A method for preparing a near-infrared second-region fluorescent probe targeting Trop-2

Abstract

The present invention discloses a method for preparing a near-infrared zone II fluorescent probe targeting Trop‑2, wherein the probe is ICG‑SG, which is used as a fluorescent navigation for guiding tumor surgical margins and a reagent for judging the benign and malignant nature of in vitro breast tissue. The present invention provides a method for preparing a near-infrared zone II fluorescent probe targeting Trop‑2, and applies the ICG‑SG probe to the fluorescent navigation for guiding tumor surgical margins, so as to realize the visualization of the breast-conserving boundary, aiming to establish a surgical navigation system for precise resection of breast tumors guided by NIR‑II fluorescence, distinguish breast tumors from normal tissues, accurately identify tumor boundaries, and improve the accuracy of breast-conserving margins for breast cancer. The ICG‑SG probe can also quickly and effectively judge the benign and malignant nature of breast tumors through in vitro specimen immersion imaging, provide guidance for intraoperative decision-making, further improve the accuracy of breast cancer surgery, reduce the positive rate of surgical margins, improve the quality of life of patients and prolong overall survival, and has important clinical significance for improving patient prognosis.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing a near-infrared second-zone fluorescent probe targeting Trop-2. Background Art

[0002] Breast cancer ranks first in the incidence and mortality of female malignant tumors worldwide, and breast-conserving surgery has become the standard surgical method for early breast cancer. The key to the success of breast-conserving surgery is the complete removal of the tumor while preserving the breast, and taking into account the postoperative aesthetics. At present, intraoperative frozen pathology is a relatively mature pathological technology for evaluating tumor margins, but intraoperative frozen pathology is a non-continuous sampling method, which is prone to missed detection and time-consuming. Another major reason for the positive margin of breast-conserving surgery is that the surgeon can only evaluate the margin status through subjective feelings such as "looking and touching" during the operation. Therefore, how to accurately and real-time determine the status of the breast-conserving surgery margin is the key to avoiding secondary surgery.

[0003] At present, intraoperative molybdenum target is mainly used in clinical practice to assist in the determination of resection margins. This method is prone to miss non-calcified lesions and has low sensitivity. Intraoperative ultrasound has a low diagnostic consistency for deep tumors and is insensitive to the detection of microcalcifications. Summary of the invention

[0004] The present invention provides a method for preparing a near-infrared second-zone fluorescent probe targeting Trop-2 to solve the above problems.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A near-infrared zone II fluorescent probe targeting Trop-2, wherein the probe is ICG-SG, wherein the structure of the probe ICG-SG is specifically as follows:

[0007]

[0008] A method for preparing a near-infrared second-zone fluorescent probe targeting Trop-2, the method comprising the following steps:

[0009] S1. Dissolve 10 mg of gosartuzumab in 1 ml of normal saline to obtain a 10 mg / ml gosartuzumab drug solution;

[0010] S2, dissolving 1.7 mg of indocyanine green maleimide in 200 μl of N,N-dimethylaminopropane, and dissolving to obtain an indocyanine green maleimide solution with a concentration of 0.01 mol / L;

[0011] S3, add 2 ml phosphate buffer, 1 ml gosartuzumab drug solution, and 200 ul indocyanine green maleimide solution into a brown light-proof reaction bottle in sequence;

[0012] S4, control the pH value of the reaction system in the reaction bottle, place the reaction bottle in a dark environment at room temperature and stir overnight, then dialyze to remove unreacted indocyanine green maleimide, and change the water once in the middle;

[0013] S5. After freeze-drying, the probe ICG-SG was obtained and stored in a refrigerator at 4°C.

[0014] Preferably, the pH value of the reaction system in the reaction bottle in step S4 is controlled at 7.0-7.4.

[0015] Preferably, the dialysis process in step S4 is specifically: dialysis at room temperature for 48 hours using a 10KDa dialysis membrane.

[0016] A near-infrared zone II fluorescent probe targeting Trop-2 is used as a fluorescent navigation agent for guiding tumor surgical margins.

[0017] A near-infrared zone II fluorescent probe targeting Trop-2 is used as a reagent for determining the benign or malignant nature of breast tissue in vitro.

[0018] After adopting the above technical solution, the present invention has the following advantages compared with the background technology:

[0019] The present invention provides a method for preparing a near-infrared zone II fluorescent probe targeting Trop-2, using ICG as a light-emitting element, and an SG antibody specifically targeting Trop-2 as a targeting element, using the tailing peak imaging of ICG in the near-infrared zone II to solve the problems of shallow penetration depth, low signal-to-noise ratio and poor resolution in the near-infrared zone I, and overcome the shortcomings of near-infrared zone II probes such as quantum dots, rare earth elements, and single-walled carbon nanotubes with high toxicity, and improving the photostability of ICG by combining with antibodies, thereby preparing a surface-modified and optimized ICG-SG fluorescent molecular probe targeting Trop-2. The ICG-SG probe is applied to the fluorescent navigation guiding the surgical margin of tumors to realize the visualization of the breast-conserving margin, aiming to establish a surgical navigation system for precise resection of breast tumors guided by NIR-II fluorescence, distinguish breast tumors from normal tissues, accurately identify tumor boundaries, and improve the accuracy of breast-conserving margins of breast cancer. In addition, the ICG-SG probe can also quickly and effectively determine the benign or malignant nature of breast tumors through in vitro specimen immersion imaging, provide guidance for intraoperative decision-making, further improve the accuracy of breast cancer surgery, reduce the positive rate of surgical margins, improve the patient's quality of life and prolong overall survival. It has important clinical significance for improving patient prognosis and provides a theoretical and technical basis for improving the survival rate and reducing the mortality rate of breast cancer patients in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the construction structure of the probe ICG-SG of the present invention;

[0021] Figure 2 This is a characterization diagram of the optical characteristics of the probe ICG-SG in an embodiment of the present invention;

[0022] Figure 3 The cytotoxicity test results of the probe ICG-SG in the embodiment of the present invention;

[0023] Figure 4 This is the cell targeting test result of the probe ICG-SG in the embodiment of the present invention;

[0024] Figure 5 The safety test results of the probe ICG-SG in the embodiment of the present invention;

[0025] Figure 6 The results of the animal targeting test of the ICG-SG fluorescent molecular probe in the MDA-MB-231 tumor-bearing mice in the embodiment of the present invention;

[0026] Figure 7 This is the test result of the ICG-SG fluorescent molecular probe in guiding the precise resection of breast cancer in vivo in an embodiment of the present invention.

[0027] Figure 8 This is the test result of co-incubation of the probe ICG-SG with human breast cancer specimens in vitro in the embodiment of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] Example

[0030] like Figure 1 As shown, the present invention discloses a near-infrared second-zone fluorescent probe targeting Trop-2, and the probe is ICG-SG.

[0031] The present invention also discloses a method for preparing a near-infrared second-region fluorescent probe targeting Trop-2, the method comprising the following steps:

[0032] S1. Dissolve 10 mg of Sacituzumab Govitecan in 1 ml of normal saline to obtain a 10 mg / ml Sacituzumab Govitecan solution.

[0033] S2, dissolving 1.7 mg of indocyanine green maleimide in 200 μl of N,N-dimethylaminopropane, and dissolving to obtain an indocyanine green maleimide solution with a concentration of 0.01 mol / L;

[0034] S3, add 2 ml phosphate buffer, 1 ml gosartuzumab drug solution, and 200 ul indocyanine green maleimide solution into a brown light-proof reaction bottle in sequence;

[0035] S4, control the pH value of the reaction system in the reaction bottle, place the reaction bottle in a dark environment at room temperature and stir overnight, then dialyze to remove unreacted indocyanine green maleimide, and change the water once in the middle;

[0036] S5. After freeze-drying, the probe ICG-SG was obtained and stored in a refrigerator at 4°C.

[0037] In step S4, the pH value of the reaction system in the reaction bottle is controlled at 7.0-7.4.

[0038] The specific dialysis process of step S4 is: using a 10KDa dialysis membrane for 48 hours at room temperature.

[0039] The present invention also discloses a near-infrared zone II fluorescent probe targeting Trop-2 as a fluorescent navigation agent for guiding the surgical margin of tumors.

[0040] The present invention also discloses a near-infrared second-zone fluorescent probe targeting Trop-2 as a reagent for judging the benign or malignant nature of breast tissue in vitro.

[0041] Experiment 1: In vitro study of ICG-SG fluorescent molecular probe

[0042] After co-incubating the ICG-SG fluorescent molecular probe with breast cancer cells MDA-MB-231 cells that highly express Trop-2 or normal breast epithelial cells MCF-10A cells that hardly express Trop-2, the optical properties, safety and targeting of the ICG-SG fluorescent molecular probe were studied by setting the probe incubation time and concentration gradient at the cellular level.

[0043] Test results: Figure 2 As shown in the figure, SG was successfully coupled to ICG by high performance liquid chromatography, and the purity can reach 99.9%; its fluorescence spectrum performance is stable, and the absorption peak and emission peak characteristics before and after modification with SG are similar, and the fluorescence tailing signal can be detected in the near infrared region II (NIR-II); Figure 3 As shown in the figure, after the same concentration of ICG-SG fluorescent molecular probe was co-incubated with MCF-10A cells, 4T1 cells, and MDA-MB-231 cells for 24 hours, the three cell lines still maintained >80% cell viability when the concentration of ICG-SG fluorescent molecular probe was as high as 50 μg / mL, indicating that the ICG-SG fluorescent molecular probe has good biosafety at the cellular level; Figure 4As shown, after the ICG-SG fluorescent molecular probe was co-incubated with breast cancer cells MDA-MB-231 cells that highly express Trop-2 and normal breast epithelial cells MCF-10A that hardly express Trop-2, the fluorescence intensity of the high MDA-MB-231 cell group was significantly higher than that of the MCF-10A group, and blocking Trop-2 could reduce the cellular uptake of ICG-SG by about 74%, proving that the ICG-SG fluorescent molecular probe has the ability to specifically target Trop-2 at the cellular level.

[0044] Experiment 2: In vivo study of fluorescent molecular probe of ICG-SG

[0045] In vivo safety study of the probe: Two groups of normal mice were injected with ICG-SG fluorescent molecular probe and PBS through the tail vein, and the body weight, blood cytology, liver function, kidney function and histological levels of various important organs of the mice were tested on 1 day, 3 days, 7 days and 28 days.

[0046] Test results: Figure 5 As shown, there was no significant difference in body weight between the two groups of mice; peripheral blood serum was collected from the eye sockets at the same time point, and ALT, AST, BUN, CR, and CK-MB in the serum were detected, and there was no significant difference between the two groups; WBC, Lymphocyte, RBC, and PLT in the whole blood were detected, and there was no significant difference between the two groups; the heart, liver, spleen, lung, and kidney were removed, and HE staining was performed to observe that there was no obvious damage in the two groups; this proves that the ICG-SG fluorescent molecular probe has good biosafety.

[0047] In vivo targeting study of the probe: MDA-MB-231-luc cells in the logarithmic growth phase were digested and centrifuged to prepare a concentration of 5×10 7 After routine disinfection, 100 μL of cell suspension was injected into the right hind limb of BALB / c-Nude female mice, with a total number of 5×10 6 cells; D-luciferin potassium salt (Beyotime, #ST196) was used to monitor tumor growth by bioluminescence every 3 days after surgery; 10 mice with subcutaneous breast cancer transplanted tumors of appropriate size were randomly divided into ICG-SG group and ICG-IgG group, with 5 mice in each group, and the optimal concentration of ICG-SG fluorescent molecular probe or ICG-IgG probe was injected into the tail vein, and NIR-II fluorescence imaging was used to collect tumor fluorescence signals at 1, 6, 12, 24, 36, 48, 72, and 96 hours, and the signal-to-background ratio between the two groups was analyzed and compared.

[0048] Test results: Figure 6As shown in the figure, after the ICG-SG fluorescent molecular probe or ICG-IgG probe was injected into the tail vein of MDA-MB-231 tumor-bearing mice, near-infrared fluorescence imaging in the second zone was performed. Compared with the ICG-IgG group, the ICG-SG group showed obvious aggregation in the tumor, and the TBR of the two groups was statistically different at the best imaging time of 36 hours. This proves that the ICG-SG fluorescent molecular probe has a good ability to specifically target Trop-2 in small animals.

[0049] Experiment 3: Evaluating the effectiveness of ICG-SG fluorescent molecular probe in accurately assessing breast cancer resection margins in living small animals

[0050] Construction of a subcutaneous breast cancer transplant mouse model:

[0051] The MDA-MB-231-luc cells in the logarithmic growth phase were digested and centrifuged to prepare a concentration of 5×10 7 After routine disinfection, 100 μL of cell suspension was injected into the right hind limb of BALB / c-Nude female mice, with a total number of 5×10 6 cells; D-luciferin potassium salt (Beyotime, #ST196) was used to monitor tumor growth by bioluminescence every 3 days after surgery;

[0052] Intraoperative NIR-II fluorescence imaging system-guided surgical navigation of subcutaneous breast cancer transplanted tumors in mice:

[0053] 1) Eight mice with subcutaneous transplanted breast cancer model were randomly divided into a white light-guided group and a NIR-Ⅱ fluorescence-guided group. After the optimal dose of probe was injected into the tail vein, the mice were anesthetized by isoflurane inhalation and fixed on a sterilized surgical cloth at the optimal imaging time. The mice in the white light group underwent breast cancer tumor resection under white light, and the wound was sutured after the surgeon judged that the tumor was completely removed. The mice in the NIR-Ⅱ fluorescence-guided group were placed under the DPM NIR-Ⅱ fluorescence imaging system for real-time imaging, and tumor resection was performed under the guidance of the DPM system until the real-time imaging results showed that there was no tumor fluorescence signal, and then the wound was sutured.

[0054] 2) Processing of postoperative specimens: ① Detection of fresh tissue specimens: The fresh tissues cut from the NIR-Ⅱ fluorescence-guided group were placed under the NIR-Ⅱ fluorescence imaging system to detect the fluorescence signals of the specimens; ③ Fluorescence detection of paraffin blocks: The fluorescence signals of the tissue paraffin blocks were collected after the tissues were fixed and embedded; ④ Fluorescence detection of paraffin sections: The tissue paraffin blocks were continuously sliced ​​into 10μm and 4μm thick tissue sections, and 10μm sections were taken for dewaxing with xylene and observed with a near-infrared scanner (LI-COR Odyssey) and an upright fluorescence microscope to collect the fluorescence signals of the sections;

[0055] 3) Comparative analysis of images from macroscopic to microscopic levels: Compare the above-mentioned in vivo and ex vivo fluorescence distributions, paraffin block and section fluorescence signals, and H&E staining results to observe whether the fluorescence distribution and pathology in the tumor can be co-localized.

[0056] 4) Follow up the local residual tumor of mice by bioluminescence and compare the survival of mice in each group: Bioluminescence imaging was performed on mice 4, 7, 14, 21, and 28 days after surgery to evaluate whether there was residual tumor and recurrence, and the weight and activity status of mice were followed up. The criteria for mouse death were: natural death, weight loss exceeding 20% ​​of the original body weight, or tumor volume exceeding 1500mm 3 Mice were judged dead during the follow-up period and then killed by cervical dislocation, and the prognosis curve was drawn.

[0057] Test results: Figure 7 As shown, injection of ICG-SG fluorescent molecular probe and NIR-II fluorescence-guided surgery can effectively identify breast tumors, satellite lesions and residual lesions during surgery, and guide precise resection, significantly improving the RFS of mice.

[0058] Experiment 4: Evaluate the effectiveness of ICG-SG fluorescent molecular probe in incubating human breast cancer tissue in vitro to determine the benign or malignant nature of breast tissue.

[0059] 1) Fluorescence imaging of fresh tissue specimens: The excised fresh tissue was incubated with the ICG-SG fluorescent molecular probe for 10 minutes, washed three times with PBS, and the fluorescence signal of the ex vivo tissue specimen was collected in the DPM fluorescence imaging system. All tissues were then subjected to subsequent pathological treatment.

[0060] 2) Fluorescence photography of tissue wax blocks and paraffin-embedded tissue sections: All tissue specimens obtained from patients were paraffin-embedded according to routine procedures, and then the wax blocks were sliced ​​continuously to obtain 10 μm tissue sections. After the tissues were dewaxed, the Odyssey fluorescence imaging system was used to scan the tissue fluorescence distribution;

[0061] 3) The paraffin blocks of tumor tissue and normal tissue around the tumor were serially sectioned, and 4 μm tissue sections were stained with H&E;

[0062] 4) Comparative analysis of images from macroscopic to microscopic levels: Compare the above-mentioned in vivo and ex vivo fluorescence distributions, paraffin block and section fluorescence signals, and H&E staining results to observe whether the fluorescence distribution and pathology in the tumor can be co-localized.

[0063] Test results: Figure 8 As shown, the ICG-SG fluorescent molecular probe was co-incubated with human breast cancer tissue specimens in vitro for 10 minutes. The near-infrared second zone fluorescence matched well with the pathology, and ICG-SG could better distinguish human breast cancer and adjacent tissues.

[0064] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a near-infrared second-region fluorescent probe targeting Trop-2, characterized in that: The method comprises the following steps: S1. Dissolve 10 mg of gosartuzumab in 1 ml of normal saline to obtain a 10 mg / ml gosartuzumab drug solution; S2, dissolving 1.7 mg of indocyanine green maleimide in 200 μl of N,N-dimethylaminopropane, and dissolving to obtain an indocyanine green maleimide solution with a concentration of 0.01 mol / L; S3, add 2 ml phosphate buffer, 1 ml gosartuzumab drug solution, and 200 ul indocyanine green maleimide solution into a brown light-proof reaction bottle in sequence; S4, control the pH value of the reaction system in the reaction bottle, place the reaction bottle in a dark environment at room temperature and stir overnight, then dialyze to remove unreacted indocyanine green maleimide, and change the water once in the middle; S5. After freeze drying, the probe ICG-SG is obtained and stored in a refrigerator at 4° C., wherein the structure of the probe ICG-SG is as follows:

2. The method for preparing a near-infrared second-region fluorescent probe targeting Trop-2 according to claim 1, characterized in that: In step S4, the pH value of the reaction system in the reaction bottle is controlled at 7.0-7.

4.

3. The method for preparing a near-infrared second-region fluorescent probe targeting Trop-2 according to claim 1, characterized in that: The dialysis process in step S4 is specifically as follows: using a 10KDa dialysis membrane for 48 hours at room temperature.

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