Sugar nucleic acid dual-targeting radioactive molecular probe as well as preparation method and application thereof

Abstract

The invention provides a sugar nucleic acid dual-targeting radioactive molecular probe for targeted therapy of bladder cancer as well as a preparation method and application of the sugar nucleic acid dual-targeting radioactive molecular probe, and belongs to the technical field of biological medicines. The probe is composed of an Sgc8 aptamer for recognizing bladder cancer cell PTK7 protein, three nitrine mannose molecules of a targeted M2 type macrophage CD206 receptor and a DOTA structure for chelating 177Lu, and precise assembly is achieved through a click chemical reaction and complementary base pairing. The probe has dual targeting ability, can act on bladder cancer cells and M2 type macrophages in a tumor microenvironment at the same time, significantly prolongs the retention time (gt, 48 hours) in the bladder, induces immune activation while killing tumor cells, and realizes the synergistic effect of radiotherapy and immunotherapy. The problems that an existing bladder cancer treatment probe is insufficient in targeting, poor in stability and limited in treatment effect are solved, a new strategy is provided for precise treatment of bladder cancer, and the bladder cancer probe has good clinical transformation value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a dual-targeting radioactive molecular probe for glyconucleotides, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Bladder cancer (BC) is one of the most common malignant tumors of the urinary system, and its incidence is increasing globally. The high recurrence rate and resistance to traditional treatments pose significant challenges to current clinical treatment. Traditional treatments, such as surgical resection, chemotherapy, and radiotherapy, while capable of controlling tumor progression to some extent, often involve significant side effects and cause considerable damage to non-target tissues. Furthermore, existing radiotherapy and chemotherapy regimens suffer from poor targeting, making it difficult to precisely target tumor cells and thus limiting treatment efficacy.

[0004] In recent years, radionuclide therapy has received widespread attention as a novel approach to cancer treatment. Among other things, 177 Due to its excellent penetration depth and therapeutic effect, Lu is widely used in the treatment of various tumors. 177 Lu (light) is a beta-ray emitter capable of causing DNA damage to tumor cells through its radioactive energy, thereby enabling precise cancer treatment. However, the targeting, stability, and retention time of radioactive probes in vivo remain major obstacles limiting their clinical translation. Developing radioactive probes with high targeting and stability is a current research focus in order to improve therapeutic efficacy and reduce damage to normal tissues.

[0005] Nucleic acid aptamers, as novel targeting molecules, have been widely used in tumor molecular imaging and therapy due to their advantages such as high affinity, low immunogenicity, and ease of modification. Compared with traditional antibody drugs, nucleic acid aptamers have smaller molecular weights, enabling them to penetrate tumor tissue more effectively without triggering an immune response. Sgc8, a nucleic acid aptamer targeting the PTK7 protein highly expressed on the surface of bladder cancer cells, has been shown to specifically recognize and bind to bladder cancer cells. Furthermore, mannose, a natural sugar molecule, has been shown to target the CD206 receptor on tumor-associated M2 macrophages. M2 macrophages have immunosuppressive effects in the tumor microenvironment, and mannose-modified probes can achieve precise localization and regulation of these immunosuppressive cells.

[0006] Against this backdrop, constructing a dual-targeting radioactive molecular probe that targets both tumor cells and immune cells using a combination of sugars and nucleic acids has significant clinical implications. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to develop a novel dual-targeting radioactive molecular probe for the precise treatment of cancers such as bladder cancer. This probe can not only improve the targeting of radionuclides to tumor cells, but also achieve synergistic therapeutic and immune activation effects by regulating immune cells in the tumor microenvironment.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A glyconucleotide dual-targeting radioactive molecular probe, characterized in that the radioactive molecular probe comprises a nucleic acid aptamer, a coupling group modified on the nucleic acid aptamer, and at least one mannose module connected to the coupling group; The nucleic acid aptamer is linked to a chelating agent through complementary base pairing, and the chelating agent chelates a radionuclide. The mannose module is azide-mannose, and the structure of the azide-mannose is as follows: ; In the formula, R is selected from: ; Where n = 1-6, m = 1-10.

[0009] In some embodiments, the coupling group is dibenzocyclooctylene (DBCO).

[0010] In some embodiments, the radionuclide is 177 Lu.

[0011] In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0012] Preferably, the nucleic acid aptamer is Sgc8.

[0013] This invention provides a method for preparing a dual-targeting radioactive molecular probe for glyconucleotides as described above, characterized by comprising the following steps: Azide-mannose and a nucleic acid aptamer chain with a coupling group are synthesized. The azide-mannose is linked to the coupling group through a click chemistry reaction. Then, a complementary chain with a chelating agent is linked through base complementarity. A radionuclide is labeled on the chelating agent to obtain a glyconucleotide dual-targeting radioactive molecular probe.

[0014] The present invention also provides the application of the glyconucleotide dual-targeting radioactive molecular probes described above, wherein the radioactive molecular probes are used to prepare relevant drugs or kits required for the diagnosis or treatment of tumors.

[0015] Furthermore, the tumor is bladder cancer.

[0016] Based on the above disclosure, this application includes the following beneficial technical effects: 1) This invention is based on the nucleic acid aptamer Sgc8, which targets the PTK7 protein of bladder cancer cells. The aptamer's original sequence was modified with mannose modules. Multiple mannose modules were precisely modified onto a nucleic acid strand containing three DBCOs via click chemistry. Then, a complementary strand containing a chelating agent was linked via base complementarity, and a radionuclide was labeled onto the chelating agent. 177 Lu constructed a novel dual-targeting glyconucleotide radioactive molecular probe, GDNP, for bladder cancer. Multi-site mannose modification enhanced the probe's selective binding ability to M2 immunosuppressive cells, enabling it to not only target tumor cells but also improve the tumor immune microenvironment. The base complementarity splicing strategy enabled controllability and diversity in probe assembly, providing a platform foundation for future multifunctional and personalized modification.

[0017] 2) The structurally optimized probe exhibits improved stability in serum, showing no significant degradation for 48 hours in a serum environment; compared to traditional single-target probes, GDNP demonstrates stronger tumor-suppressive activity, higher survival benefits, and extremely low systemic toxicity; through 177 This Lu-mediated beta radiation probe enables tumor cell killing and M2 macrophage clearance, while simultaneously inducing antigen-presenting cell (DC) maturation and T cell activation. It synergistically integrates radiotherapy and immune remodeling, forming a closed-loop tumor treatment feedback network. This invention fills a technological gap in dual-targeted therapy design using radioactive nucleic acid probes and has promising prospects for clinical translation.

[0018] 3) Based on this probe, by utilizing a wide range of DNA aptamer libraries and modifying different DNA aptamers with sugar modules, targeted molecular imaging probes for various tumors can be easily produced.

[0019] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the synthesis of azidomannose Man-N3 as shown in Example 1 of the present invention.

[0022] Figure 2 This is the mass spectrum of azidomannose Man-N3 shown in Example 2 of the present invention.

[0023] Figure 3 This is a schematic diagram of the structure of the glyconucleotide dual-targeting radioactive molecular probe Sgc8-Man3 shown in Example 2 of the present invention.

[0024] Figure 4 The mass spectrum of the dual-targeting radioactive molecular probe Sgc8-Man3 of glyconucleotides shown in Example 2 of this invention is shown.

[0025] Figure 5 The in vitro serum stability of the glyconucleotide dual-targeting radioactive molecular probe Sgc8-Man3 shown in Example 2 of this invention.

[0026] Figure 6 This illustrates the binding and endocytosis of the glyconucleotide molecular probe with bladder cancer cells, as shown in Example 2 of the present invention.

[0027] Figure 7 This study investigates the in vivo retention imaging of the glyconucleotide molecular probe Sgc8-Man3 shown in Example 2 of this invention.

[0028] Figure 8 As shown in Embodiment 3 of the present invention 177 Lu-labeled RNA molecular probe GDPN ( 177 Lu-Sgc8-Man3) and 177 Labeling efficiency detection of Lu-Sgc8.

[0029] Figure 9 This refers to the bladder instillation injection performed on C57 bladder cancer orthotopic model mice as shown in Example 3 of the present invention. 177 Image showing the effect of 14-day treatment with Lu-labeled RNA chain molecular probe GDPN.

[0030] Figure 10 This refers to the bladder instillation injection performed on C57 bladder cancer orthotopic model mice as shown in Example 3 of the present invention. 177H&E staining image 14 days after treatment with the Lu-labeled RNA chain molecular probe GDPN.

[0031] Figure 11 This refers to the bladder instillation injection performed on C57 bladder cancer orthotopic model mice as shown in Example 3 of the present invention. 177 Immunological assay 14 days after treatment with the Lu-labeled RNA chain molecular probe GDPN. Detailed Implementation

[0032] To better describe the present invention, specific embodiments are provided below for further explanation. Unless otherwise specified, the methods in the following embodiments are conventional methods.

[0033] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0034] The following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental conditions not specifically stated in the examples are generally performed under conventional conditions or as recommended by the reagent company; reagents, consumables, etc., used in the following examples are commercially available unless otherwise specified.

[0035] Example 1: Synthesis of azidomannose (Man-N3): like Figure 1 As shown, using α-D-pentaacetic acid mannoester as a raw material, azide-mannose for glycosylation modification was successfully obtained through a two-step chemical reaction.

[0036] The specific steps are as follows: 1.1 Preparation of Compound 1 (Mannose Ether): 1.47 g of dried α-D-mannose pentaacetate was dissolved in 30 mL of anhydrous DCM. Under argon protection, 1.00 g of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethanol was added to the solution, and the mixture was stirred for 30 min. 2.32 mL of boron trifluoride diethyl ether solution was added to the solution in an ice bath. The reaction was allowed to proceed for 16 h, and the reaction was terminated by adding saturated sodium bicarbonate aqueous solution. The low-boiling solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to obtain 1.22 g of compound 1.

[0037]

[0038] Characterized by NMR and MS as follows:1 H NMR (400 MHz, CDCl3) δ 5.36 (dd, J = 10.0,3.4 Hz, 1H), 5.33 – 5.29 (m, 1H), 5.27 (dd, J = 3.3, 1.7 Hz, 1H), 4.88 (d, J= 1.7 Hz, 1H), 4.30 (dd, J = 12.4, 5.2 Hz, 1H), 4.08 (m, 2H), 3.82 (m, 1H), 3.68 (dd, J = 5.5, 2.8 Hz, 13H), 3.40 (t, J = 5.0 Hz, 2H), 2.16 (s, 3H), 2.11 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 170.71, 170.05,169.90, 169.74, 97.71, 72.46, 70.74, 70.70, 70.66, 70.03, 69.98, 69.56,69.08, 68.37, 67.38, 66.12, 62.39, 61.77, 50.67, 20.92, 20.78, 20.73, 20.71. 1.2 Preparation of compound 2 (Man-N3, azidomannose): 400 mg of dried compound 1 was dissolved in 14 mL of MeOH. 2 mL of H₂O and 2 mL of TEA were added to the solution, and the mixture was stirred overnight. The residue was removed by vacuum distillation to obtain 230 mg of compound 2.

[0039]

[0040] Characterization was performed using NMR and MS. 1H NMR (400 MHz, CDCl3) δ 4.87 (s, 1H), 4.54 (s,4H), 3.99 – 3.90 (m, 3H), 3.84 (dd, J = 9.7, 3.0 Hz, 1H), 3.80 – 3.74 (m,2H), 3.70 – 3.60 (m, 13H), 3.56 (d, J = 9.6 Hz, 1H), 3.40 (t, J = 5.0 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 100.16, 77.24, 72.23, 71.55, 70.86, 70.65,70.58, 70.11, 70.02, 66.72, 66.30, 61.04, 53.45, 50.65. Its mass spectrum is as follows: Figure 2 As shown.

[0041] Example 2 Synthesis and Detection of Sgc8-Man3 2.1 Synthesis of Sgc8-Man3: Purchase Sgc8 nucleic acid strands with three DBCO groups from Sangon Biotech (Shanghai) Co., Ltd. Prepare a 100 μmol / L solution of the Sgc8-DBCO nucleic acid strand with PBS according to experimental requirements. Simultaneously, weigh out 300 μmol / L of azidomannose (purity ≥95%). Slowly add the dissolved azidomannose solution to the Sgc8-DBCO nucleic acid strand solution while gently stirring to ensure thorough mixing. Incubate the mixture at 4°C overnight for 12-16 hours. During the reaction, gently shake the reaction vessel every 4-6 hours to promote the reaction. After the reaction, transfer the solution to ultrafiltration centrifuge tubes with a molecular weight cutoff of approximately 3 kDa. Centrifuge the tubes at 4000-6000 rpm for 15-20 minutes. After centrifugation, carefully remove and discard the lower layer of unlinked small molecule azide-mannose solution; the upper layer of linked product solution is the desired product.

[0042] like Figure 3 The diagram shows the structure of the synthesized glyconucleotide molecular probe Sgc8-Man3.

[0043] like Figure 4 The mass spectrum of the synthesized glyconucleotide molecular probe Sgc8-Man3 is shown in the figure.

[0044] 2.2 Performance Testing: Comparison of in vitro serum stability before and after modification: The glyconucleotide chain Sgc8-Man3 and the nucleic acid aptamer Sgc8 were incubated in 10% fetal bovine serum at 37°C for 0, 1, 2, 4, 8, 12, 24, and 48 h, respectively. The degradation of the two chains Sgc8-Man3 and Sgc8 after incubation was detected by 10% polyacrylamide gel electrophoresis. Figure 5 As shown in the bands, Sgc8 is rapidly degraded over time, while Sgc8-Man3, although degraded, still exhibits stable bands. This demonstrates that the azidomannose-modified nucleic acid chains effectively enhance the serum stability of the nucleic acid aptamers.

[0045] The binding and endocytosis of the glyconucleotide probe Sgc8-Man3, as well as Sgc8 and the control strand, with three different types of bladder cancer cells. Figure 6 As shown, the Sgc8-Man3 glyconucleotide probe exhibited stronger binding and endocytosis with bladder cancer cells than Sgc8 and the control strand.

[0046] A study on the in vivo retention of the glyconucleotide probe Sgc8-Man3. We injected the glyconucleotide probe Sgc8-Man3 and the standalone Sgc8 nucleic acid strand into the bladders of mice with orthotopic bladder cancer at a concentration of 250 nM. In vivo imaging of the small animals was performed after 0.5, 1, 4, 6, 12, 24, and 48 hours. Figure 7 As shown, the Sgc8 nucleic acid strand no longer exhibited fluorescence after 24 hours, while the Sgc8-Man3 glyconucleotide probe group still showed fluorescence after 48 hours. This clearly demonstrates that the Sgc8-Man3 glyconucleotide probe has a longer in vivo retention time.

[0047] Example 3 177 Preparation and detection of Lu-labeled probes (GDNP) 177 Radio-TLC labeling efficiency was measured after Lu-labeled glyconucleotide probes. First, partial amounts of nucleic acid precursors Sgc8-Linker and Sgc8-Man3-Linker were purchased from Sangon Biotech Co., Ltd. (Shanghai). Then, azidomannose was modified via chemical click reaction, and the nuclide chelator DOTA was modified according to the base complementarity principle, ultimately yielding Sgc8-DOTA and Sgc8-Man3-DOTA. Using... 177 Lu generator, prepared by elution with 0.1 mol / L hydrochloric acid solution. 177 Lu solution. Prepared... 177The Lu solution was mixed thoroughly with both Sgc8-DOTA and Sgc8-Man3-DOTA, ensuring complete contact between the two. The mixture was then incubated in a 100°C water bath with shaking at 300 rpm for 10 minutes to promote the labeling reaction. After incubation, the reaction mixture was cooled to room temperature for 10 minutes to terminate the reaction.

[0048] To remove unreacted 177 Lu and other impurities were removed, and the reaction product was purified using a PD-10 column. The purified product was then analyzed by radio-TLC with 10 mM EDTA as the mobile phase to determine the labeling efficiency. Figure 8 As shown, the purified radiolabeled product 177 The labeling efficiency of Lu-Sgc8-Man3 (GDNP) reached over 98%.

[0049] like Figure 9 As shown, RNA nucleoside drugs 177 Schematic diagram of Lu-Sgc8-Man3 (GDNP) in vivo treatment. First, an in situ bladder model is established one week in advance, and then we use a glyconucleotide nucleoside drug. 177 Lu-Sgc8-Man3 (GDNP) was administered in two doses via bladder instillation on days 0 and 7, with each dose being 100 µCi. After treatment, the mice were dissected, and tumor areas from the heart, liver, spleen, lungs, and kidneys were collected for H&E staining. Figure 10 As shown, glyconucleotides 177 In the Lu-Sgc8-Man3 (GDNP) treatment group (Group 2), the necrotic area in the tumor region was larger than that of the nucleic acid nuclide alone. 177 Lu-Sgc8 treatment group (Group 1).

[0050] Flow cytometry immunoassay: Tumor tissue and spleen tissue were collected 14 days after treatment, ground, and then subjected to immunoassay. Figure 11 As shown, we observed an increase in the number of T cells after treatment and that it activated the maturation of dendritic cells. The number of M2 macrophages decreased and the number of M1 macrophages increased after treatment. This indicates that the glyconucleotide probe of this invention can also activate an immune response, achieving a synergistic therapeutic effect.

[0051] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0052] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A radioactive molecular probe that targets both glyconucleotides, characterized in that, The radioactive molecular probe includes a nucleic acid aptamer, a coupling group modified on the nucleic acid aptamer, and at least one mannose module connected to the coupling group; The nucleic acid aptamer is linked to a chelating agent through complementary base pairing, and the chelating agent chelates a radionuclide. The mannose module is azide-mannose, and the structure of the azide-mannose is as follows: ； In the formula, R is selected from: ； Where n = 1-6, m = 1-10.

2. The dual-targeting radioactive molecular probe for glyconucleotides as described in claim 1, characterized in that, The coupling group is dibenzocyclooctylene.

3. The dual-targeting glyconucleotide radioactive molecular probe as described in claim 1, characterized in that, The radioactive nuclide is 177 Lu.

4. The dual-targeting glyconucleotide radioactive molecular probe as described in claim 1, characterized in that, The chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

5. The dual-targeting radioactive molecular probe for glyconucleotides as described in claim 1, characterized in that, The nucleic acid aptamer is Sgc8.

6. A method for preparing a dual-targeting radioactive molecular probe for glyconucleotides as described in any one of claims 1-5, characterized in that, Includes the following steps: Azide-mannose and a nucleic acid aptamer chain with a coupling group are synthesized. The azide-mannose is linked to the coupling group through a click chemistry reaction. Then, a complementary chain with a chelating agent is linked through base complementarity. A radionuclide is labeled on the chelating agent to obtain a glyconucleotide dual-targeting radioactive molecular probe.

7. The application of a dual-targeting radioactive molecular probe for glyconucleotides as described in any one of claims 1-5, characterized in that, The radioactive molecular probes are used to prepare relevant drugs or kits for the diagnosis or treatment of tumors.

8. The application as described in claim 7, characterized in that, The tumor is bladder cancer.

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