Optical Molecular Imaging Probe for Osteosarcoma Detection, Its Preparation Method and Application

Abstract

The present invention discloses an optical molecular imaging probe for osteosarcoma detection, its preparation method and application. The probe is a fluorescent molecular probe in which a monoclonal antibody targeting a membrane protein is conjugated with a fluorescent dye, and the targeted membrane protein is expressed on the cell membrane of osteosarcoma. The osteosarcoma-targeted fluorescent probe proposed by the present invention can specifically bind to osteosarcoma, so that it emits fluorescence under the irradiation of a light source with a specific wavelength during the operation, thereby providing real-time osteosarcoma localization information for surgeons and guiding the resection of osteosarcoma. This method is expected to significantly improve the accuracy and safety of the operation, reduce the recurrence rate, improve the quality of life of patients. The probe can also label the tumor area in the specimen by soaking the osteosarcoma specimen, and is expected to solve the problem of intraoperative pathological diagnosis in bone tumor surgery.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedical probes and molecular imaging, and particularly relates to an optical molecular imaging probe targeting CD276 (B7H3) for osteosarcoma detection, and a preparation method and application thereof. Background Art

[0002] Osteosarcoma (OS) is the most common primary malignant tumor in bone tissue, which occurs frequently at the distal femur, tibia and proximal femur, and mainly occurs in children and adolescents. Osteosarcoma has a very high degree of malignancy, and local tumor recurrence and lung metastasis are still the main causes of death of osteosarcoma patients. At present, the treatment of osteosarcoma patients usually adopts the treatment mode of neoadjuvant chemotherapy before surgery - resection during surgery - chemotherapy after surgery. However, the 5-year survival rate of osteosarcoma patients is only 66.2%, and patients still face treatment difficulties such as local tumor recurrence and distant metastasis. Obtaining a more accurate resection margin of osteosarcoma, detecting distant metastasis foci earlier, completely removing the tumor and retaining normal tissues as much as possible helps to improve the curative effect of osteosarcoma. However, due to the complex anatomical structure involved in the location of osteosarcoma, and the tumor lesion edges are often indistinguishable from normal tissues, it is difficult for surgeons to control the resection margin range during tumor resection, and some small lesions are difficult to observe with the naked eye during surgery, which brings difficulties to the diagnosis and treatment of the disease. Therefore, accurately marking the tumor area and visualizing the tumor during surgery are important guarantees for the precise resection of osteosarcoma. In addition, during tumor surgery, rapid pathological diagnosis of the surgical margin is crucial for judging whether the resection margin is sufficient. However, in bone tumor surgery, the surgical resection margin often includes hard tissues such as cortical bone, and frozen sections cannot be performed, which brings great difficulties to the judgment of the osteosarcoma surgical margin.

[0003] With the rapid development of fluorescence molecular imaging technology, the research and development of molecular probe drugs for intraoperative imaging of osteosarcoma, visualizing osteosarcoma during surgery, is beneficial for surgeons to accurately identify the tumor lesion edges during surgery, accurately calibrate the surgical resection range, and at the same time discover small lesions that are difficult to detect with the naked eye, which is beneficial to improving the treatment effect of patients, prolonging the event-free survival period and the quality of life after surgery.

[0004] B7H3 belongs to the members of the B7 family of the immunoglobulin superfamily. B7H3 is not only inducibly expressed in immune cells, but also constitutively highly expressed in a variety of tumor cells. In addition, there are few current studies on osteosarcoma fluorescence probes, and existing fluorescence probes basically have characteristics such as poor imaging effect, high price, and large toxicity, and are far from clinical application. Summary of the Invention

[0005] To address the deficiencies in the prior art described above or in other aspects, the present invention provides an optical molecular imaging probe targeting B7H3 for osteosarcoma detection and a preparation method thereof. B7H3 is lowly expressed in normal tissues and highly expressed in osteosarcoma tissues. Therefore, it can specifically image tumor tissues, mark the tumor boundary, has a high signal-to-noise ratio, and has a series of applications such as intraoperative navigation and intraoperative rapid pathological diagnosis.

[0006] The present invention provides an optical molecular imaging probe for osteosarcoma detection. The probe is a fluorescent molecular probe in which a monoclonal antibody targeting a membrane protein is conjugated with a fluorescent dye, and the membrane protein targeted is expressed on the cell membrane of osteosarcoma.

[0007] Among them, the fluorescent dye part is the IRDye-800CW fluorescent dye.

[0008] Among them, the monoclonal antibody targeting the cell membrane protein is a monoclonal antibody targeting B7H3.

[0009] The preparation method of the optical molecular imaging probe for osteosarcoma detection includes the following steps:

[0010] 1) Mix the monoclonal antibody targeting the membrane protein and the fluorescent dye to obtain a reaction solution;

[0011] 2) React the reaction solution obtained in step 1) under stirring, and then purify it to obtain a probe solution.

[0012] Among them, in step 1), the molar mass ratio of the fluorescent dye to the monoclonal antibody targeting the membrane protein is 5:1 - 10:1.

[0013] Among them, in step 2), the reaction conditions are carried out under dark conditions at 4°C, and the reaction time is 4 - 8 hours.

[0014] Among them, in step 2), the stirring speed is 300 - 600 revolutions per minute.

[0015] Among them, the fluorescent dye part is the IRDye-800CW fluorescent dye.

[0016] Among them, the monoclonal antibody targeting the cell membrane protein is a monoclonal antibody targeting B7H3.

[0017] Application of the above optical molecular imaging probe or the optical molecular imaging probe prepared by the above method in the preparation of reagents, kits of reagents or test kits for osteosarcoma detection.

[0018] Among them, the osteosarcoma detection includes precise fluorescence imaging of the osteosarcoma region, marking osteosarcoma, and detecting micro-cancer foci.

[0019] Use of the above-mentioned optical molecular imaging probe or the optical molecular imaging probe prepared by the above-mentioned method in the preparation of a reagent or kit for in vivo detection of tumors or in vitro pathological staining of tumor tissues.

[0020] The present invention further provides an osteosarcoma imaging reagent prepared from the above-mentioned optical molecular imaging probe, which is used for intraoperative fluorescence imaging of osteosarcoma after in vivo injection, accurately marking the tumor margin, detecting micro-cancer foci, and even pre-cancerous lesion tissues.

[0021] The present invention further provides the use of the above-mentioned osteosarcoma imaging reagent in the preparation of a reagent or kit for in vivo detection of tumors or in vitro pathological staining of tumor tissues.

[0022] The beneficial effects of the present invention are as follows: The present invention aims to develop a targeted fluorescence probe for bone tumor surgery to solve a major problem in current bone tumor surgery: the accurate determination of the resection boundary. The surgical treatment of bone tumors faces the challenge of how to remove as much tumor tissue as possible while retaining as much normal tissue function as possible. It is difficult for the prior art to accurately distinguish tumors from normal tissues during surgery. In addition, hard tissues often exist in the bone tumor site, which greatly limits the intraoperative pathological diagnosis. Therefore, it is difficult to know whether a negative resection margin has been achieved during osteosarcoma surgery, thus affecting the surgical effect and the prognosis of patients. The osteosarcoma-targeted fluorescence probe proposed by the present invention can specifically bind to osteosarcoma, making it emit fluorescence under the irradiation of a light source with a specific wavelength during surgery, thereby providing real-time osteosarcoma localization information for surgeons and guiding the resection of osteosarcoma. This method is expected to significantly improve the accuracy and safety of surgery, reduce the recurrence rate, and improve the quality of life of patients. The probe can also mark the tumor area in the specimen by soaking the osteosarcoma specimen, and is expected to solve the problem of intraoperative pathological diagnosis in bone tumor surgery. Through this innovative technology, it is expected to bring a revolutionary progress to the surgical treatment of osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Reaction route for preparing the optical molecular imaging probe provided by the present invention

[0024] Figure 2 Absorption spectra of the optical molecular imaging probe provided by the present invention and IRDye-800CW in the ultraviolet, visible, and near-infrared light ranges, as well as fluorescence emission spectra of the optical molecular imaging probe, IRDye-800CW, and anti-B7H3 antibody.

[0025] Figure 3 Expression profiles of B7H3 protein in four osteosarcoma cell lines and two normal cells.

[0026] Figure 4 Targeting effect of the optical molecular imaging probe provided by the present invention on the 143B osteosarcoma cell line.

[0027] Figure 5 The continuous imaging effect of the optical molecular imaging probe provided by the present invention on a subcutaneous osteosarcoma model in mice.

[0028] Figure 6 The imaging effect of the optical molecular imaging probe provided by the present invention on an orthotopic osteosarcoma model in mice.

[0029] Figure 7 The imaging effect of the optical molecular imaging probe provided by the present invention on a pulmonary metastatic osteosarcoma model in mice.

[0030] Figure 8 The comparison between the tumor regions shown by the imaging of the optical molecular imaging probe provided by the present invention on fresh human osteosarcoma specimens and the tumor regions shown by pathological sections. Detailed implementation manners

[0031] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0032] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0033] B7H3 (CD276) is a member of the B7 family. Multiple studies have shown that B7H3 is mainly expressed on the surface of tumor cells, rarely expressed in normal tissues, and the abnormal expression of B7H3 is related to the occurrence, development and metastasis of various cancers. A large amount of evidence indicates that its high expression is associated with poor prognosis of various malignant tumors. The B7H3 monoclonal antibody is a monoclonal antibody targeting B7H3 and can specifically bind to B7H3. The B7H3 monoclonal antibody is purchased from Univ-Bio and Abcam.

[0034] IRDye-800CW is a widely used near-infrared fluorescent dye. Under near-infrared light excitation, it can generate fluorescence, and the maximum excitation and emission wavelengths are 774 nm and 800 nm respectively. IRDye-800CW has good safety and stability. IRDye800CW NHS ester is purchased from Xi'an Ruixi Biotechnology and LI-COR.

[0035] According to the general inventive concept of the present invention, an optical molecular imaging probe for osteosarcoma detection is provided. The probe (B7H3-IRDye800CW) is a conjugate of a monoclonal antibody targeting membrane protein (B7H3 monoclonal antibody) and a near-infrared fluorescent dye (IRDye-800CW).

[0036] The inventors developed a novel B7H3 monoclonal antibody conjugated with IRDye-800CW probe detection reagent. Through the research of the inventors, it was found that the detection reagent will produce very important and significant effects during the detection of osteosarcoma.

[0037] The optical molecular imaging probe for osteosarcoma detection provided by the present invention can be used as a tumor detection reagent for osteosarcoma, as well as for precise fluorescence imaging of the osteosarcoma region, marking the edge of osteosarcoma and detecting tiny cancer foci.

[0038] The present invention further provides an application of the above-mentioned osteosarcoma imaging reagent in the preparation of a reagent or kit for in vivo tumor detection or in vitro pathological staining of tumor tissues.

[0039] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available through conventional commercial channels unless otherwise specified.

[0040] In the following examples, the 143B, U2OS, MG62, and KHOS osteosarcoma cell lines are from the ATCC company, human BMSC and HUVEC are from the National Experimental Cell Resource Sharing Platform, the 143B-LUC cell line is from Beijing Zhuangmeng International Biotechnology Co., Ltd., BALB / C-nude mice are from Beijing Vital River Laboratory Animal Technology Co., Ltd., MEM medium (11090081, Gibco), McCoy's 5A medium (16600082, Gibco), and DMEM medium (11965092, Gibco), a high-sensitivity optical imaging system (PerkinElmer product, product model IVIS Spectrum, USA), and osteosarcoma specimens are from the Department of Orthopedic Oncology, Peking University People's Hospital.

[0041] Example 1 Probe Preparation:

[0042] The IRDye-800CW active ester was mixed with the PBS solution containing the anti-B7H3 monoclonal antibody to obtain a reaction solution. We explored the conditions of the synthesis reaction, set two molar ratios of IRDye800CW activated ester to anti-B7H3 monoclonal antibody (5:1 or 10:1), added magnetic beads to the reaction solution, and stirred it using a magnetic stirrer. The stirring speed was set at 300 or 600 revolutions per minute. The reaction was carried out under dark conditions at 4 °C for 4 or 8 hours. Finally, the reaction solution was purified using a desalting column to obtain the above-mentioned optical molecular imaging probe. If the probe needs to be concentrated, an ultrafiltration tube with a cut-off molecular weight of 30 kDa was used to ultrafilter the optical molecular imaging probe. The ultrafiltration was carried out at 4 °C, in the dark, and under 2500 gravitational accelerations until the required probe concentration was obtained. The reaction parameters of the specific probe synthesis and the corresponding probe numbers are shown in Table 1.

[0043] Table 1. Probe synthesis reaction conditions and probe numbers

[0044]

[0045]

[0046] The reaction equation is as Figure 1 shown. The IRDye800CW active ester is connected to the anti-B7H3 monoclonal antibody to form the B7H3-IRDye800CW optical molecular imaging probe.

[0047] Performance detection of the probe. The absorption spectra in the ultraviolet, visible, and near-infrared light ranges were detected for the 10 μg / mL IRDye800CW solution, anti-B7H3 monoclonal antibody, and B7H3-IRDye800CW 1-8 solution (the absorption spectra were detected using a Shimadzu UV-2600 instrument). The results are as Figure 2 shown. Figure 2 This is the absorption spectra of the optical molecular imaging probe B7H3-IRDye800CW and IRDye-800CW provided by the present invention in the ultraviolet, visible, and near-infrared light ranges. It can be seen that the anti-B7H3 monoclonal antibody has been successfully conjugated with IRDye800CW. The fluorescence emission spectra of the optical molecular imaging probe, IRDye-800CW, and anti-B7H3 antibody are shown in the figure. The B7H3-IRDye800CW 1-8 probe has near-infrared fluorescence like IRDye800CW. The following takes the B7H3-IRDye800CW 1 probe as an example to further conduct experiments.

[0048] Example 2

[0049] 1. Cell culture

[0050] Prepare the complete medium required for cell culture. The complete medium is obtained by adding 10% fetal bovine serum and 1% penicillin-streptomycin solution to the medium. Cells such as 143B, 143B-GFP, 143B-LUC, BMSC, and HUVEC are cultured using DMEM medium, MG63 and KHOS cells are cultured using MEM medium, and U2OS cells are cultured using McCoy's 5A. The cells grow in a cell culture incubator at 37°C containing 5% CO 2 and all reagents, pipette tips, culture flasks, etc. used in cell culture are sterile items.

[0051] 2. Cell digestion and passage

[0052] After the cells reach confluence, aspirate the old medium, rinse the cells twice with PBS, digest the cells with 0.25% (w / v) trypsin for 1 - 2 minutes, then add medium to terminate digestion and pipette the cell suspension to mix evenly, and then add fresh medium to continue culturing the cells.

[0053] 3. Western-blot detection of cell sample B7H3

[0054] Add protein extraction reagent and protease inhibitor to 143B, MG63, KHOS, U2OS, BMSC, and HUVEC cells, incubate on ice for 10 min, then centrifuge using a refrigerated centrifuge (4°C, 13000 rpm / min, 10 min), aspirate the supernatant and aliquot it into EP tubes to obtain the total protein solution.

[0055] Use a BCA protein assay kit and bovine serum albumin standard to plot a standard curve, use a BCA protein concentration assay kit to measure the protein concentration in tissue specimens, adjust the protein concentrations of each sample to be the same, and add loading buffer.

[0056] Prepare SDS-PAGE stacking gel and separating gel, load samples into the sample wells, with a loading volume of 20 μg protein per well and 5 μL of protein marker loaded. Connect the power supply and start electrophoresis. Set the voltage to 80V when the protein marker is in the stacking gel, and change to 120V electrophoresis when the protein marker reaches the separating gel. Stop electrophoresis when the protein marker reaches the bottom of the separating gel.

[0057] Cut the whole separating gel and place it in pre-cooled transfer buffer. Arrange the transfer sponge, 3 pieces of filter paper, PVDF membrane, separating gel, 3 pieces of filter paper, and transfer sponge in sequence from the positive electrode to the negative electrode in the way of sandwiching. After removing the air bubbles between layers, fill the transfer buffer to cover the transfer sponge and perform wet transfer at 100V for 60 minutes. Take out the PVDF membrane and wash it 3 times with 0.1% TBST.

[0058] Add skim milk to the PVDF membrane, seal it on a shaker at room temperature for 60 min, wash the PVDF membrane 3 times with 0.1% TBST, add the primary antibody (rabbit anti-B7H3 monoclonal antibody) diluted 1:1000, and incubate overnight at 4°C. After recovering the primary antibody, wash the PVDF membrane 3 times with TBST, incubate with the secondary antibody (HRP-goat anti-rabbit antibody) diluted 1:5000, and incubate on a shaker at room temperature for 1 h, then wash 3 times with TBST again.

[0059] Mix solution A and solution B in the ECL luminescent agent in a ratio of 1:1, drop it onto the protein side of the PVDF membrane, expose it in a darkroom, adjust the exposure parameters according to the light intensity, and develop the image.

[0060] The results are as Figure 3 shown. Figure 3 For the expression of B7H3 protein in four osteosarcoma cell lines and two normal cells, it can be seen that the expression level of B7H3 in osteosarcoma cell lines is significantly higher than that in normal cells.

[0061] Example 3 Targeting of 143B osteosarcoma cell line with B7H3-IRDye800CW probe

[0062] Seed the 143B osteosarcoma cell line on cell slides. When the cells reach 50% confluence, fix them with paraformaldehyde, perform immunofluorescence staining of the cell slides with 5 μg / mL B7H3-IRDye800CW, then counterstain with DAPI, and observe under a fluorescence microscope after repeated washing. The results are as Figure 4 shown. Figure 4 For the targeting effect of the optical molecular imaging probe provided by the present invention on the 143B osteosarcoma cell line, it can be seen that the B7H3-IRDye800CW probe can effectively stain the osteosarcoma cell line.

[0063] Example 4 Establishment of a murine subcutaneous osteosarcoma model

[0064] Inject 10 6 143B cells into the subcutaneous tissue of the right back of BALB / C-nude mice. When the tumors on the back of the mice grow to a diameter of 5 - 10 mm, the murine subcutaneous osteosarcoma model is successfully established.

[0065] Imaging of the murine subcutaneous osteosarcoma model with an optical molecular imaging probe

[0066] Inject the B7H3-IRDye800CW probe into the mice through the tail vein at a dose of 1 μg per g of mouse body weight. Image the mice using a near-infrared camera at 1 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h to observe whether the probe can light up the subcutaneous osteosarcoma in the mice. The results are as Figure 5 shown.

[0067] Figure 5 The continuous imaging effect of the optical molecular imaging probe provided by the present invention on a mouse subcutaneous osteosarcoma model shows that at 12 h, the tumor can be well distinguished from the surrounding tissues. At 24 h - 72 h, there is a good tumor-to-background ratio, which can be used for guiding the resection of osteosarcoma during surgery. At the same time, it can also be observed that the tumor area labeled by the probe is consistent with the position of the subcutaneous tumor observed by the naked eye.

[0068] Example 5 Establishment of a mouse orthotopic osteosarcoma model

[0069] Inject 10 6 143B-LUC cells into the right tibial bone marrow cavity of BALB / C-nude mice. After the osteosarcoma in the right tibia of the mice grows to a diameter of 5 - 10 mm, the mouse orthotopic osteosarcoma model is successfully established.

[0070] Evaluation of the imaging accuracy of the optical molecular imaging probe for a mouse orthotopic osteosarcoma model

[0071] Inject the B7H3-IRDye800CW probe into the mice via the tail vein at a dose of 1 μg per g of mouse body weight. Image the mice at 24 h using a near-infrared camera to observe whether the probe can light up the mouse orthotopic osteosarcoma. Subsequently, inject the luciferase substrate to perform bioluminescence imaging on the mice to obtain the actual position of the tumors in the mice. Finally, compare the tumor positions labeled by the probe and the tumor positions labeled by the bioluminescence imaging. The results are as Figure 6 shown.

[0072] Figure 6 The imaging effect of the optical molecular imaging probe provided by the present invention on a mouse orthotopic osteosarcoma model shows that the tumor area shown by bioluminescence is consistent with the tumor area shown by the probe, indicating that the probe can accurately label the mouse orthotopic osteosarcoma model.

[0073] Example 6 Establishment of a mouse pulmonary metastatic osteosarcoma model

[0074] Inject 10 6 143B-LUC cells into BALB / C-nude mice via the tail vein. After 10 - 20 days, perform bioluminescence imaging on the mice. If obvious bioluminescence signals are observed in the chest of the mice, it is considered that the mouse pulmonary metastatic osteosarcoma model is successfully established.

[0075] Evaluation of the imaging accuracy of the optical molecular imaging probe for a mouse pulmonary metastatic osteosarcoma model

[0076] The B7H3-IRDye800CW probe was injected into mice via the tail vein at a dose of 1 μg per g of mouse body weight. Twenty-four hours after the probe injection, the luciferase substrate was injected. Then, the chest cavity of the mouse was opened, and bioluminescence imaging was performed on the mouse to obtain the actual location of the mouse tumor. At the same time, the mouse was also imaged using a near-infrared camera at 24 h. Finally, the tumor location marked by the probe was compared with the tumor location marked by the bioluminescence imaging.

[0077] Figure 7 The imaging effect of the optical molecular imaging probe provided by the present invention on the mouse lung metastatic osteosarcoma model was observed. It was found that the tumor area shown by bioluminescence was consistent with the tumor area shown by the probe, indicating that the probe could accurately label the mouse lung metastatic osteosarcoma model.

[0078] Example 7: Application of the optical molecular imaging probe in rapid intraoperative pathological diagnosis of osteosarcoma

[0079] Freshly resected human osteosarcoma specimens were cut in half from the coronal plane (n = 5). Specimens with a thickness of 3 mm were sawn off from the coronal plane, and the specimens were rinsed with physiological saline to remove sawdust. Then, the specimens were immersed in Kerbs-Henseleit solution containing 2% fetal bovine serum. After 5 min, the specimens were incubated in B7H3-IRDye800CW solution (PBS solution containing 50 μg / mL B7H3-IRDye800CW and 30% PEG-300) for 30 min. They were washed 3 times in PBS solution containing 30% PEG-300, 2 minutes each time. The samples were dried with absorbent paper, and near-infrared fluorescence images of the samples were captured.

[0080] The specimens were cut into tissue blocks of 2.5 cm × 2 cm using a hard tissue microtome. The tissue blocks were fixed with 4% paraformaldehyde, decalcified with formic acid, embedded in wax, sectioned into 5-μm thickness, stained with H&E, scanned with a pathological section scanner, and the tissue sections were stitched into a whole. The pathological tumor area was compared with the tumor area shown by NIR-II fluorescence imaging.

[0081] Figure 8 The comparison of the tumor areas shown by the imaging of the optical molecular imaging probe provided by the present invention on fresh human osteosarcoma specimens and the tumor areas shown by pathological sections is presented. The preoperative x-ray, CT, and MRI images of osteosarcoma patients are shown in Figure 8 A. After surgical resection, the specimen was further sawn into 3-mm thickness ( Figure 8 B). After the B7H3-IRDye800CW soaking and washing steps, NIR-II fluorescence imaging was performed, and strong fluorescence signals were visible in the black coil area ( Figure 8C), the fluorescence intensity of adjacent tissues was weak; including the growth plate, articular cartilage, ligaments, cortical bone near the bone axis, and cancellous bone near the joint, etc. Next, the whole specimen was stained with HE to understand the tissue composition of the fluorescence-highlighted area, and the pathological tumor invasion area was outlined in black on the high-power HE staining image ( Figure 8 D), we can see that the tumor area marked by fluorescence is almost the same as the tumor area confirmed by pathological evidence. The area 1 with obvious abnormal near-infrared high fluorescence signal and MRI was confirmed as osteosarcoma by HE staining ( Figure 8 E), the area 5 with no obvious change in near-infrared high fluorescence signal and MRI signal was also confirmed as osteosarcoma, and the area 3 with low near-infrared fluorescence signal and normal MRI signal was normal cortical bone. High fluorescence signal can be seen at the distal end of the growth plate (circled in blue and area 4) ( Figure 8 C). We further searched for this area on the HE staining and found multiple micro-lesions of osteosarcoma in this area ( Figure 8 D). In the area circled in black, we can see some areas with relatively low fluorescence intensity (circled in yellow and area 2), and the HE staining of area 2 showed a necrotic osteosarcoma area.

[0082] In summary, B7H3-IRDye800CW can accurately label the osteosarcoma area, even the micro-lesion area of osteosarcoma, by simple soaking, which indicates that the B7H3-IRDye800CW probe has great potential in the preparation of rapid pathological diagnosis-related reagents or equipment for osteosarcoma.

[0083] At present, the intraoperative fluorescence navigation probe for osteosarcoma is blank. Currently, clinically, only preoperative imaging data such as X-ray, CT, and MRI can be combined with the experience of surgeons during the operation to judge the boundary of osteosarcoma. This method cannot display the tumor boundary in real time during the operation, and is highly subjective and easily interfered by various factors such as a narrow surgical field and bleeding in the surgical field. The osteosarcoma probe developed by the present invention can display the tumor site in real time under near-infrared fluorescence imaging, guide doctors to resect the tumor, and obtain better surgical results.

[0084] Hard tissues, such as hard cortical bone, are often included near the osteosarcoma tumor mass, and the cryostat cannot section hard tissues. Therefore, intraoperative rapid pathological diagnosis of the osteosarcoma resection margin often cannot be performed, and conventional tissue sectioning after decalcification takes a long time and cannot judge the benign or malignant nature of the resection margin in time during the operation. The osteosarcoma probe provided by the present invention can judge the benign or malignant nature of the resection margin through simple soaking operation, which helps to improve the survival of patients after bone tumor surgery.

[0085] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under similar parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, in accordance with the principle of the present invention, this application is intended to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art. The application of some basic features can be made within the scope of the following appended claims.

Claims

1. An optical molecular imaging probe for osteosarcoma detection, characterized in that: The probe is a fluorescent molecular probe of a membrane protein-targeting monoclonal antibody coupled with a fluorescent dye, wherein the membrane protein-targeting protein is expressed on the osteosarcoma cell membrane; the fluorescent dye part is an IRDye-800CW fluorescent dye; and the cell membrane protein-targeting monoclonal antibody is a B7H3-targeting monoclonal antibody.

2. The method for preparing the optical molecular imaging probe for osteosarcoma detection according to claim 1, characterized in that: The steps include: 1) Mixing the membrane protein-targeting monoclonal antibody and the fluorescent dye to obtain a reaction solution; 2) reacting the reaction solution obtained in step 1) under stirring, and then purifying to obtain a probe solution; The fluorescent dye part is IRDye-800CW fluorescent dye; the monoclonal antibody targeting cell membrane protein is a monoclonal antibody targeting B7H3.

3. The method according to claim 2, characterized in that In the step 1), the molar mass ratio of the fluorescent dye to the membrane protein targeting monoclonal antibody is 5:1-10:

1.

4. The method according to claim 2, characterized in that In the step 2), the reaction is carried out at 4° C. in the dark, and the reaction time is 4-8 hours.

5. The method according to claim 2, characterized in that: In the step 2), the stirring speed is 300-600 revolutions per minute.

6. Use of the optical molecular imaging probe according to claim 1 or the optical molecular imaging probe prepared according to any one of the methods of claims 2 to 5 in the preparation of a reagent or kit for osteosarcoma detection.

7. The use according to claim 6, characterized in that: The osteosarcoma detection includes precise fluorescence imaging of the osteosarcoma area, marking of osteosarcoma and detection of tiny cancer foci.

8. Use of the optical molecular imaging probe according to claim 1 or the optical molecular imaging probe prepared according to any one of the methods of claims 2 to 5 in the preparation of a reagent or kit for in vivo tumor detection or in vitro pathological staining of tumor tissue, wherein the tumor is osteosarcoma.

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