be used for tumor diagnosis. Background Art

[0002] Some cancers are difficult to diagnose due to the limitations of traditional detection methods, such as triple-negative breast cancer (TNBC). Triple-negative breast cancer is a subtype of breast cancer that lacks the expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2). Imaging examinations play a central role in the screening and diagnosis of breast cancer. At present, mammography, breast ultrasound, and magnetic resonance imaging are commonly used in clinical practice to perform imaging tests on triple-negative breast cancer. However, these clinical detection methods all have some limitations: mammography is easily affected by breast density, and for patients with high breast density, the effect is poor, and it may be difficult to distinguish between lumps and normal tissues; breast ultrasound is not suitable for full breast examination, especially in evaluating the overall morphology and structure of the breast, and is not as effective as mammography; magnetic resonance imaging is not specific for triple-negative breast cancer, and it is difficult to accurately distinguish between malignant masses and benign lesions. These traditional diagnostic methods are often limited in sensitivity and specificity, resulting in difficulties in early diagnosis and a high misdiagnosis rate.

[0003] In recent years, nuclear imaging technologies including positron emission tomography (PET) and single photon emission computed tomography (SPECT) have made steady progress. They can non-invasively visualize, characterize and quantify biological processes of organisms at the molecular level. Among them, PET / CT technology combined with computed tomography (CT) can simultaneously display the biological function and anatomical structure of tissues, which helps to diagnose lesions more accurately. Currently, fluorodeoxyglucose (FDG) is commonly used in clinical practice. 18 F-FDG) is used as a PET / CT tumor imaging agent to achieve its enrichment and targeted imaging in the tumor area, and is used for breast cancer staging, distant metastasis detection and prognosis prediction. However, 18 The enrichment of F-FDG is non-specific and is affected by breast tissue density. Dense normal breast tissue will also absorb 18 F-FDG, resulting in an increase in background signals and a weakening of the imaging diagnostic effect. In recent years, the development of radionuclide-labeled molecular probes based on small molecules, antibodies and other molecules has also provided new tools and strategies for the detection and treatment of tumors. Antibody-based molecular imaging probes are also being widely studied. However, due to the cumbersome preparation of antibodies and their high price, they have great application limitations.

[0004] Aptamers have similar functions to antibodies without the need for expensive and cumbersome screening processes. Compared with antibodies, aptamers can tolerate non-physiological conditions, high temperatures, extreme pH values, and organic solvents. Since their discovery, aptamers have been developed for various clinical applications. However, despite their great potential for clinical application, the clinical application of aptamer-based PET / CT molecular probes still faces many challenges. Therefore, it is urgent to develop new PET / CT molecular probes with high specificity and stability to achieve accurate diagnosis and treatment of some cancers. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a sugar nucleic acid molecular imaging probe, which comprises a DNA nucleic acid aptamer sequence and one or more sugar nucleoside modules, and is labeled with a radionuclide, wherein: the DNA nucleic acid aptamer sequence can specifically target tumor cells after functional folding; the sugar nucleoside module is a sugar-modified nucleoside derivative, which is precisely modified at the 5' end or / and 3' end of the nucleic acid aptamer through DNA solid phase synthesis. It can be understood that whether it is modified at the 5' end or the 3' end, as long as it does not affect the functional folding of the nucleic acid aptamer, even if only a single sugar nucleoside module is modified, the dual targeting and stability improvement of the nucleic acid aptamer can be achieved.

[0006] Preferably, the sugar group of the glycosyl nucleoside module is derived from one or more of glucose, mannose and galactose. Based on the tumor's need for sugar enrichment, the presence of these sugar groups will improve the tumor targeting ability of the probe of the present invention.

[0007] Preferably, the DNA aptamer is AS1411 aptamer or Sgc8 aptamer. Both are aptamers with tumor-specific targeting, wherein the nucleolin protein bound by the AS1411 aptamer is a binding protein for several ligands related to angiogenesis and tumorigenesis; the Sgc8 aptamer recognizes the PTK7 protein on the surface of tumor cells, and PTK7 is related to tumor growth and infiltration.

[0008] Preferably, the radionuclide selected is 68 Ga, 18 F. 177 Lu, 64 Cu, 89 Zr, 99 Tc, 123 I. 125 I. 131 I. 11 C. 13 N or 15 Any one of O.

[0009] Preferably, the sugar nucleic acid molecular imaging probe comprises at least 3 consecutively arranged sugar nucleoside modules.

[0010] The present invention also provides a method for preparing a sugar nucleic acid molecule imaging probe, comprising the following steps:

[0011] Step 1: preparing a glycosyl nucleoside module, wherein the glycosyl nucleoside module has a glycosyl group derived from one or more of glucose, mannose, and galactose;

[0012] Step 2: Designing nucleic acid aptamers with tumor-specific targeting;

[0013] Step 3: using DNA solid phase synthesis technology, one or more sugar nucleoside modules are connected to the 3' and / or 5' end of the nucleic acid aptamer to prepare a sugar nucleic acid molecular probe;

[0014] Step 4: The sugar nucleic acid molecular probe obtained in step 3 is connected to a metal chelator (NOTA / DOTA) for radionuclide labeling to obtain a sugar nucleic acid molecular imaging probe.

[0015] The present invention also provides a method for using the sugar nucleic acid molecule imaging probe, wherein the sugar nucleic acid molecule imaging probe is used for the preparation of related drugs or kits required for PET / CT imaging of tumor tissue, wherein the background of the tumor tissue is muscle, blood, heart or brain.

[0016] Preferably, the tumor tissue is triple-negative breast cancer (TNBC).

[0017] The present invention modularizes sugar molecules and successfully synthesizes and prepares sugar nucleoside modules through four-step chemical reactions. The module is suitable for precise nucleotide connection technologies such as DNA solid phase synthesis technology. The present invention uses precise connection technology to accurately modify the sugar module of nucleic acid aptamers, so that multiple sugar nucleoside modules can be continuously, accurately and controllably modified to one or both ends of the nucleic acid aptamer. The preparation method modularizes the sugar modification of nucleic acid aptamers, which is very convenient, safe and low-cost. It can also adapt to the needs of different nucleic acid aptamers to realize continuous modification of multiple modules and is widely used; the sugar nucleic acid molecular probe prepared by it has good biosafety, its stability in serum is significantly improved, and it has excellent specific targeting ability at the cell and animal levels.

[0018] The present invention uses a radionuclide-labeled sugar nucleic acid molecular imaging probe with high sensitivity and high resolution imaging characteristics, which can be rapidly enriched and sensitively traced at the tumor site, and thus has excellent diagnostic effects on some cancers that are difficult to diagnose with traditional methods. With the help of a small animal PET / CT imager, the present invention verifies the tumor targeting effect of the mannose nucleic acid molecular imaging probe in TNBC tumor-bearing mice and the in vivo dynamic metabolic process. PET / CT imaging shows that the new molecular probe can specifically target tumor cells and can achieve sensitive tracing of the tumor site. The radionuclide-labeled new sugar nucleic acid molecular probe of the present invention has important application potential and clinical transformation value in the diagnosis of some cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Schematic diagram of the synthesis of glycosyl nucleoside modules.

[0020] Figure 2 : Mass spectrum of sugar nucleic acid molecular probe MA02.

[0021] Figure 3 : HPLC chromatogram of sugar nucleic acid molecular probe MA02.

[0022] Figure 4 : In vitro serum stability test of sugar nucleic acid molecular probe MA02.

[0023] Figure 5 : Flow cytometry of the sugar nucleic acid molecular probe MA02 binding to 4T1 cells.

[0024] Figure 6 : H&E staining results of in vivo toxicity of sugar nucleic acid molecular probe MA02.

[0025] Figure 7 : 68 Ga-labeled sugar nucleic acid molecular probe MA02 ( 68 The labeling efficiency of Ga-NOTA-MA02 was detected.

[0026] Figure 8 : 68 Ga-labeled sugar nucleic acid molecular probe MA02 ( 68 PET / CT molecular images within 2 hours after injection of Ga-NOTA-MA02 into the tail vein of 4T1 tumor-bearing mice.

[0027] Fig. 9 : Injection into the tail vein of 4T1 tumor-bearing mice 68 Ga-labeled sugar nucleic acid molecular probe MA02 ( 68 Biodistribution diagram of the radioactivity in each organ after 0.5 hour, 1 hour, and 2 hours of Ga-NOTA-MA02.

[0028] Fig.10 : Injection into the tail vein of 4T1 tumor-bearing mice 68 Ga-labeled sugar nucleic acid molecular probe MA02 ( 68 Comparison of the signal intensity ratio between the tumor site and muscle tissue after 0.5 hour, 1 hour, and 2 hours between the Ga-NOTA-MA02 and control group probes. DETAILED DESCRIPTION

[0029] Unless otherwise specified, the experimental methods used in the following implementation methods are all conventional methods. The reagents and consumables used in the examples can be purchased from normal commercial channels unless otherwise specified.

[0030] 1. Preparation of sugar nucleoside modules

[0031] like Figure 1 As shown, using α-D-pentaacetic acid mannoside as raw material, through four steps of chemical reaction, the mannosyl nucleoside module for DNA solid phase synthesis was finally successfully obtained.

[0032] First, under the protection of argon, 5-hexyn-1-ol and boron trifluoride etherate were slowly added to α-D-pentaacetic acid mannosyl ester in an ice bath, stirred for 30 min, and allowed to react at room temperature overnight to purify and separate compound 3 (1.86 g, yield 85%). At the same time, under the protection of argon, 5-iodo-2'-deoxyuridine dissolved in anhydrous pyridine was stirred and reacted with 4,4'-bismethoxytrityl chloride at room temperature for 4 h to purify and obtain compound 6 (4.83 g, yield 51%). Next, compound 6, tetrakis(triphenylphosphine)palladium and cuprous iodide were dissolved in anhydrous N,N-dimethylformamide, and N,N-diisopropylethylamine was added thereto, and stirred at room temperature for 10 min. Compound 3 dissolved in anhydrous N,N-dimethylformamide was added dropwise to the reaction bottle within 1 h, and then reacted at room temperature in the dark overnight to purify and separate compound 7 (7.01 g, yield 84%). Finally, compound 7 was dissolved in anhydrous dichloromethane, and N,N-diisopropylethylamine was added thereto under argon protection and stirred for 5 min. 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (compound 8) was added to the reaction bottle under ice bath conditions, stirred at room temperature for 2 h, and purified to obtain product 9 (75.8 mg, 63%), i.e., the mannoside nucleoside module.

[0033] It is understandable that the above preparation process belongs to the conventional preparation of nucleoside compounds and their derivatives. In addition to the above scheme, those skilled in the art can adopt other existing synthesis methods to modify the sugar group to the nucleoside derivative to form a glycosyl nucleoside module that meets the conditions of DNA solid phase synthesis. At the same time, based on the above steps, the mannose glycosyl raw material can be replaced by sugar esters of other sugar molecules such as glucose, mannose, galactose, etc., which can realize the construction of the glycosyl nucleoside module of the present invention and exert the corresponding technical effects.

[0034] 2. Preparation and properties of sugar nucleic acid molecular probe MA02

[0035] 2.1 Preparation of sugar nucleic acid molecular probe MA02

[0036] Using DNA solid phase synthesis technology, three mannose modules were continuously added to the 5' end of the nucleic acid aptamer AS1411 base sequence to accurately synthesize the sugar nucleic acid chain molecule MA02. The control group T-AS141 added three thymine nucleotides to the 5' end of the AS1411 base sequence using the same DNA solid phase synthesis technology.

[0037] DNA solid phase synthesis technology is used for efficient connection of single nucleotides at the 5' end or 3' end of oligonucleotide chains. With the support of this technology, the glycosyl nucleosides of the present invention can be used as functional modules to batch modify various nucleic acid aptamers, and accurately control and realize the glycosylation of nucleic acid aptamers.

[0038] It can be understood by those skilled in the art that, whether modified at the 5' end or the 3' end, the modification of the nucleic acid aptamer with only a single sugar nucleoside module can achieve the improvement of the dual targeting and stability properties of the nucleic acid aptamer, which is determined by the principle of the present invention and can be reasonably inferred by those skilled in the art based on the data obtained by the present invention. The probe scheme modified with three sugar nucleoside modules used in the embodiments of the present invention and its accompanying drawings is a preferred embodiment of the present invention. Although AS1411 is selected in this embodiment, the technical effect intended to be achieved in this embodiment can also be achieved in a nucleic acid aptamer with tumor-specific targeting (such as the Sgc8 nucleic acid aptamer).

[0039] 2.2 Property detection:

[0040] (1) Molecular weight of sugar nucleic acid probe and liquid chromatography results

[0041] We verified the molecular weight of the sugar nucleic acid molecular probe MA02 prepared above by mass spectrometry. Figure 2 The molecular weight of the sugar nucleic acid molecular probe MA02 was verified by mass spectrometry to be 16004.8, which was consistent with the expected result.

[0042] The HPLC chromatogram of sugar nucleic acid molecular probe MA02 is shown in Figure 3As shown, the sugar nucleic acid molecular probe MA02 was separated and purified by HPLC, and its retention time was found to be about 15.5-16.3 minutes.

[0043] (2) In vitro serum stability of sugar nucleic acid molecular probes

[0044] The sugar nucleic acid molecular probe MA02 and nucleic acid aptamer AS1411 were incubated in 10% fetal bovine serum at 37°C for 0, 1, 2, 4, 8, 12, 24, 48, and 72 h, respectively. The degradation of the two chains of MA02 and AS1411 after incubation was detected by 10% polyacrylamide gel electrophoresis.

[0045] The results are as follows Figure 4 As shown in the figure, AS1411 was rapidly degraded over time, while MA02 was degraded, but there were still stable bands after 24h or even 72h. It can be seen that the modification of the mannose module significantly enhanced the in vitro stability of the nucleic acid aptamer.

[0046] (3) In vitro targeting of sugar nucleic acid molecular probes

[0047] 250 nM MA02-Cy5 and AS1411-Cy5 were incubated with mouse breast cancer cell line 4T1 cells in binding buffer at 4°C for 30 min, washed twice with washing buffer, and then detected using flow cytometry.

[0048] The results are as follows Figure 5 As shown. The results show that compared with the nucleic acid aptamer AS1411-Cy5, the mannose-modified MA02-Cy5 sugar nucleic acid chain has a stronger binding ability with 4T1 cells, and the fluorescence intensity is significantly shifted compared with the control chain; further statistical analysis and comparison of the average fluorescence intensity corresponding to the two probes can more clearly show that the fluorescence intensity of 4T1 cells binding to MA02-Cy5 is also significantly higher than that of AS1411-Cy5. This is because on the basis of the targeted nucleic acid aptamer, the sugar group of the sugar nucleoside can also target tumor cells, so that the sugar nucleic acid molecular probe of the present invention actually has a "dual targeting" targeting effect on tumor cells.

[0049] (4) In vivo biosafety testing

[0050] Healthy 5-week-old female BALB / c mice were divided into a control group (DPBS, 100 μL) and an experimental group (MA02 probe, 2nmol, 100 μL) for major organ toxicity studies. After one week, the major organs (heart, liver, spleen, lung and kidney) were collected and the in vivo toxicity was evaluated by H&E staining.

[0051] The results are as follows Figure 6All tissue structures were intact, and the physiological morphology of the heart, liver, spleen, lung, and kidney showed no obvious abnormalities, indicating that the sugar nucleic acid molecular probe MA02 had basically no long-term organ toxicity.

[0052] 3. Radionuclide-labeled sugar nucleic acid molecular imaging probe

[0053] 3.1 Preparation 68 Ga-labeled sugar nucleic acid molecular probe MA02 ( 68 Ga-NOTA-MA02 probe)

[0054] First, on the basis of the solid phase synthesis obtained above nucleic acid chain, by modifying C6NH2 at the 5' end of the nucleic acid chain to connect with the metal chelator NOTA, the precursors MA02-NOTA and AS1411-T-NOTA that can be used for radionuclide labeling are obtained, and then the radionuclide is carried out. 68 It is understood that the radioactive labeled nuclides that can be used here are not limited to 68 Ga, can also be 18 F. 177 Lu, 64 Cu, 89 Zr, 99 Tc, 123 I. 125 I. 131 I. 11 C. 13 N. 15 Any one of O.

[0055] 68 Ga solution through 68 Ge / 68 The Ga generator was eluted with 0.1 mol / L hydrochloride solution. 68 The Ga solution was mixed with MA02-NOTA, incubated at 100°C with shaking at 300 rpm for 10 min, and then cooled at room temperature for 10 min. After purification using a PD-10 column, 10 mM EDTA was used as the mobile phase to purify the radionuclide-labeled MA02 and control nucleic acid chain molecules.

[0056] Radio-TLC was used to determine the radiolabeling efficiency. Figure 7 As shown, the purified radiolabeled product 68 The labeling efficiency of Ga-NOTA-MA02 reached over 99%.

[0057] 3.2 Property detection

[0058] (1) Radionuclide imaging in mice at different times

[0059] 200 μCi of probe was injected into 4T1 tumor-bearing mice 68 After Ga-NOTA-MA02, dynamic PET-CT scanning imaging was performed for 2 h, and the imaging at the time points of 5, 10, 20, 30, 60, 90, and 120 min was intercepted for analysis.

[0060] The results are as follows Figure 8 As shown in the figure, 68 Ga-NOTA-MA02 began to rapidly accumulate in the tumor site 10 minutes after injection (circled area on the right side of the mouse), and after 20 minutes, it showed an imaging radiation intensity significantly higher than that of the surrounding muscle tissue. At the same time, as time went on, the radioactive signal of the probe in the tumor site also increased, indicating that the probe of the present invention is also very stable in the body and is not easily degraded. At the same time, it has excellent targeting and enrichment effects on the tumor site.

[0061] (2) Detection of radioactivity distribution in major organs and tissues

[0062] 100 µCi 68 Ga-NOTA-MA02 was injected into the 4T1 tumor mouse model via the tail vein. After 0.5 h, 1 h, and 2 h, the main organ tissues (blood, brain, heart, liver, spleen, lung, kidney, bone, large intestine, small intestine, stomach, muscle) and tumors of the mice were collected for in vitro radioactivity detection.

[0063] The results are as follows Fig. 9 The distribution of radioactivity in various organs shows that 68 Ga-NOTA-MA02 is highly radioactive in various metabolic organs because it can be metabolized. In the tumor site, it has radioactive signal enrichment at 0.5 h, about 0.9~1.41%ID / g, and there is still a high radioactive signal after 2 h, which corresponds to the experimental results in the above (1); 68 The radiation signal of Ga-NOTA-MA02 is very weak in the blood, muscles, heart, and brain. There is a clear difference in the signal intensity at the tumor compared with the signals of these organs.

[0064] The dynamic metabolic process of the probe of the present invention in vivo shown in this experiment once again verifies that the modification of the glycosyl nucleoside module effectively enhances the stability, targeting ability and enrichment ability of AS1411 tumors in vivo; on the other hand, the background of TNBC tumor cells in mice is muscle, and the probe of the present invention can effectively diagnose TNBC that is difficult to diagnose by traditional means. At the same time, the results of this experiment also show that the signal of the probe of the present invention is clearly distinguished from the tumor in multiple in vivo sites, because this also reflects that the probe of the present invention has excellent recognition and diagnostic differentiation when applied to radioactive imaging of tumor cells with blood, muscle, heart and brain as the background, and therefore has a broad application space.

[0065] (3) Comparison of radioactivity intensity between tumor and muscle

[0066] The probe of the present invention was injected into the tail vein of 4T1 tumor-bearing mice. 68 Ga-NOTA-MA02 and control probes 68 Ga-NOTA-T-AS1411 was added, and the ratio of the radioactive signal intensity between the tumor site and the muscle tissue was detected at 0.5 h, 1 h and 2 h after injection.

[0067] The results are as follows Fig.10 Although the radionuclide-labeled AS1411 nucleic acid aptamer has a certain degree of targeting, due to insufficient uptake, weak targeting, and easy degradation of the nucleic acid aptamer, the probe tumor / muscle contrast ratio is about 1, and it is completely impossible to distinguish between tumors and muscles. 68 The glycosyl nucleoside module of Ga-NOTA-MA02 gives it better targeting and aggregation efficiency. It can form a nearly 5-fold strong contrast between TNBC tumors and muscles in just half an hour, and the signal lasts for two hours without attenuation.

[0068] This experiment shows that after nucleic acid aptamers are modified with simple and convenient sugar nucleosides continuously, their in vivo stability, tumor targeting, enrichment and other properties have undergone significant changes. The molecular imaging probe based on them can sensitively and effectively show tumor tissues that were difficult to detect in the past, and their targeting, sensitivity, signal strength and retention time are all beyond expectations. This also reflects that for some tumors with blood or muscle as the background that are difficult to detect by traditional means, the technical solution of the present invention provides a powerful means for their future PET / CT imaging diagnosis.

[0069] Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to modify the above-mentioned embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection required by the present invention.

Claims

1. A sugar nucleic acid molecular imaging probe, characterized in that: The sugar nucleic acid molecular imaging probe comprises a DNA nucleic acid aptamer sequence and three consecutively arranged sugar nucleoside modules and is labeled with a radionuclide, wherein: The DNA aptamer sequence can specifically target tumor cells after functional folding, and the DNA aptamer is an AS1411 aptamer; The sugar nucleoside module is a mannose nucleoside module, which is precisely modified at the 5' end or / and the 3' end of the nucleic acid aptamer through DNA solid phase synthesis.

2. A sugar nucleic acid molecular imaging probe as claimed in claim 1, characterized in that: The radionuclides selected were 68 Ga, 18 F. 177 Lu, 64 Cu, 89 Zr, 99 Tc, 123 I. 125 I. 131 I. 11 C. 13 N or 15 Any one of O.

3. A method for preparing a sugar nucleic acid molecule imaging probe as claimed in claim 1, characterized in that: The following steps are involved: Step 1: preparing a glycosyl nucleoside module, wherein the sugar group of the glycosyl nucleoside module is mannose; Step 2: Designing nucleic acid aptamers with tumor-specific targeting; Step 3: using DNA solid phase synthesis technology, connecting three consecutively arranged sugar nucleoside modules to the 3' and / or 5' end of the nucleic acid aptamer to prepare a sugar nucleic acid molecular probe; Step 4: After connecting the sugar nucleic acid molecular probe obtained in step 3 with a metal chelator, labeling with a radioactive nuclide is performed to obtain a sugar nucleic acid molecular imaging probe.

4. An application of the sugar nucleic acid molecular imaging probe as claimed in claim 1, characterized in that: The sugar nucleic acid molecule imaging probe is used for preparing relevant drugs or kits required for PET / CT imaging of tumor tissue, wherein the background of the tumor tissue is muscle, blood, heart or brain.

5. The use of a sugar nucleic acid molecular imaging probe as claimed in claim 4, characterized in that: The tumor tissue is triple-negative breast cancer.

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