PET and NIR bimodal diagnosis and treatment integrated molecular probe as well as preparation and application thereof
By using a PET/NIR dual-modal diagnostic and therapeutic molecular probe, combined with PET imaging and fluorescence imaging, targeting the CD206 receptor on M2 tumor-associated macrophages, the limitations of a single modality in bladder cancer diagnosis and treatment have been overcome, enabling precise diagnosis and treatment, especially with significant effects in intraoperative tumor boundary localization and treatment of multiple metastatic tumors.
Patent Information
- Application Number
- CN202610117602.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-28
- Publication Date
- 2026-05-05
AI Technical Summary
In the current technology, the single imaging mode in the clinical treatment of bladder cancer limits doctors from developing personalized and precise treatment plans, and the accuracy and sensitivity of tumor imaging are not high, especially in the assessment of tumor margins during surgery and the treatment of multiple metastatic tumors.
A PET/NIR dual-modal diagnostic and therapeutic molecular probe was developed, which combines PET imaging and fluorescence imaging to achieve precise preoperative diagnosis and intraoperative localization by targeting the CD206 receptor on M2 tumor-associated macrophages. It can also be used in combination with therapeutic radionuclides for radiotherapy of multi-metastatic tumors.
It improves the accuracy and sensitivity of tumor imaging, enables preoperative PET/CT system diagnosis to guide surgical routes, allows for precise localization of tumor boundaries through intraoperative fluorescence imaging, and can be used for radiotherapy of multiple metastatic tumors, thus improving treatment outcomes.
Smart Images
Figure CN121974975A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of medical imaging technology, and in particular to a dual-modal molecular probe for diagnosis and treatment using PET and NIR, its preparation, and its application. Background Technology
[0002] Currently, the clinical treatment of bladder cancer primarily involves surgical resection, supplemented by chemotherapy, radiotherapy, and immunotherapy. Positive surgical margins and missed diagnoses are among the main causes of high recurrence rates in bladder cancer, resulting in significant psychological and financial burdens for patients. Therefore, early, comprehensive, accurate, and dynamic detection of primary and metastatic lesions of bladder cancer is crucial for guiding accurate clinical staging, prognosis assessment, treatment selection, and evaluation of treatment efficacy.
[0003] Compared to targeting tumor cells, targeting tumor-associated macrophages (TAMs), which are more numerous, have stronger cellular uptake capabilities, and are distributed near tumor blood vessels, is more advantageous. Among them, M2-type TAMs are closely related to the occurrence and development of cancer, and designing molecular probes targeting M2-type TAMs is a highly promising diagnostic and therapeutic approach. The CD206 receptor is a biomarker of M2-type TAMs, which is highly expressed at the lesion site and is highly correlated with the malignancy of bladder cancer, making it a potential target for probe design.
[0004] The limitations of a single imaging modality restrict physicians from developing personalized and precise clinical treatment plans for patients. Therefore, integrating the advantages of different imaging technologies and developing multimodal clinical approaches can overcome the limitations of a single modality, thereby improving the accuracy and sensitivity of diagnosis. Summary of the Invention
[0005] This invention provides a PET / NIR dual-modal therapeutic molecular probe for tumors. This probe features visualization of dynamic changes in lesions, enabling preoperative PET imaging and providing precise imaging references for intraoperative tumor margin assessment. Furthermore, the probe can be used for intraoperative fluorescence surgical navigation, combined with preoperative PET imaging, to accurately trace tumor boundaries during surgery. After chelating therapeutic radionuclides, this dual-modal therapeutic molecular probe can also be used for radiotherapy of inoperable sites in multi-metastatic tumors. The probe in this invention improves the accuracy and sensitivity of in vivo imaging, thereby overcoming the limitations of existing single-modal tumor imaging technologies, which suffer from low accuracy and sensitivity.
[0006] According to a first aspect of the present invention, a PET / NIR dual-modal diagnostic and therapeutic molecular probe is provided, the structure of which is shown in Formula I:
[0007] ; Formula I Wherein, R is a nuclide chelating group; F is a fluorescent group.
[0008] Preferably, the radionuclide chelating group is DOTA or NOTA, with the following structural formula: .
[0009] Preferably, the fluorescent group is a near-infrared fluorescent group.
[0010] Preferably, the fluorescent group is a group formed from ICG, Cy7, Cy7.5, IR780, IR800, IR-820 or FD1080.
[0011] According to another aspect of the present invention, a method for preparing the PET / NIR dual-modal diagnostic and therapeutic molecular probe is provided, comprising the following steps: (1) The fluorescent molecule was subjected to an amino coupling reaction with the compound N-α-fluorenylmethoxycarbonyl-N-ε-tert-butoxycarbonyl-L-lysine, and then the 9-fluorenylmethoxycarbonyl group was removed. The compound was purified by silica gel column chromatography to obtain compound 1. (2) Compound 1 was subjected to an amino coupling reaction with azidoacetic acid, and then the tert-butyloxycarbonyl group was removed. The compound 2 was purified by silica gel column chromatography. (3) Compound 2 was coupled with a radionuclide chelating agent and then dialyzed to obtain compound 3; (4) Compound 3 was subjected to a click chemistry reaction with the DBCO-modified M2pep polypeptide, the amino acid composition of which is shown in SEQ ID NO:1, and the structural formula of the DBCO is as follows. After purification by high performance liquid chromatography, a PET / NIR dual-modal diagnostic and therapeutic molecular probe was obtained.
[0012] According to another aspect of the present invention, the application of a PET / NIR dual-modal diagnostic and therapeutic molecular probe in the preparation of tumor imaging agents is provided.
[0013] Preferably, the imaging is PET imaging and / or fluorescence imaging.
[0014] Preferably, the molecular probe is labeled with a radionuclide before being used for imaging; Preferably, the radionuclide is a diagnostic radionuclide or a therapeutic radionuclide; Preferably, the diagnostic radionuclide is... 68 Ga、 64 Cu、 18 F, 86 Y、 89 Zr or 111 In; the therapeutic radionuclide is 177 Lu、 161 Tb,211 At、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bior 212 Pb.
[0015] Preferably, the tumor is a bladder cancer tumor, a breast cancer tumor, or a glioblastoma; Preferably, the tumor is a bladder cancer tumor.
[0016] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages: (1) This invention develops a type of PET / NIR dual-modal diagnostic and therapeutic molecular probe for tumors, which is a molecular probe of the CD206 receptor on the surface of M2 tumor-associated macrophages. This probe has the characteristics of rapid tumor imaging, long-term retention within the tumor, and rapid clearance from normal organs.
[0017] (2) A PET / NIR dual-modal diagnostic and therapeutic molecular probe of the present invention is preferably used for the precision diagnosis and treatment of bladder cancer. The probe can be used for preoperative PET / CT imaging to systematically diagnose lesions, guide the planning of surgical routes, and accurately locate tumor boundaries during intraoperative fluorescence imaging, thereby improving postoperative efficacy. The probe can also be used for radiotherapy of unresectable sites in multi-metastatic tumors (such as bladder cancer) after chelating therapeutic radionuclides. Attached Figure Description
[0018] Figure 1 This is an LC-MS result diagram of ICG-DOTA-M2pep in Embodiment 1 of the present invention.
[0019] Figure 2 This is the result of in vitro cell targeting in Example 2 of the present invention.
[0020] Figure 3 This is an NIR-II fluorescence imaging of mice with subcutaneous bladder tumors in Example 3 of the present invention.
[0021] Figure 4 This is the in vivo blocking result of subcutaneous bladder tumor in mice in Example 4 of the present invention.
[0022] Figure 5 This is the surgical navigation of mice with subcutaneous bladder tumors under NIR-II fluorescence guidance in Example 5 of the present invention. Detailed Implementation
[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.
[0024] This invention discloses a PET / NIR dual-modal diagnostic and therapeutic molecular probe for bladder cancer, having the structure shown in Formula I.
[0025] Formula I Wherein, R is a nuclide chelating group; F is a fluorescent group.
[0026] Preferably, the radionuclide chelating group is a group formed by a bifunctional chelating agent, and the bifunctional chelating agent is either DOTA or NOTA.
[0027] Preferably, the fluorescent group is a near-infrared fluorescent group, including but not limited to at least one of indocyanine green (ICG), Cy7, Cy7.5, IR780, IR800, IR-820, and FD1080. Near-infrared dyes are more suitable for surgical navigation due to their advantages such as longer emission wavelengths, deeper tissue penetration, and less fluorescence scattering.
[0028] Preferably, the CD206-targeted dual-modal molecular probe ICG-DOTA-M2pep has the structure shown in Formula II;
[0029] Formula II The present invention provides a synthetic route for the ICG-DOTA-M2pep probe with the structure shown in Formula II, specifically including the following steps:
[0030] (1) Compound ICG-NH2 was subjected to an amino coupling reaction with compound N-α-fluorenylmethoxycarbonyl-N-ε-tert-butoxycarbonyl-L-lysine. After washing, 9-fluorenylmethoxycarbonyl was removed directly by 20% piperidine solution, and the compound ICG-Lys-Boc-NH2 was purified by silica gel column chromatography. (2) The compound ICG-Lys-Boc-NH2 was subjected to an amino coupling reaction with the compound azidoacetic acid. After washing, the compound was directly de-tert-butyloxycarbonylated by 50% trifluoroacetic acid solution and purified by silica gel column to obtain the compound ICG-Lys-NH2-N3. (3) The compound ICG-Lys-NH2-N3 was subjected to an amino coupling reaction with the compound DOTA-NHS, and after dialysis, the compound ICG-DOTA-N3 was obtained; (4) The compound ICG-DOTA-N3 was subjected to a click chemical reaction with the polypeptide DBCO-M2pep. The amino acid sequence of the M2pep polypeptide is shown in SEQ ID NO:1 (YEQDPWGVKWWY). After purification by high performance liquid chromatography (HPLC), the compound ICG-DOTA-M2pep was obtained.
[0031] This invention relates to a PET / NIR dual-modal diagnostic and therapeutic molecular probe for tumors, which is obtained by radionuclide labeling.
[0032] Preferably, the radionuclide can be a diagnostic radionuclide or a therapeutic radionuclide.
[0033] Preferably, the diagnostic radionuclide includes, but is not limited to, those used in diagnostics. 68 Ga、 64 Cu、 18 F, 86 Y、 89 Zr and 111 At least one of In; the therapeutic radionuclide includes, but is not limited to, 177 Lu、 161 Tb, 211 At、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.
[0034] In some preferred embodiments, the radionuclide is preferably... 68 Ga、 64 Cu、 89 Zr and 177 Any one of Lu.
[0035] The tumor PET / NIR dual-modal diagnostic and therapeutic molecular probe of this invention can be used for any one of bladder cancer, breast cancer, and glioblastoma. It is preferably used for bladder cancer.
[0036] The following are specific embodiments. Example 1 The synthesis of ICG-DOTA-M2pep: HATU, DIPEA, ICG-NH2, and N-α-fluorenemethoxycarbonyl-N-ε-tert-butyloxycarbonyl-L-lysine were dispersed in 10 mL of DMF at a molar ratio of 1.5:3:1:1.2 and stirred for 24 h. The mixture was then concentrated and redispersed in CH2Cl2, and washed with NaHCO3 and HCl. Extraction was then performed with CH2Cl2, followed by drying with anhydrous magnesium sulfate. No further purification was required; the 9-fluorenemethoxycarbonyl group was removed by stirring in 20% piperidine solution for 20 min. Finally, purification was achieved by elution with a silica gel column using a mixed solvent of CH2Cl2 and MeOH. The resulting product was ICG-Lys-Boc-NH2. HATU, DIPEA, azidoacetic acid, and ICG-Lys-Boc-NH2 were dispersed in 10 mL of DMF at a molar ratio of 1.5:3:1.2:1 and stirred for 24 h. The mixture was concentrated, redispersed in CH2Cl2, and washed with NaHCO3 and HCl. Extraction was then performed with CH2Cl2, followed by drying with anhydrous magnesium sulfate. No further purification was required; the product was directly purified by stirring in 50% trifluoroacetic acid solution for 0.5 h to remove the tert-butyloxycarbonyl group. Finally, purification was achieved by elution with a silica gel column using a mixed solvent of CH2Cl2 and MeOH. The resulting product was ICG-Lys-NH2-N3. DIPEA, DOTA-NHS and ICG-Lys-NH2-N3 were dispersed in 10 mL of DMF at a molar ratio of 3:1.2:1 and stirred for 12 h. After concentration and dialysis purification, the product obtained was ICG-DOTA-N3. ICG-DOTA-N3 and DBCO-M2pep were dispersed in 10 mL of DMSO at a molar ratio of 1.2:1 and stirred for 12 h. After purification by HPLC, the product obtained was ICG-DOTA-M2pep. The results of liquid chromatography-mass spectrometry (LC-MS) are as follows: Figure 1 As shown.
[0037] Example 2 In vitro cell targeting experiments: RAW264.7 cells were treated with 20 ng / mL IL-4 for 24 hours to obtain M2-type TAMs. Different samples were diluted with DMEM medium to a final ICG concentration of 10 μM and co-incubated with M2-type TAMs for 1, 2, or 4 hours. Intracellular ICG fluorescence was then analyzed by flow cytometry. To further verify targeting specificity, M2-type TAMs were co-incubated with 5 μg / mL M2pep for 4 hours, and then the medium was replaced with ICG-DOTA-M2pep. After another 4 hours of co-incubation, analysis was performed by flow cytometry. In vitro cell targeting results are shown below. Figure 2 As shown.
[0038] Example 3 NIR-II in vivo fluorescence imaging: Mice were anesthetized with 2% isoflurane gas throughout the procedure. Following intravenous injection of either Free ICG or ICG-DOTA-M2pep (ICG = 0.2 mM, 0.1 mL) into the tail vein of mice bearing subcutaneous bladder tumors, NIR-II in vivo fluorescence imaging was performed at 5 min, 1 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h. Imaging conditions: 808 nm laser, 75 mW / cm² laser power. 2 The exposure time was 200 ms, and a 1300 nm long-pass filter was used. The NIR-II in vivo fluorescence imaging results are as follows: Figure 3 As shown.
[0039] Example 4 In vivo blocking experiments: Two groups of mice bearing subcutaneous bladder cancer were administered equal amounts of ICG-DOTA-M2pep (ICG = 0.2 mM, 0.1 mL) via tail vein injection. For the M2pep blocking experiment, unlabeled M2pep (350 μg, approximately 100 times the dose of ICG-DOTA-M2pep) was injected into the mice via tail vein one hour before the ICG-DOTA-M2pep injection. NIR-II in vivo fluorescence imaging conditions and parameters were the same as in Example 3. The in vivo blocking experiment results are as follows... Figure 4 As shown.
[0040] Example 5 Surgical navigation for bladder tumors in mice: Bladder cancer cells were subcutaneously implanted on the lateral hind limb of mice. When the tumors grew to 4-6 mm, ICG-DOTA-M2pep (ICG = 0.2 mM, 0.1 mL) was injected via the tail vein. At the optimal imaging time of 48 hours, the bladder tumors were surgically removed with the assistance of an NIR-II imaging device. The NIR-II in vivo fluorescence imaging conditions and parameters were the same as in Example 3. The results of the bladder tumor surgical navigation experiment in mice are as follows: Figure 5 As shown.
[0041] The ICG-DOTA-M2pep prepared in this implementation case was characterized and tested, and the results are as follows: Figures 1 to 5 As shown.
[0042] See Figure 1 LC-MS results showed that the ICG-DOTA-M2pep bimodal molecule was successfully synthesized.
[0043] See Figure 2Cellular uptake results showed that ICG-DOTA-M2pep uptake in M2-type TAMs was much higher than that in the FreeICG group.
[0044] See Figure 3 NIR-II in vivo fluorescence imaging results showed that ICG-DOTA-M2pep rapidly accumulated in mouse tumors and remained there for a long time, while being rapidly cleared from normal organs.
[0045] See Figure 4 The blocking results showed that ICG-DOTA-M2pep mainly achieves rapid accumulation in tumors by actively targeting the CD206 receptor on M2-type TAMs through M2pep.
[0046] See Figure 5 Surgical navigation results showed that after ICG-DOTA-M2pep was injected into the tail vein, the tumor was completely removed under NIR-II fluorescence guidance, the resection margin was clear, the mice recovered well after surgery, and there was no recurrence.
[0047] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A PET / NIR dual-modal diagnostic and therapeutic molecular probe, characterized in that, The structure of the probe is shown in Formula I: ； Formula I Wherein, R is a nuclide chelating group; F is a fluorescent group.
2. The PET / NIR dual-modal diagnostic and therapeutic molecular probe as described in claim 1, characterized in that, The chelating group of the radionuclide is DOTA or NOTA, with the following structural formula: 。 3. The PET / NIR dual-modal diagnostic and therapeutic molecular probe as described in claim 1, characterized in that, The fluorescent group is a near-infrared fluorescent group.
4. The PET / NIR dual-modal diagnostic and therapeutic molecular probe as described in claim 3, characterized in that, The fluorescent group is a group formed from ICG, Cy7, Cy7.5, IR780, IR800, IR-820 or FD1080.
5. The method for preparing the PET / NIR dual-modal diagnostic and therapeutic molecular probe according to any one of claims 1-4, characterized in that, Includes the following steps: (1) The fluorescent molecule was subjected to an amino coupling reaction with the compound N-α-fluorenylmethoxycarbonyl-N-ε-tert-butoxycarbonyl-L-lysine, and then the 9-fluorenylmethoxycarbonyl group was removed. The compound was purified by silica gel column chromatography to obtain compound 1. (2) Compound 1 was subjected to an amino coupling reaction with azidoacetic acid, and then the tert-butyloxycarbonyl group was removed. The compound 2 was purified by silica gel column chromatography. (3) Compound 2 was coupled with a radionuclide chelating agent and then dialyzed to obtain compound 3; (4) Compound 3 was subjected to a click chemistry reaction with the DBCO-modified M2pep polypeptide, the amino acid composition of which is shown in SEQ ID NO:1, and the structural formula of the DBCO is as follows. After purification by high performance liquid chromatography, a PET / NIR dual-modal diagnostic and therapeutic molecular probe was obtained.
6. The application of the PET / NIR dual-modal diagnostic and therapeutic molecular probe as described in any one of claims 1-4 in the preparation of tumor imaging agents.
7. The application as described in claim 6, characterized in that, The imaging is PET imaging and / or fluorescence imaging.
8. The application as described in claim 6 or 7, characterized in that, The molecular probe is labeled with a radionuclide before being used for imaging.
9. The application as described in claim 8, characterized in that, The radionuclide is a diagnostic radionuclide or a therapeutic radionuclide; Preferably, the diagnostic radionuclide is... 68 Ga、 64 Cu、 18 F, 86 Y、 89 Zr or 111 In; the therapeutic radionuclide is 177 Lu、 161 Tb, 211 At、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bior 212 Pb.
10. The application as described in claim 6 or 7, characterized in that, The tumor is either bladder cancer, breast cancer, or glioblastoma. Preferably, the tumor is a bladder cancer tumor.