A NIR / CT dual-modal imaging probe targeting CD146 and its application

Abstract

The present invention relates to a preparation method of a NIR / CT dual-modal imaging probe targeting CD146, comprising: mixing a compound specifically binding to CD146 with hydroxyethyl cellulose to obtain CD146 monoclonal antibody-modified hydroxyethyl cellulose; using L-glutathione, formamide, and gadolinium chloride as raw materials, and preparing near-infrared fluorescent gadolinium(III)-doped carbon dots by a solvothermal synthesis method; mixing CD146 monoclonal antibody-modified hydroxyethyl cellulose with near-infrared fluorescent gadolinium(III)-doped carbon dots to obtain a probe; dissolving the probe in methanol, adding potassium iodide and sodium hydroxide for reaction, and filtering to obtain the NIR / CT dual-modal imaging probe targeting CD146. The present invention prepares a NIR / CT dual-modal imaging probe with high sensitivity and specificity for CD146 molecules, which integrates the characteristics of high sensitivity of NIR and high spatial resolution of CT for the detection of small metastatic tumor lesions, extracts more imaging details through multi-dimensional information, and provides more comprehensive and accurate tumor detection and evaluation.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and particularly relates to the preparation of a novel near-infrared fluorescence (NIR) / computed tomography (CT) dual-modal imaging probe targeting CD146 and its application in the diagnosis of metastatic lung cancer. Background Art

[0002] Lung cancer is a relatively common malignant tumor in the lungs. According to statistics, the number of people who die from lung cancer globally is 1 million per year on average. In the past few decades, although the diagnosis and treatment technologies of lung cancer have been developed, when there are no obvious symptoms, for lung cancer patients found in the advanced stage or with metastases to other parts, the cure rate is relatively low (less than 30%), and the prognosis is poor.

[0003] If lung cancer can be diagnosed at an early stage, the survival rate can be significantly improved (more than 80%). Currently, the diagnostic methods for lung cancer include: ① imaging methods, such as imaging techniques like CT and MRI (magnetic resonance imaging). The diagnosis of lung cancer highly relies on imaging methods, but as an early diagnosis method for lung cancer, there are limitations in identifying early or tiny metastatic lesions, with insufficient sensitivity and specificity. Although the positron emission tomography (PET) method has a certain sensitivity, its ability to identify specific tumor markers is limited; ② tissue examination, specifically including bronchoscopy, sputum cytology examination, fine needle aspiration examination, mediastinoscopy, etc. These methods of examining living tissues, such as pathology and cytology, are invasive and not easy to perform repeated detections; ③ tumor marker examination. As a non-invasive examination method that can predict prognosis and judge the treatment effect, it is widely used. The tumor marker examination is based on antigen detection methods. Most tumor markers use the standard that when the detected concentration exceeds a certain threshold, it is judged as cancer, otherwise it is judged as the normal reference value, and there are limitations in the accuracy of distinguishing patients from non-patients. Specifically, the change amount of tumor markers in the blood shows an unclear change trend in the cancer onset stages 0-3, and there is a quantitative difference in stage 4. Using only the tiny change characteristics as the tumor marker examination method, its specificity (about 89%) and sensitivity (about 43%) are still not high, especially in the cancer stages 0-1, it is very difficult to distinguish between normal and cancerous.

[0004] CD146, also known as melanoma cell adhesion molecule (MCAM) or cell surface glycoprotein MUC18, is a single-chain transmembrane glycoprotein and belongs to the members of the immunoglobulin superfamily (Igsf). CD146 is a Ca 2+A membrane glycoprotein of non-dependent cell adhesion molecule, which was first identified as a tumor marker because of its high expression in melanoma and no expression in normal control groups. This discovery has attracted the attention of the tumor research field to CD146. A large number of studies have confirmed that CD146 is expressed in melanoma, prostate cancer, breast cancer, liver cancer, urothelial cancer and gynecological tumors and is closely related to the occurrence, development, metastasis, treatment and prognosis of these tumors. In breast cancer cells, knocking down the expression level of CD146 molecule significantly reduces the metastatic potential of breast cancer cells and prevents the formation of secondary tumor metastases in the lungs by breast cancer cells. Another study reported that as a ligand of S100A8 / A9 protein, CD146 can induce the metastatic progression of malignant melanoma through NF-κB activation and ROS accumulation. Previous studies have shown that CD146 is significantly highly expressed in metastatic lung cancer.

[0005] The principle of fluorescence imaging is to use light in the range from ultraviolet to infrared to excite molecules. After being activated, the molecules will emit photons with longer wavelengths, and special sensors are used to detect these photons. The interference in the near-infrared (NIR) (700 nm - 2000 nm) spectrum is less than that in the visible light (400 nm - 700 nm) spectrum. Therefore, the NIR region is considered as the "optical or therapeutic window", where the light has the maximum penetration depth and the highest tissue transparency. Fluorescent probes are based on spectrochemical and optical waveguide and measurement technologies. Affected by the surrounding environment, in a certain system, after a chemical or physical interaction with a certain substance, the fluorescence signal of the molecule can change accordingly, so as to collect the characteristics of the surrounding environment or a certain specific information existing in the environment. By using the contrast between the signal changes of specific molecular probes and the intrinsic properties of in-vivo tissues, imaging effects can be obtained. Fluorescence imaging can show a very high spatial resolution in vivo. Fluorescent probes can respond to a variety of analytes. When the analytes are various disease indicators, the fluorescence changes with the disease indicators, thus helping people to image and diagnose related diseases. The structures of the fluorescent probes provided by the existing technologies include fluorophores (luminescent substances), connecting parts and recognition parts. The fluorophores are mainly served by inorganic fluorescent materials and organic small molecule fluorescent dyes. The near-infrared wavelength range is outside the range of tissue autofluorescence, and the tissue penetration depth can reach 5 - 10 mm. Therefore, almost all optical imaging agents used in clinics are NIR materials.

[0006] Existing imaging techniques have problems of insufficient specificity and sensitivity in the diagnosis of metastatic lung cancer. Especially for cases where the tumor volume is small, there are micrometastases, or it is in the early stage, it is difficult for existing imaging techniques to distinguish tumor cells from normal tissues. In addition, the existing imaging modes are single, and most existing probes only support a single imaging mode, which is not conducive to comprehensive evaluation and accurate positioning. CN110237272A discloses a dual-modal tumor imaging nanoprobe applicable to MRI / NIR-II, a preparation method and applications thereof. Its fluorescence emission wavelength is in the second near-infrared region and can be used for detection imaging of different diseases such as tumors. However, this method is not very suitable for early detection of lung cancer.

[0007] Therefore, it is necessary to develop a technique for early diagnosis of metastatic lung cancer. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, a human phage antibody library constructed based on PBMC of metastatic lung cancer patients with high expression of CD146 molecules is used in the present invention, and 23 unique sequence antibodies are screened and enriched. Further results show that: MM21 shows high binding affinity and binding specificity with the CD146 protein. Based on this, in the first aspect of the present invention, a compound specifically binding to CD146 is provided, which is selected from the group consisting of antibodies, antibody fragments, and antibody derivatives. The compound includes: a light chain variable domain, and the amino acid sequence of the light chain variable domain is as shown in SEQ ID NO:1; and a heavy chain variable domain, and the amino acid sequence of the heavy chain variable domain is as shown in SEQ ID NO:2.

[0009] According to a preferred embodiment, the amino acid sequence SEQ ID NO:1 of the light chain variable domain is: SYELTQPLSVSAAPGQMVTMSCSGSSSNIGNNYVFWHQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLAITGLQTGDEADYYCGTWDSSLSGGVFGGGTKLTVL.

[0010] According to a preferred embodiment, the amino acid sequence SEQ ID NO:3 of the light chain constant region is:

[0011] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS.

[0012] According to a preferred embodiment, the amino acid sequence SEQ ID NO: 2 of the heavy chain variable domain is: QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLIRGAFDIWGQGTLVTVSS.

[0013] According to a preferred embodiment, the amino acid sequence SEQ ID NO: 4 of the heavy chain constant region is:

[0014] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0015] According to a preferred embodiment, the compound that specifically binds to CD146 provided in the first aspect of the present invention can be a monoclonal antibody.

[0016] The second aspect of the present invention provides a NIR / CT dual-modal imaging probe targeting CD146, comprising: the compound that specifically binds to CD146 provided in the first aspect of the present invention, and near-infrared fluorescent gadolinium (III)-doped carbon dots.

[0017] The third aspect of the present invention provides a method for preparing a NIR / CT dual-modal imaging probe targeting CD146, comprising: mixing the compound specifically binding to CD146 provided in the first aspect of the present invention with hydroxyethyl cellulose to obtain CD146 monoclonal antibody-modified hydroxyethyl cellulose; using L-glutathione, formamide, and gadolinium chloride as raw materials, preparing near-infrared fluorescent gadolinium (III)-doped carbon dots by a solvothermal synthesis method; mixing the CD146 monoclonal antibody-modified hydroxyethyl cellulose with the near-infrared fluorescent gadolinium (III)-doped carbon dots to obtain a probe; dissolving the probe in methanol, adding potassium iodide and sodium hydroxide for reaction, and filtering to obtain the NIR / CT dual-modal imaging probe targeting CD146.

[0018] The fourth aspect of the present invention provides a composition, which comprises the compound provided in the first aspect of the present invention.

[0019] The fifth aspect of the present invention provides a nucleic acid, which encodes the compound provided in the first aspect of the present invention.

[0020] The sixth aspect of the present invention provides a vector, which comprises the nucleic acid provided in the fifth aspect of the present invention.

[0021] The seventh aspect of the present invention provides a host cell, which comprises the vector provided in the sixth aspect of the present invention.

[0022] The eighth aspect of the present invention provides a method for preparing the compound provided in the first aspect of the present invention, the method comprising: culturing the host cell provided in the seventh aspect of the present invention to produce a compound specifically binding to the CD146 protein.

[0023] The ninth aspect of the present invention provides an electrochemical biosensor, which is obtained by disposing the probe provided in the third aspect of the present invention on the surface of a working electrode.

[0024] The tenth aspect of the present invention provides the use of the compound specifically binding to the CD146 protein, the NIR / CT dual-modal imaging probe targeting CD146, the method for preparing the NIR / CT dual-modal imaging probe targeting CD146, the composition, the nucleic acid, the vector, the host cell, the electrochemical biosensor, and the method for preparing the compound specifically binding to the CD146 protein in the preparation of a metastatic lung cancer diagnostic product or reagent.

[0025] Technical effects:

[0026] The present invention provides a novel high-affinity detection antibody for specifically recognizing metastatic lung cancer. Specifically, the NIR / fluorescent imaging probe can specifically recognize the CD146 molecule with metastatic characteristics, effectively solving the problems of insufficient specificity and sensitivity in the prior art for metastatic lung cancer.

[0027] The present invention prepares a NIR / CT dual-modal imaging probe with high sensitivity and specificity for CD146 molecules. It integrates the characteristics of high sensitivity of NIR and high spatial resolution of CT for the detection of tiny metastatic tumor lesions, extracts more imaging details through multi-dimensional information, and provides more comprehensive and accurate tumor detection and evaluation.

[0028] Compared with traditional "continuous emission" probes, the probe prepared by the present invention combines NIR and fluorescence imaging technologies, significantly reduces background fluorescence, enhances sensitivity and image contrast. It can clearly distinguish different tissues, shows excellent photostability and chemical stability, and has more accurate and comprehensive imaging results, etc., improving the diagnostic efficiency and accuracy of metastatic lung cancer.

[0029] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, although the applicant studied a large number of literatures and patents when making the present invention, all details and contents are not listed in detail due to space limitations. However, this does not mean that the present invention does not possess the features of these prior arts. On the contrary, the present invention already possesses all the features of the prior arts, and the applicant reserves the right to add relevant prior arts in the background art. Brief Description of the Drawings

[0030] Figure 1 It is a schematic diagram of the phage display vector provided by the present invention;

[0031] Figure 2 It is the detection result of the binding activity between the MM21 antibody and the CD146 antigen provided by the present invention;

[0032] Figure 3 It is the in-vivo imaging detection result provided by the present invention. Detailed Description of the Invention

[0033] The following is a detailed description in conjunction with the drawings. Example 1

[0034] This example provides a method for constructing and screening a detection antibody against CD146 protein.

[0035] 1. Construction of a CD146-specific phage display library.

[0036] Collect the peripheral blood of metastatic lung cancer patients with high CD146 expression, isolate peripheral blood mononuclear cells (PBMCs), and extract total lymphocyte RNA according to the ratio of Trizol: chloroform = 5:1. Refer to the human full set of antibody gene sequence information provided by the V-BASE website to design primers for PCR amplification of the human full set of antibodies. Obtain cDNA by reverse transcription, and construct light chain full-length and heavy chain fragment gene libraries respectively. Using the pMD19-T vector as a template, design and construct light chain and heavy chain fragment storage vectors. After digestion with Sfi I and BsmB I enzymes, the target gene is ligated with the pComb-3XSSSB-Fab phage surface display vector and linker fragment in a ratio of 1:1:1:1 to construct a CD146-specific phage surface display Fab antibody gene library, and calculate the library capacity and diversity.

[0037] 2. Screen phage antibodies that specifically recognize the CD146 protein from the phage library.

[0038] Based on the principle of biotin-avidin binding reaction, use biotin-labeled CD146 antigen and streptavidin-labeled magnetic beads for the enrichment and screening of anti-CD146 antibodies. The specific steps are as follows: Bind the biotinylated CD146 antigen to streptavidin-coupled magnetic beads in the liquid phase, and use the action of the magnetic field to screen phage antibodies. Take 1 ml of phage library solution, add PBST solution containing 2% skim milk by final concentration and 80 μl of streptavidin-coupled magnetic beads (tube 1), and incubate for 0.5 h on a rotary shaker for blocking; aspirate the solution in tube 1 into a new EP tube, add 1 ml of 5 μg / ml biotinylated CD146 antigen, shake and mix well for 2 h, then add 80 μl of magnetic beads, and incubate for 0.5 h on a rotary shaker at room temperature. Wash 4 times each with 1×PBST and 1×PBS; add 300 μl of glycine-hydrochloric acid, shake and mix well for 15 min; take the supernatant and add it to a new EP tube, neutralize it with 110 μl of buffer with pH = 8.0, and infect 5 ml of TG1 competent cells; take the infected bacterial solution and perform 10-fold serial dilutions, set 8 gradients, and the settings of the 8 gradients are: starting with 10 μl of bacterial solution + 90 μl of PBS as the initial dilution concentration, and label it as tube A; from the mixed liquid in tube A, take 10 μl of the mixed solution + 90 μl of PBS, and label it as tube B;... and so on, set 8 concentration gradients, spread them on LB solid medium, and culture overnight at 37°C to calculate the library capacity. Take 2 ml of the remaining bacterial solution twice and incubate it on LB solid medium overnight at 37°C; take an appropriate amount of colonies from the LB solid medium and dilute and culture them in 2-YT liquid medium containing ampicillin and glucose (OD is about 0.1), shake the bacteria at 37°C until OD 600 = 0.3, add 6×10 13Helper phage infection, incubate in a 37 °C incubator for 0.5 h, then shake and mix for another 0.5 h; after centrifugation to discard the supernatant, resuspend the bacterial pellet with 100 ml of 2-YT liquid medium, and incubate overnight at 30 °C and 250 r / min; precipitate the phage with PEG8000, dissolve the precipitate with 7% DMSO, and store at -80 °C. Perform 2 to 4 rounds of enrichment screening according to the above method, but the CD146 antigen concentration in the 2 to 4 rounds is 1 μg / ml, and there is no need to amplify the phage neutralizing solution eluted after the first round. The screening and enrichment of the Fab phage display library against the CD146 antigen are shown in Table 1.

[0039] Table 1 Screening and enrichment of the Fab phage display library against the CD146 antigen

[0040]

[0041] 3. Affinity screening of phage supernatant activity.

[0042] After four rounds of screening, randomly select 96 clones on LB solid medium, inoculate them into 2-YT liquid medium respectively, culture at 37 °C for 8 h, centrifuge to discard the supernatant, resuspend the pellet with 2-YT liquid medium, and shake overnight at 30 °C and 250 r / min. Determine the affinity of the phage supernatant after overnight incubation with the CD146 antigen by ELISA. The results of ELISA identification of phage supernatant activity are as Figure 1 shown, Figure 1 in which the abscissa 1 to 12 represents the column, and A to H represent the rows in the 96-well plate. The ordinate reflects the binding degree of the antibody to the corresponding ligand. The higher the value, the stronger the binding strength, that is, the higher the affinity. Select positive clones with higher affinity for sequencing analysis. Finally, 23 positive clones with ELISA readings > 2 were obtained. After amino acid sequence alignment, the CD146-specific monoclonal antibody with the highest affinity was finally named MM21.

[0043] 4. Antibody expression and purification.

[0044] Using the plasmid DNA of the extracted MM21 antibody as a template, PCR amplification was performed on the light chain gene and heavy chain gene of the antibody; an appropriate amount of BsmB I and Sfi I were used to digest the antibody heavy chain gene and antibody light chain gene overnight respectively, and separation and purification were carried out by 1% agarose gel electrophoresis; the mammalian cell surface IgG1 full-length antibody display vector pDGB4 was digested with Sfi I and BsmB I successively. The digested antibody heavy chain gene and antibody light chain gene were inserted between the corresponding restriction sites of pDGB4-IgG, and then chemically competent Escherichia coli TG1 was transformed. Random monoclonal colonies were picked and inoculated into LB culture medium for cultivation. When the Escherichia coli was amplified to an appropriate concentration of OD = 2, plasmid extraction was carried out using a plasmid DNA extraction kit. FCHO cells in the logarithmic growth phase were inoculated into 12-well plates in advance. 1 μg of the antibody expression vector and 2.5 μl of the transfection reagent at 1 μg / μl were both diluted with 50 μl of Hanm's-F12 culture medium, and then the two were vortexed and mixed evenly. After 30 min, they were added to the FCHO cells, and the medium was changed after 6 h of cultivation and continued to be cultured for 72 h; to purify the antibody protein, the culture supernatant of FCHO cells collected by centrifugation was added to AmMagᵀᴹ Protein A magnetic beads and incubated overnight, and the antibody bound to the magnetic beads was eluted using a magnetic separation device.

[0045] 5. Detection of the affinity between the antibody and CD146 antigen by ELISA method.

[0046] The indirect ELSIA method was used to coat CD146 protein as the antigen on a 96-well enzyme-linked immunosorbent assay plate. After blocking with the blocking buffer for 1 h, the specific antibody (Ab1) and anti-idiotypic antibody (Ab2) were added to the ELISA plate and incubated with shaking for 1 h; the plate was washed 3 times with PBST, and the HRP enzyme-labeled secondary antibody was added and incubated at room temperature for 30 min; the plate was washed 3 times, the TMB substrate solution was added, and the reaction was terminated after incubating at room temperature for 15 min; the absorbance at 450 nm was read using an enzyme-linked immunosorbent assay reader. The data were analyzed and the curve was fitted using GraphPad Prism8 software to calculate the EC50 value. Figure 2 In it, the abscissa is the corresponding detection concentration of the MM21 antibody, with the unit of (μg / ml). This figure shows the relationship between different MM21 (denoted as MACM-2 in the figure) antibody concentrations and the absorbance at 450 nm. The change in absorbance reflects the binding of the antibody to the specific antigen. As the antibody concentration increases, the absorbance also increases, indicating an increase in the binding of the antibody to the antigen. EC50 is the antibody concentration corresponding to 50% of the maximum absorbance, Figure 2 and is the point where the slope of the curve begins to increase significantly. Specifically, Figure 2The abscissa ranges from 0.01 µg / ml to 10 µg / ml, and the entire reaction process from low concentration to high concentration can be observed. When the antibody concentration is between 0.01 and 0.1 µg / ml, the dose-response curve rises rapidly. Between 0.1 and 10 µg / ml, the dose-response curve gradually flattens. The EC50 value of the MM21 monoclonal antibody in this experimental result is low, at 0.054 μg / ml, and a relatively small amount of antibody can achieve a high binding level, indicating that the MM21 monoclonal antibody has a high affinity for the CD146 antigen. Example 2

[0047] This example provides the exploration process of the NIR / CT dual-modal imaging probe.

[0048] 1. Design and preparation of the NIR / CT imaging probe targeting CD146.

[0049] According to the functional requirements of the probe for targeting in small metastatic lung cancer tissues, long-wave emission, and NIR / CT dual-modal imaging, the CD146 monoclonal antibody was mixed with CMC (carboxymethyl cellulose) and stirred in a phosphate buffer solution at pH = 7.2 for 2 h to prepare the modified CMC of the CD146 monoclonal antibody. Subsequently, 60 ml of HAuCl4 (1 mM) was added to a 250 ml beaker, and 30 ml of 25 mM AgNO3 solution was added. While stirring, 100 ml of 10 mM TPP solution was added. After reacting for 30 min, the supernatant was removed by centrifugation. 100 ml of water was added again, and the supernatant was removed by centrifugation. This step was repeated 3 times, followed by washing with water and removing the supernatant by centrifugation. Finally, 5 ml of 0.01 M PAA solution was added for stabilization treatment. Near-infrared fluorescent gadolinium (III)-doped carbon dots were accurately prepared by a solvothermal synthesis method using L-glutathione, formamide, and GdCl3 (anhydrous gadolinium chloride) as raw materials. In a three-necked flask, 500 µl of the modified CMC of the CD146 monoclonal antibody was added, then 50 µl of OPD and 50 µl of DEA were added. After standing at room temperature for 30 min, 200 µl of PEG was added, and it was left standing for another 10 min. 100 µl of HCl was added, followed by precipitation and washing. Finally, 100 µl of APS was added, and the supernatant was removed by centrifugation. The probe targeting CD146 was dissolved in methanol, and KI and NaOH were added to make their concentrations 6 mM and 0.8 mM respectively. After drying with nitrogen, 1 ml of EG and 4 ml of EtOAc were added. It was vibrated at room temperature for 15 min and then dried with nitrogen. The sample was dissolved in pure water and filtered through a filter membrane to obtain the NIR / CT dual-modal imaging probe.

[0050] 2. Investigation and characterization of the structure and performance of the NIR / CT dual-modal imaging probe targeting CD146.

[0051] (1) By controlling the reaction conditions, intermediates and carbon dots with different degrees of polymerization and carbonization are prepared. The above products are purified by methods such as column chromatography, ultrafiltration, and dialysis. The NIR / CT dual-modal imaging probe targeting CD146 is measured and characterized by techniques such as ultraviolet, infrared, X-ray photoelectron spectroscopy, nuclear magnetic resonance, mass spectrometry, liquid chromatography, absorption cross-section, fluorescence lifetime, quantum yield, and electron microscopy. Parameters such as its photostability and hydrophilicity-hydrophobicity are analyzed to investigate the relationship between its structure and performance. Based on the above, further combined with means such as nuclear magnetic resonance titration and mass spectrometry, the corresponding recognition mechanism of carbon dots for the analyte is verified, providing a theoretical basis for the precise preparation of fluorescent carbon dots and their imaging applications.

[0052] (2)In tumor cell models prone to lung metastasis such as HOS-8603 and H460, the immunobiological activity and cytotoxicity of the NIR / CT dual-modal imaging probe targeting CD146 were detected by CCK-8 or MTT assay. Using BALB / c nude mice as the animal model, the animal injection dose (mg / kg) was calculated according to the following formula: animal injection dose (mg / kg) = cell injection dose (μg / mL) × fluid uptake (mL / kg) ÷ animal body weight (kg). The NIR / CT dual-modal imaging probe targeting CD146 was injected via the tail vein, once every other day, for three consecutive times. After that, the mice were sacrificed at 24 h, and the blood of the mice was collected to detect BNP, liver function (ALT, AST, ALP), and renal function (BUN) to evaluate the safe dose range of its application in vivo in mice. The specific results are shown in Table 2. The main organs (heart, liver, spleen, lung, kidney) of the mice were collected for H&E staining, and no organic changes were found. The experiments in Table 2 included a normal saline group (blank control), a dual-modal imaging probe group at 5 mg / kg, and a dual-modal imaging probe group at 10 mg / kg. The BNP, ALT, AST, ALP, BUN and other indicators of each group were recorded at 6, 12, 24, and 48 h. The results in Table 2 specifically showed the safety of different dose groups at different time points. BNP is used to evaluate cardiac function, especially the situation of heart failure. In Table 2, the BNP levels remained normal at all doses and time points, indicating that cardiac function was not significantly affected. ALT is a sensitive indicator for evaluating hepatocyte damage, AST mainly reflects hepatocyte damage, and ALP can reflect liver biliary obstruction or bone diseases. The ALT, AST, and ALP levels in the two dose groups remained normal at all time points and showed no significant difference compared with the normal saline group. There was no obvious cell damage in the livers of the mice in the dose groups, indicating that the dose of 5 - 10 mg / kg was safe in vivo in mice. BUN is an indicator for evaluating renal function. There was no significant difference between the two dose groups in Table 2 and the normal saline group, and the BUN levels at different time points were normal, indicating normal renal excretion function. Therefore, according to the results in Table 2, the BNP, ALT, AST, ALP, and BUN indicators of the CD146-specific NIR / CT dual-modal imaging probe group were all normal, indicating that the CD146-specific NIR / CT dual-modal imaging probe has high safety, and its use concentration range can be 5 - 10 mg / kg.

[0053] Table 2 Safety analysis of CD146-specific NIR / CT dual-modal imaging probe

[0054]

[0055] Dual-modal imaging and molecular recognition of a NIR / CT dual-modal imaging probe targeting CD146 in metastatic lung cancer. A metastatic lung cancer mouse model was constructed using HOS-8603 cells with luciferase; the NIR / CT dual-modal imaging probe targeting CD146 and the Gd-DTPA chelate contrast agent were injected via the tail vein for NIR or CT imaging. The imaging effects in different tumor animal models were compared, the information that could be provided by NIR and CT imaging was contrasted, and image fusion analysis was performed. The specific experimental results are summarized in Table 3. Table 3 lists different types of tumors and their lung metastasis situations, including osteosarcoma, pancreatic cancer, breast cancer, renal cell carcinoma, melanoma, and colon cancer. Among them, the positive number represents the number of tumor metastases successfully identified by the imaging method. Compared with the case numbers of osteosarcoma, pancreatic cancer, breast cancer, renal cell carcinoma, melanoma, and colon cancer, both the positive number of CT imaging and the positive number of NIR imaging were significantly reduced, indicating that the sensitivity or specificity of these two imaging techniques was relatively low and their effects in detecting lung metastasis were limited. However, the positive number of the NIR / CT dual-modal imaging probe significantly exceeded both the positive number of CT imaging and the positive number of NIR imaging, and basically all approached the corresponding case numbers. Therefore, the results in Table 3 show that in different tumor types (such as lung metastasis of osteosarcoma, lung metastasis of pancreatic cancer, lung metastasis of breast cancer, lung metastasis of renal cell carcinoma, lung metastasis of melanoma, and lung metastasis of colon cancer), the recognition specificity of the NIR / CT dual-modal imaging probe was significantly higher than that of CT imaging and NIR imaging, showing a higher positive number in the recognition of multiple tumor types. This indicates that the NIR / CT dual-modal imaging probe has higher sensitivity or specificity and is more effective in detecting lung metastasis.

[0056] Figure 3 In the results, the high luminescence brightness property of the NIR / CT dual-modal imaging probe ensured that the probe could provide clear and bright signals during imaging even at extremely low concentrations, greatly improving the sensitivity and signal-to-noise ratio of imaging. This property was particularly important in the early diagnosis of diseases (especially in tumor detection) and the detection of minute lesions, allowing doctors to accurately identify them at the initial stage of pathological changes. The targeting advantage of the NIR / CT dual-modal imaging probe in this example was reflected in its ability to specifically bind to target cells or tissues, reducing the background noise caused by non-specific binding. This high degree of specificity allowed the probe to penetrate the complex biological environment and directly locate to the target position or biomarker of interest. The imaging effect of the NIR / CT dual-modal imaging probe was good, and this advantage was achieved through the balance of the luminescence property and biocompatibility of the probe. Figure 3In this case, the probe exhibits excellent performance in different imaging modes. Whether it is fluorescence microscopy, confocal microscopy, or in vivo imaging, it can provide high-quality images, clearly revealing the details of biological processes. Photostability is another major feature of the NIR / CT dual-modal imaging probe. During the long imaging process, the NIR / CT dual-modal imaging probe shows good photostability, reducing the impact of photobleaching and ensuring the persistence and reliability of signals during imaging. This is particularly important for detections that require long exposure or continuous imaging, ensuring the stability and repeatability of the detection data. Therefore, the NIR / CT dual-modal imaging probe targeting CD146 provided in this embodiment has the characteristics of high luminescence brightness, good targeting, good imaging effect, and good photostability, and is suitable for the specific recognition of early metastatic lung cancer. In addition, the NIR / CT dual-modal imaging probe targeting CD146 will play an important role in the fields of precision medicine, drug screening, and biological monitoring.

[0057] Table 3 Specific recognition of CD146-specific NIR / CT dual-modal imaging probe and early lung metastatic tumor tissues of different tumor types by fluorescence imaging and CT imaging

[0058]

[0059] It should be noted that the above specific embodiments are exemplary. Those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also fall within the scope of the disclosure of the present invention and within the protection scope of the present invention. Those skilled in the art should understand that the description of the present invention and its drawings are illustrative and do not constitute a limitation on the claims. The protection scope of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts. For example, "preferably" and "according to a preferred embodiment" indicate that the corresponding paragraphs disclose an independent concept. The applicant reserves the right to file divisional applications according to each inventive concept. Throughout the text, the features guided by "preferably" are only optional ways and should not be understood as must be set. Therefore, the applicant reserves the right to waive or delete relevant preferred features at any time.

Claims

1. A NIR / CT dual-modal imaging probe targeting CD146, characterized in that, It is prepared by the following method: Mix the compound specifically binding to CD146 with hydroxyethyl cellulose to obtain CD146 monoclonal antibody modified hydroxyethyl cellulose; Using L-glutathione, formamide and GdCl3 (gadolinium(III) chloride anhydrous) as raw materials, precisely prepare near-infrared fluorescent gadolinium(III)-doped carbon dots by solvothermal synthesis method, Mix CD146 monoclonal antibody modified hydroxyethyl cellulose with near-infrared fluorescent gadolinium(III)-doped carbon dots to obtain an NIR / CT dual-modal imaging probe, Among them, the compound specifically binding to CD146 is a CD146 monoclonal antibody, including: A light chain variable domain, the amino acid sequence of the light chain variable domain is as shown in SEQ ID NO: 1; A light chain constant region, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 3; A heavy chain variable domain, the amino acid sequence of the heavy chain variable domain is as shown in SEQ ID NO: 2, And a heavy chain constant region, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 4, Among them, the EC50 of the compound is 0.054 μg / ml.

2. A preparation method of a NIR / CT dual-modal imaging probe targeting CD146, characterized in that, Including: Mix the compound specifically binding to CD146 with hydroxyethyl cellulose to obtain CD146 monoclonal antibody modified hydroxyethyl cellulose; Using L-glutathione, formamide and gadolinium chloride as raw materials, prepare near-infrared fluorescent gadolinium(III)-doped carbon dots by solvothermal synthesis method; Mix CD146 monoclonal antibody modified hydroxyethyl cellulose with near-infrared fluorescent gadolinium(III)-doped carbon dots to obtain a probe; Dissolve the probe in methanol, add potassium iodide and sodium hydroxide to react, and After filtration, obtain an NIR / CT dual-modal imaging probe targeting CD146, Among them, the compound specifically binding to CD146 includes: A light chain variable domain, the amino acid sequence of the light chain variable domain is as shown in SEQ ID NO: 1; A light chain constant region, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 3; A heavy chain variable domain, the amino acid sequence of the heavy chain variable domain is as shown in SEQ ID NO: 2, And a heavy chain constant region, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 4, Among them, the EC50 of the compound is 0.054 μg / ml.

3. Application of the probe according to claim 1 and the preparation method according to claim 2 in the preparation of metastatic lung cancer diagnostic products.

4. The application according to claim 3, characterized in that, The metastatic lung cancer diagnostic product is a metastatic lung cancer diagnostic reagent.

Images