B7-H3-based bimodal imaging probe as well as preparation method and application thereof

Abstract

The invention discloses a bimodal imaging probe based on B7-H3 and a preparation method and application thereof.The bimodal imaging probe comprises a B7-H3 affinity body, the C end of the B7-H3 affinity body is connected with near-infrared fluorescent dye IR-Dye 800CW, the N end of the B7-H3 affinity body is connected with a Gd-DOTA chelate, the Gd-DOTA chelate is chelated with gadolinium ions Gd < 3 + > for magnetic resonance imaging, and the B7-H3 affinity body is connected with a fluorescent dye IR-Dye 800CW. The near-infrared fluorescent dye IR-Dye 800CW is used for near-infrared two-region fluorescence imaging. The bimodal imaging probe integrates the advantages of magnetic resonance imaging and near-infrared two-region fluorescence imaging, can be used for both magnetic resonance imaging and near-infrared two-region fluorescence imaging, and overcomes the limitation of a single detection means.

Description

Technical Field

[0001] The present application generally relates to the technical field of biomedicine. More specifically, the present application relates to a B7-H3-based dual-modality imaging probe, preparation method, and use. Background Art

[0002] Prostate cancer is one of the most common malignancies in men worldwide. Currently, its diagnosis relies primarily on invasive prostate biopsy. However, noninvasive, highly sensitive, and accurate detection methods are a hot topic in current research. While existing noninvasive methods such as prostate MRI and PET-CT have some accuracy, they also have limitations. Prostate MRI has an accuracy of approximately 80%, while PSMA-PET approaches 90%. However, PSMA expression decreases during prostate cancer progression, affecting diagnostic sensitivity.

[0003] In terms of treatment, radical prostatectomy is the main treatment for intermediate- and high-risk localized prostate cancer. However, postoperative problems such as positive surgical margins and complications can occur. Therefore, finding new methods to assist with precise resection during surgery is clinically critical. Previous studies have used intraoperative injection of indocyanine green (ICG) for real-time fluorescence imaging to identify prostate cancer lesion boundaries and positive lymph nodes. However, due to the lack of tumor specificity of ICG, it is difficult to accurately delineate boundaries and cannot meet practical needs. Near-infrared zone II fluorescence molecular imaging uses targeted fluorescent probe imaging to visualize tumors during surgery.

[0004] At present, a single imaging method is difficult to meet clinical needs. Magnetic resonance imaging has high resolution and deep tissue penetration capabilities, but it is expensive and has limited display of fine structures; near-infrared second-zone fluorescence imaging has high sensitivity and high temporal resolution, but has limited tissue penetration depth. Therefore, developing a technology that integrates the advantages of multiple imaging methods to overcome the limitations of a single detection method is an urgent problem that needs to be solved. In addition, existing fluorescent probes also have deficiencies in specificity, stability, and imaging effects, which make it difficult to meet the clinical needs for accurate diagnosis and treatment of prostate cancer. Summary of the Invention

[0005] In order to at least solve one or more of the technical problems mentioned above, the present application proposes a dual-modal imaging probe based on B7-H3, a preparation method and a use scheme in multiple aspects.

[0006] In a first aspect, the present application provides a dual-modality imaging probe based on B7-H3, wherein the dual-modality imaging probe comprises a B7-H3 affibody, wherein the C-terminus of the B7-H3 affibody is connected to a near-infrared fluorescent dye IR-Dye800CW, and the N-terminus of the B7-H3 affibody is connected to a Gd-DOTA chelate, wherein the Gd-DOTA chelate chelates the gadolinium ion Gd 3+For magnetic resonance imaging, the near-infrared fluorescent dye IR-Dye800CW is used for near-infrared second-zone fluorescence imaging.

[0007] In some embodiments, the structure of the dual-modality imaging probe is as follows:

[0008]

[0009] In other embodiments, the B7-H3 affibody has a molecular weight of 8 kDa.

[0010] In yet other embodiments, the dual-modality imaging probe has an excitation wavelength of 782 nm and an emission wavelength of 807 nm.

[0011] In some embodiments, the T1 relaxation time of the dual-modality imaging probe in an aqueous solution gradually decreases as the concentration of the dual-modality imaging probe increases.

[0012] In a second aspect, the present application provides a preparation method for the aforementioned dual-modal imaging probe, comprising the following steps: dissolving DOTA-NHS and B7-H3 affinity in an organic solvent, adding triethylamine, and stirring the reaction under an inert gas atmosphere to obtain a first intermediate having DOTA connected to the N-terminus of the B7-H3 affinity; dissolving the first intermediate and IRDye800CW-maleimide in an aqueous solution containing an organic solvent, and stirring the reaction under an inert gas atmosphere to obtain a second intermediate having IR-Dye800CW connected to the C-terminus of the B7-H3 affinity; dissolving the second intermediate and GdCl3 in deionized water, and reacting under stirring conditions to obtain a dual-modal imaging probe based on B7-H3.

[0013] In other embodiments, the organic solvent is N,N-dimethylformamide; and / or the inert gas comprises nitrogen; and / or the second intermediate and GdCl3 are dissolved in deionized water at a pH of 6.0.

[0014] In some other embodiments, the preparation method further comprises: purifying the first intermediate after obtaining the first intermediate; and / or purifying the second intermediate after obtaining the second intermediate; and / or purifying the dual-modal imaging probe.

[0015] In a third aspect, the present application provides a use of the aforementioned dual-modality imaging probe in preparing a prostate cancer diagnostic reagent.

[0016] In some embodiments, the diagnostic agent is used for at least one of: preoperative tumor localization by magnetic resonance imaging; and / or real-time navigation of prostate cancer tumor boundaries during surgery; and / or screening patients suitable for B7-H3 targeted therapy for prostate cancer.

[0017] Through the B7-H3-based dual-modal imaging probe provided above, IR-Dye800CW and Gd-DOTA chelate are respectively connected to the C-terminus and N-terminus of the B7-H3 affinity body, thereby integrating the advantages of magnetic resonance imaging and near-infrared second-zone fluorescence imaging. It can be used for both magnetic resonance imaging and near-infrared second-zone fluorescence imaging, overcoming the limitations of a single detection method. Therefore, it can be used for precise positioning of prostate cancer tumors before surgery, improving the diagnostic accuracy of prostate cancer, and real-time guidance of surgical resection during surgery, helping to accurately remove tumors and reduce complications. It can also be used to screen patients suitable for B7-H3 targeted therapy. The dual-modal imaging probe of the embodiment of the present application has the characteristics of high specificity and stability, as well as good biocompatibility and operability, and has significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and other objects, features and advantages of the exemplary embodiments of the present application will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present application are shown in an exemplary and non-limiting manner, and the same or corresponding reference numerals represent the same or corresponding parts, wherein:

[0019] Figure 1 shows a structural diagram of a dual-modality imaging probe according to an embodiment of the present application;

[0020] Figure 2 shows an excitation spectrum diagram of a dual-modality imaging probe according to an embodiment of the present application;

[0021] Figure 3 shows an emission spectrum diagram of a dual-modality imaging probe according to an embodiment of the present application;

[0022] Figure 4 shows a T1 relaxation time variation trend diagram of the dual-modality imaging probe according to an embodiment of the present application;

[0023] Figure 5 shows magnetic resonance imaging images of mice injected with a dual-modality imaging probe;

[0024] Figure 6 Shown are near-infrared second-zone fluorescence images of mice injected with the dual-modality imaging probe. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of this application.

[0026] It should be understood that the terms "include" and "comprising" used in the description and claims of this application indicate the presence of described features, wholes, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or collections thereof.

[0027] It should also be understood that the terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit this application. As used in this specification and claims, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. It should also be further understood that the term "and / or" as used in this specification and claims refers to any and all possible combinations of one or more of the associated listed items, including and including these combinations.

[0028] As used in this specification and claims, the term "if" can be interpreted as "when" or "upon" or "in response to determining" or "in response to detecting," depending on the context. Similarly, the phrase "if it is determined" or "if [described condition or event] is detected" can be interpreted as meaning "upon determination" or "in response to determining" or "upon detection of [described condition or event]" or "in response to detecting [described condition or event]," depending on the context.

[0029] The applicant has discovered that B7-H3, as an immunomodulatory glycoprotein that is specifically and highly expressed in prostate cancer, has important clinical value. Antibody-drug conjugates targeting B7-H3 have also shown good results in early clinical trials. Therefore, enhanced MR examinations carrying B7-H3 molecular markers are expected to significantly improve the detection rate of prostate cancer. At the same time, near-infrared zone II fluorescence molecular imaging can be used for tumor visualization during surgery. If it can be combined with the special value of B7-H3 in prostate cancer, it will bring opportunities for precise surgery of prostate cancer. Based on this, the present application aims to provide a dual-modal imaging probe that can effectively integrate enhanced MR imaging with near-infrared zone II fluorescence imaging technology with high sensitivity and high temporal resolution, so as to overcome the limitations of a single detection method and meet the clinical needs for precise diagnosis and treatment of prostate cancer. The specific implementation methods of the present application are described in detail below with reference to the accompanying drawings.

[0030] In a first aspect, the present application provides a dual-modality imaging probe based on B7-H3, which comprises a B7-H3 affibody, wherein the C-terminus of the B7-H3 affibody is connected to a near-infrared fluorescent dye IR-Dye800CW, and the N-terminus of the B7-H3 affibody is connected to a Gd-DOTA chelate, wherein the Gd-DOTA chelate chelates the gadolinium ion Gd. 3 +For magnetic resonance imaging, the near-infrared fluorescent dye IR-Dye800CW is used for near-infrared second-zone fluorescence imaging.

[0031] B7-H3 is an immunomodulatory glycoprotein highly expressed in prostate cancer. It is closely associated with processes such as cancer immune escape and accelerated tumor growth, making it a key target for prostate cancer diagnosis and treatment. B7-H3 Affibodies are molecules that bind to the B7-H3 protein specifically expressed on the surface of prostate cancer cells.

[0032] In some embodiments, the sequence of the B7-H3 Affibody is:

[0033] AEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQGGG C (SEQ ID NO: 1).

[0034] The dual-modal imaging probe prepared from the B7-H3 affibody can be represented as:

[0035] (Gd-DOTA)2-KAEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQGGGC-(IRDye 800CW Mal) (SEQ ID NO: 2).

[0036] The N-terminus and C-terminus of the B7-H3 Affibody are two important parts in its molecular structure. The N-terminus generally refers to the amino terminus of a protein or polypeptide chain, while the C-terminus is the carboxyl terminus, i.e., the N-terminus has an amino group (-NH2) and the C-terminus has a carboxyl group (-COOH). In the present application, the C-terminus of the B7-H3 Affibody is connected to a near-infrared fluorescent dye IR-Dye800CW, while the N-terminus is connected to a Gd-DOTA chelate. This connection method is usually achieved by covalently binding IR-Dye800CW and Gd-DOTA chelate to the corresponding endpoints of the B7-H3 Affibody.

[0037] The N-terminus and C-terminus of the B7-H3 affibody have different chemical properties. In comparison, the C-terminus may, in some cases, be involved in the folding, stability, and formation of the binding site of the molecule. If a larger molecule (such as a Gd-DOTA chelate) is attached to the C-terminus, the three-dimensional structure of the B7-H3 affibody may be altered, thereby affecting the binding ability of the B7-H3 affibody to the B7-H3 protein. Therefore, the Gd-DOTA chelate is attached to the N-terminus of the B7-H3 affibody, and the C-terminus is attached to the highly stable fluorescent group IRDye800CW to avoid affecting the binding activity of the B7-H3 affibody to the B7-H3 protein, thereby reducing interference with the structure and function of the B7-H3 affibody, ensuring the effectiveness of fluorescence imaging, and ensuring that the dual-modal imaging probe can effectively bind to the target protein.

[0038] Gd-DOTA chelate is a magnetic resonance imaging (MRI) contrast agent. DOTA is a chelating agent that can bind to gadolinium ions (Gd 3 +) stably binds. Gadolinium ion is a paramagnetic contrast agent commonly used in clinical MRI. In this application, Gd-DOTA chelate is connected to the N-terminus of B7-H3 affinity body, by chelating gadolinium ion Gd 3 +, so that the dual-modal imaging probe has magnetic resonance imaging function. In some embodiments, the N-terminus of the B7-H3 affibody can be connected to multiple Gd-DOTA chelates, which is beneficial to improving the magnetic resonance imaging effect. The spatial structure of multiple Gd-DOTA chelates is relatively large. In this application, by connecting them to the N-terminus of the B7-H3 affibody instead of the C-terminus, it will not affect the binding activity of the B7-H3 affibody to the B7-H3 protein. Different groups are connected to the two ends of the B7-H3 affibody, and the groups will not affect each other and affect the imaging function.

[0039] In some embodiments, the structure of the dual-modality imaging probe is as follows:

[0040]

[0041] In other embodiments, the structure of the dual-modality imaging probe is as follows:

[0042]

[0043] In other embodiments, the B7-H3 affibody has a molecular weight of 8 kDa (kilodaltons). This relatively small molecular weight enables the B7-H3 affibody to have good tissue permeability and stability in vivo, enabling rapid distribution and penetration of tissues, facilitating timely intraoperative imaging. Furthermore, the affibody is easy to manipulate during synthesis and purification, facilitating large-scale production and clinical application.

[0044] Figure 1FIG. 1 shows a structural diagram of a dual-modality imaging probe according to an embodiment of the present application. Figure 1 As shown in , the dual-modal imaging probe includes a B7-H3 affibody 101, a near-infrared fluorescent dye IR-Dye800CW102, and a Gd-DOTA chelate 103. The specific structures of the near-infrared fluorescent dye IR-Dye800CW102 and the Gd-DOTA chelate 103 can be found in Figure 1 The partially enlarged structure is shown in the dotted box. Figure 1 As can be seen in the figure, the Gd-DOTA chelate 103 can be bonded to the N-terminus of the B7-H3 affibody 101 via an amide bond, and the near-infrared fluorescent dye IR-Dye800CW102 can form a thioether bond with the C-terminus of the B7-H3 affibody 101 to achieve a bonding connection.

[0045] Excitation and emission spectroscopy tests:

[0046] In this test case, the FLS980 spectrometer was used to place the dual-modal imaging probe solution in the sample cell. The appropriate excitation wavelength was then selected through a monochromator, and the dual-modal imaging probe was excited using a xenon lamp or laser. The fluorescence emitted by the dual-modal imaging probe was collected, split by another monochromator, and finally the fluorescence intensity was detected by a photomultiplier tube, and the emission spectra at different wavelengths were recorded. At the same time, the software automatically plotted the excitation spectrum to show the excitation and emission characteristics of the dual-modal imaging probe. The test results are as follows: Figure 2 and Figure 3 shown. Figure 2 shows an excitation spectrum diagram of a dual-modality imaging probe according to an embodiment of the present application; Figure 3 The emission spectrum of the dual-modality imaging probe according to the embodiment of the present application is shown.

[0047] The excitation wavelength refers to the wavelength of light used to excite the fluorescent dye to emit fluorescence. Figure 2 As shown in the figure, the excitation wavelength of the dual-modality imaging probe of the embodiment of the present application is 782nm. This means that when the dual-modality imaging probe is illuminated by 782nm light, the IR-Dye800CW molecules in the dual-modality imaging probe absorb the light energy of this wavelength, thereby entering an excited state. The 782nm excitation light can effectively excite the IR-Dye800CW, causing it to emit bright fluorescence, providing a good signal foundation for subsequent fluorescence imaging.

[0048] The emission wavelength refers to the wavelength of fluorescence emitted by the fluorescent dye after being excited. Figure 3As shown in , the emission wavelength of the dual-modal imaging probe of the embodiment of the present application is 807nm, indicating that the IR-Dye800CW in the dual-modal imaging probe will emit fluorescence with a wavelength of 807nm under 782nm light excitation. The dual-modal imaging probe also has a high fluorescence signal in the near-infrared second band (900-1200nm). The fluorescence in this band has a large penetration depth in biological tissues and can achieve high-resolution imaging of deep tissues. In addition, the 807nm fluorescence signal has low scattering and absorption in the tissue, which can produce clearer images, helping to accurately identify tumor boundaries and positive lymph nodes in real-time navigation during surgery, providing doctors with clear visual guidance and improving the accuracy and success rate of surgery.

[0049] In intraoperative application scenarios, by using 782nm excitation light irradiation, the fluorescence signal of the near-infrared second band of 900-1200nm can be detected in real time, providing doctors with real-time tumor boundary and positive lymph node location information, helping doctors to more accurately remove tumor tissue while preserving normal tissue as much as possible, reducing surgical complications, and improving patients' postoperative quality of life.

[0050] T1 relaxation time test:

[0051] In nuclear magnetic resonance imaging, the T1 relaxation time is the time required for the longitudinal magnetization intensity to return to the equilibrium state. In this test example, a probe solution with a gradient concentration range of 0 to 0.4 mM (mmol / L) was prepared. Specific concentrations may include 0 mM (blank control), 0.1 mM, 0.2 mM, 0.3 mM, and 0.4 mM. Deionized water or physiological saline is usually used as a solvent in the probe solution to ensure that the pH value and ionic strength of the probe solution are consistent with the actual application conditions. Use a standard NMR sample tube to ensure that the inner diameter and length of the sample tube are suitable for the probe of the MRI instrument. The prepared probe solutions of different concentrations are placed in the sample tubes separately, ensuring that the amount of solution in each sample tube is the same and there are no bubbles.

[0052] Use an MRI machine with a field strength of 7.0T or higher to ensure adequate resolution and sensitivity. Select a T1-weighted imaging sequence, such as spoiled gradient recalled echo (SPGR) or fast low angle shot (FLASH). Set the following imaging parameters: (1) Repetition time (TR): Set to a longer time, such as 500-1000 ms, to ensure sufficient T1 relaxation; (2) Echo time (TE): Set to a shorter time, such as 2-5 ms, to reduce the influence of T2 relaxation; (3) Flip angle (FA): Select an appropriate flip angle, such as 90° or 30°, to optimize signal intensity; (4) Matrix size: Set to 256×256 or higher to ensure image resolution; (5) Field of view (FOV): Set an appropriate field of view, such as 20-30 mm, based on the size of the sample tube.

[0053] Place sample tubes containing probe solutions of varying concentrations sequentially in the MRI instrument's probe, ensuring consistent positioning of the sample tubes to minimize the impact of positional differences on the measurement results. Start the MRI instrument and acquire a T1-weighted image for each sample. Ensure consistent imaging conditions for each sample to ensure data comparability. Calculate the T1 relaxation time using the following formula:

[0054] S=S0(1-e -TR / T1 )

[0055] (Formula 1);

[0056] Where S is the measured signal intensity, S0 is the maximum signal intensity (usually measured when TR is much greater than T1), TR is the repetition time, and T1 is the T1 relaxation time.

[0057] Substitute the signal intensity data at different TR values into the above formula and calculate the T1 relaxation time using a nonlinear fitting method (such as the least squares method). Figure 4 As shown in , the T1 relaxation time of the dual-modal imaging probe in aqueous solution gradually shortens with the increase of the concentration of the dual-modal imaging probe, which indicates that the Gd-DOTA chelate of the dual-modal imaging probe can effectively shorten the T1 relaxation time of the aqueous solution and enhance the MRI signal.

[0058] Figure 4 r1=10.43s -1 mM -1 It represents the T1 relaxation rate, which is an indicator to measure the relaxation ability of the probe contrast agent. The T1 relaxation rate is related to the concentration and relaxation time of the probe contrast agent, and its calculation formula is:

[0059]

[0060] Where T1 is the relaxation time and C is the concentration of the probe contrast agent. Figure 4 In the middle, r1 = 10.43s -1 mM -1 This means that as the concentration of the probe contrast agent increases, the T1 relaxation time decreases, and this relationship is linear. Specifically, for every 1 mM increase in the probe contrast agent concentration, the T1 relaxation time decreases by 1 / 10.43 ms. This indicates that the probe contrast agent can cause a significant change in the T1 relaxation time at a unit concentration, reflecting the probe's contrast effect. In magnetic resonance imaging, higher r1 values generally indicate better contrast effects because they can achieve significant signal changes at lower contrast agent concentrations, helping to improve image contrast and clarity.

[0061] In a second aspect, the present application provides a preparation method for the aforementioned dual-modal imaging probe, comprising the following steps: dissolving DOTA-NHS and B7-H3 affinity in an organic solvent, adding triethylamine, and stirring the reaction under an inert gas atmosphere to obtain a first intermediate having DOTA connected to the N-terminus of the B7-H3 affinity; dissolving the first intermediate and IRDye800CW-maleimide in an aqueous solution containing an organic solvent, and stirring the reaction under an inert gas atmosphere to obtain a second intermediate having IR-Dye800CW connected to the C-terminus of the B7-H3 affinity; dissolving the second intermediate and GdCl3 in deionized water, and reacting under stirring conditions to obtain a dual-modal imaging probe based on B7-H3.

[0062] In some embodiments, DOTA-NHS and the B7-H3 affibody can be dissolved in an organic solvent at a mass ratio of 2:1. In other embodiments, the organic solvent is N,N-dimethylformamide. In still other embodiments, the inert gas comprises nitrogen. In some embodiments, the second intermediate and GdCl3 are dissolved in deionized water at a pH of 6.0.

[0063] In some other embodiments, the above-mentioned preparation method further comprises: after obtaining the first intermediate, purifying the first intermediate; and / or after obtaining the second intermediate, purifying the second intermediate; and / or purifying the dual-modal imaging probe. In some embodiments, the purification treatment can be performed by preparative high performance liquid chromatography (HPLC). Preparative high performance liquid chromatography (HPLC) purification treatment refers to the process of separating and purifying the target compound in the mixture using HPLC technology. HPLC is a solution-phase-based chromatography technology, the principle of which is to dissolve the mixture to be separated in a mobile phase solution, and to achieve separation of different components through distribution and redistribution processes on the stationary phase.

[0064] Example 1:

[0065] Step 1) DOTA-NHS (10 mg, Xi'an Ruixi Biotechnology Co., Ltd.) and B7-H3 affibody (5 mg, Qiangyao Biotechnology Co., Ltd.) were dissolved in 5 mL of 10% N,N-dimethylformamide (DMF) in deionized water by volume, and then triethylamine (Et3N, 2.6 mg) was added to the solution, and the mixed solution was continuously stirred at 20°C under a nitrogen atmosphere for 12-16 hours; the solvent in the mixed solution was removed under reduced pressure, and the product was purified by preparative high performance liquid chromatography (HPLC) to obtain a first intermediate with DOTA connected to the N-terminus of the B7-H3 affibody.

[0066] Step 2) Dissolve 4.2 mg of the first intermediate and 7.5 mg of IRDye800CW-maleimide (from Xi'an Ruixi Biotechnology Co., Ltd.) in 5 mL of deionized water containing 10% v / v DMF, stir at 20°C under a nitrogen atmosphere for 8-12 hours, evaporate the solvent under reduced pressure, and purify the product by HPLC to obtain a second intermediate with IR-Dye800CW attached to the C-terminus of the B7-H3 affinity body.

[0067] Step 3) Dissolve 4.9 mg of the second intermediate and 6.3 mg of GdCl3·6H2O in deionized water at a pH of 6.0, stir the resulting mixture at 20°C for 72 hours, and then remove the solvent under reduced pressure to obtain a dual-modal imaging probe based on B7-H3. The dual-modal imaging probe can be purified by preparative HPLC to obtain the final product. In addition, by using o-cresol orange as an indicator, it can be confirmed that there is no free Gd in the dual-modal imaging probe. 3+ The presence of ions.

[0068] To facilitate understanding of the above steps, the reaction formula corresponding to each step is listed below:

[0069] Step 1):

[0070]

[0071] Step 2):

[0072]

[0073] Step 3):

[0074]

[0075] In a third aspect, the present application also provides a use of the aforementioned dual-modality imaging probe in the preparation of a prostate cancer diagnostic reagent. In some embodiments, the diagnostic reagent is used for at least one of the following: preoperative tumor localization via magnetic resonance imaging; and / or real-time navigation of prostate cancer tumor boundaries during surgery; and / or screening patients suitable for B7-H3-targeted therapy for prostate cancer.

[0076] Specifically, the dual-modal imaging probe of the present application can provide high-resolution tissue structure information through magnetic resonance imaging (MRI) modality, helping doctors to accurately locate prostate cancer tumors before surgery, clarify the size, location and boundaries of the tumor, and provide an important basis for the formulation of surgical plans.

[0077] In prostate cancer surgery, real-time navigation is crucial to ensure complete tumor resection and reduce damage to normal tissue. Near-infrared zone II fluorescence imaging (NIR-II) has high sensitivity and high temporal resolution, and can provide real-time visualization information of tumor boundaries and positive lymph nodes. The dual-modality imaging probe of this application can guide surgical resection in real time during surgery through the near-infrared zone II fluorescence imaging modality, helping doctors to identify and remove tumor tissue more accurately, while preserving normal tissue as much as possible, reducing surgical complications, and improving the patient's quality of life after surgery.

[0078] B7-H3 is an immunomodulatory glycoprotein that is specifically and highly expressed in prostate cancer and is a key target for the diagnosis and treatment of prostate cancer. By detecting the expression of B7-H3 in patients, patients suitable for B7-H3 targeted therapy can be screened. The dual-modality imaging probe of this application can effectively bind to the B7-H3 protein and determine whether the patient is suitable for B7-H3 targeted therapy based on the imaging results, thereby achieving personalized treatment plans, improving treatment effects, and avoiding unnecessary treatment side effects and economic burdens.

[0079] In summary, the dual-modal imaging probe of the present application not only improves the accuracy of prostate cancer diagnosis by integrating the advantages of magnetic resonance imaging and near-infrared second-zone fluorescence imaging, but also provides an effective means for intraoperative navigation and B7-H3 targeted therapy patient screening, and has significant clinical application value.

[0080] Effect experiment example:

[0081] 1. Cell culture

[0082] Human prostate cancer cells 22RV1 were cultured in RPMI-1640 medium supplemented with 10 wt% fetal bovine serum at 37°C, 5% (volume percentage) CO2, and saturated humidity. Cells were subcultured every 2 to 3 days to ensure cell viability and proliferation, providing a sufficient cell source for subsequent tumor model establishment.

[0083] 2. Tumor model establishment and probe injection

[0084] 5×10 6 The 22RV1 cells were inoculated subcutaneously in the right groin of mice. When the tumors reached week 2 to 3 of growth, the mice were injected with the dual-modality imaging probe prepared in the present invention at a dose of 0.002 mol / kg via the tail vein. This step aims to deliver the dual-modality imaging probe into the mice, allowing it to accumulate in the tumor tissue and prepare for subsequent imaging observations.

[0085] 3. Imaging observation

[0086] Mice injected with the dual-modality imaging probe were observed in a 7.0T magnetic resonance imaging system and a near-infrared zone II in vivo imaging system. Magnetic resonance imaging can obtain high-resolution images of tumor tissue, which can be used to accurately determine the location, size, and boundaries of the tumor. Near-infrared zone II fluorescence imaging can provide real-time visualization of tumor boundaries and positive lymph nodes, facilitating intraoperative navigation and precise tumor resection.

[0087] Figure 5 Magnetic resonance imaging images of mice injected with dual-modality imaging probes are shown. Figure 5 As shown in , T2-weighted imaging (T2WI) before injection shows the actual location of the tumor. Figure 5 The figure also shows the dynamic changes in T1-weighted imaging (T1WI) before (Pre) and 0.5 to 4 hours after injection of the dual-modality imaging probe. Before injection (Pre), the tumor area showed a uniform low signal intensity. 0.5 hours after injection, the tumor boundary began to blur and the signal intensity increased slightly. 1 hour later, scattered high-signal foci appeared within the tumor. At 2 hours, the high-signal area significantly expanded and merged into a single piece. At 4 hours, the overall tumor signal intensity reached its peak, and the contrast with the surrounding normal tissue was significantly enhanced.

[0088] from Figure 5 It can be further seen that the signal in the tumor area gradually increases over time, while normal tissues such as muscle and fat always maintain low signals, confirming that the dual-modality imaging probe achieves tumor-specific enrichment by targeting the B7-H3 molecule, which is consistent with the high spatial resolution advantage of MRI as a preoperative positioning method.

[0089] Figure 6 Figure 2 shows the near-infrared fluorescence imaging of mice injected with dual-modality imaging probes. Figure 6 As shown in , it shows the white light image before injection and the actual location of the tumor by tumor bioluminescence imaging (BLI), as well as the dynamic process of NIR-II fluorescence imaging from 0.5h to 4h after injection. Figure 6It can be seen that at 0.5h, weak fluorescence signals began to appear in the tumor area, mainly distributed at the edge of the tumor; at 1h, the fluorescence intensity was significantly enhanced, the tumor boundary was clearly visible, and the signal in the central area was slightly lower than that in the periphery; at 2h, the fluorescence signal reached its peak, and the interior of the tumor was uniformly bright, forming a sharp contrast with the surrounding normal tissue; at 3-4h, the tumor fluorescence intensity decreased slightly, but still maintained high contrast, and excretion fluorescence signals appeared in the liver and kidney areas.

[0090] This shows that the fluorescence signal is always confined to the tumor area, and the intensity change is consistent with the enrichment and metabolic process of the probe in the tumor (first accumulation and then slow clearance). At 4 hours, the liver and kidney fluorescence signals increased, indicating that the probe is excreted through the hepatobiliary system and kidney dual channels, meeting clinical safety requirements. No nonspecific fluorescence appeared in normal tissues (such as muscle and bone), verifying the high tumor penetration and low autofluorescence interference characteristics of the IRDye800CW fluorescent dye. At the same time, the fluorescence signal reached its peak at 1-2 hours, which is highly consistent with the optimal time window for surgical resection, providing an experimental basis for real-time visualization of tumor boundaries during surgery.

[0091] Combine Figure 5 and Figure 6 It can be seen that the MRI of mice injected with the dual-modality imaging probe still maintained high contrast 4 hours after injection, indicating that the dual-modality imaging probe of this application is suitable for precise positioning of tumors before surgery; NIR-II fluorescence reaches a peak at 1-2 hours, indicating that the dual-modality imaging probe of this application is suitable for real-time navigation of tumors during surgery, and the time windows of the two modalities are closely connected. This experimental result further verifies that the dual-modality imaging probe of this application can simultaneously meet the dual needs of preoperative diagnosis (MRI) and intraoperative navigation (NIR-II).

[0092] Although multiple embodiments of the present application have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art can conceive of many changes, modifications, and alternatives without departing from the thought and spirit of the present application. It should be understood that in the process of practicing the present application, various alternatives to the embodiments of the present application described herein can be adopted. The accompanying claims are intended to define the scope of protection of the present application and therefore cover equivalents or alternatives within the scope of these claims.

Claims

1. A dual-modality imaging probe based on B7-H3, characterized in that: The dual-modality imaging probe includes a B7-H3 affibody, wherein the C-terminus of the B7-H3 affibody is connected to a near-infrared fluorescent dye IR-Dye800CW, and the N-terminus of the B7-H3 affibody is connected to a Gd-DOTA chelate, wherein the Gd-DOTA chelate chelates the gadolinium ion Gd 3 +For magnetic resonance imaging, the near-infrared fluorescent dye IR-Dye800CW is used for near-infrared second-zone fluorescence imaging.

2. The dual-modality imaging probe according to claim 1, characterized in that: The structure of the dual-modality imaging probe is as follows:

3. The dual-modality imaging probe according to claim 1 or 2, characterized in that: The molecular weight of the B7-H3 affibody is 8 kDa.

4. The dual-modality imaging probe according to claim 1 or 2, characterized in that: The excitation wavelength of the dual-modality imaging probe is 782 nm, and the emission wavelength is 807 nm.

5. The dual-modality imaging probe according to claim 1 or 2, characterized in that: The T1 relaxation time of the dual-modality imaging probe in aqueous solution gradually shortens as the concentration of the dual-modality imaging probe increases.

6. A method for preparing the dual-modality imaging probe according to any one of claims 1 to 5, characterized in that: The following steps are involved: Dissolve DOTA-NHS and B7-H3 affimer in an organic solvent, add triethylamine, and react with stirring under an inert gas atmosphere to obtain a first intermediate in which DOTA is connected to the N-terminus of the B7-H3 affimer; The first intermediate and IRDye800CW-maleimide are dissolved in an aqueous solution containing an organic solvent, and the mixture is stirred under an inert gas atmosphere to react to obtain a second intermediate in which IR-Dye800CW is connected to the C-terminus of the B7-H3 affimer; The second intermediate and GdCl3 are dissolved in deionized water and reacted under stirring conditions to obtain a dual-modal imaging probe based on B7-H3.

7. The preparation method according to claim 6, characterized in that The organic solvent is N,N-dimethylformamide; and / or The inert gas comprises nitrogen; and / or The second intermediate and GdCl 3 were dissolved in deionized water at pH 6.

0.

8. The preparation method according to claim 6 or 7, characterized in that The preparation method further comprises: After obtaining the first intermediate, purifying the first intermediate; and / or After obtaining the second intermediate, purifying the second intermediate; and / or The dual-modality imaging probe is purified.

9. Use of the dual-modality imaging probe according to any one of claims 1 to 5 in the preparation of a diagnostic reagent for prostate cancer.

10. The use according to claim 9, characterized in that The diagnostic reagent is used for at least one of the following: Preoperative tumor localization by magnetic resonance imaging; and / or Intraoperative real-time navigation of prostate cancer tumor margins; and / or Screening patients for B7-H3 targeted therapy in prostate cancer.

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