A pd-l1 guided specific targeting ctc detection binding tumor boundary distinguishing biological probe and a preparation method thereof

Abstract

The application belongs to the technical field of biological probes, and relates to a PD-L1 guided specific targeted CTC detection combined with tumor boundary distinguishing biological probe and a preparation method thereof. The PD-L1 guided specific targeted CTC detection combined with tumor boundary distinguishing biological probe comprises a core, a covalent organic framework and a PD-L1 antibody, the core comprises a superparamagnetic nanoparticle, the covalent organic framework is coated on the surface of the core, and the PD-L1 antibody is combined on the surface of the covalent organic framework. The PD-L1 guided specific targeted CTC detection combined with tumor boundary distinguishing biological probe prepared by the application can be transported into the body across the membrane, smoothly enter a PD-L1 positive malignant tumor lesion, and perform Raman imaging to microscopically distinguish the tumor boundary.

Description

Technical Field

[0001] This invention belongs to the field of biological probe technology, and relates to a PD-L1-guided biological probe for specific targeting of CTC detection combined with tumor boundary differentiation and its preparation method. Background Technology

[0002] The incidence and mortality rates of cancer in middle-aged and elderly patients are increasing year by year. Chemotherapy and surgery severely reduce the quality of life for patients in their later years. Therefore, research on immunotherapy has become a major trend in cancer treatment to alleviate patient suffering. Existing research has demonstrated that early diagnosis and accurate differentiation of tumor boundaries play an important role in the treatment and prognostic assessment of malignant tumors. Among them, programmed cell death ligand 1 (PD-L1) plays a unique role. PD-L1 refers to cytokines that can bind to programmed cell death receptor 1. It can transmit inhibitory signals, inhibit the proliferation of CD8+ T cells, and indirectly regulate the Bcl-2 gene, inhibiting the aggregation of antigen-specific T cells in lymph nodes.

[0003] Currently, in clinical practice, to clarify the diagnosis of malignant tumors, assess prognosis, formulate treatment plans, and evaluate efficacy, most cases require puncture biopsy to obtain pathological specimens. However, because puncture biopsy is an invasive procedure, it can sometimes lead to bleeding and infection, and the removal of too much tissue affects appearance, making it unacceptable to some patients. Furthermore, puncture biopsy is difficult to dynamically evaluate the progression of malignant lesions and treatment effectiveness. In particular, the breast is an organ rich in glandular and lymphatic tissue, making imaging examinations prone to false negatives. When clinical manifestations and laboratory tests show abnormalities, the pathological changes in the breast may have already entered the metastatic stage and become irreversible. With rising living standards, the incidence of colorectal cancer is also increasing, and it is often diagnosed with systemic metastatic symptoms. Therefore, clinical practice needs a non-invasive, sensitive, patient-acceptable evaluation method that can dynamically monitor the pathological changes of malignant tumors. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a PD-L1-guided biological probe for the detection of specific targeted CTCs combined with tumor boundary differentiation, and its preparation method.

[0005] One objective of this invention is achieved through the following technical solution:

[0006] A PD-L1-guided specific targeted CTC detection and tumor boundary differentiation biological probe includes a core, a covalent organic framework, and a PD-L1 antibody. The core comprises superparamagnetic nanoparticles, the covalent organic framework is coated on the surface of the core, and the PD-L1 antibody is bound to the surface of the covalent organic framework.

[0007] Preferably, the superparamagnetic nanoparticles are oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm.

[0008] Preferably, the preparation method of the oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm includes the following steps:

[0009] A polar solvent, cis-9-octadecenoic acid, and an inorganic base were mixed to obtain a mixture. Then, an aqueous solution of ferrous ammonium sulfate was added to the mixture, and the mixture was heated at 100–500 °C for 1–20 h. The supernatant was removed, and the precipitate was dispersed in a non-polar solvent. After washing, the precipitate was stored in a non-polar solvent to obtain oil-phase superparamagnetic Fe3O4 nanoparticles.

[0010] Preferably, the polar solvent includes one or more of anhydrous ethanol, methanol, acetonitrile, and acetone;

[0011] Preferably, the inorganic base includes one or more of sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, and copper hydroxide;

[0012] Preferably, the nonpolar solvent includes one or more of cyclohexane, hexane, benzene, carbon tetrachloride, dichloroethane, and dichloromethane.

[0013] Preferably, the covalent organic framework is TAPB-BTCA COF, which is prepared from monomers comprising TAPB and BTCA.

[0014] Preferably, the step of coating the core surface with a covalent organic framework includes: adding the core, TAPB and BTCA to acetonitrile, followed by adding acetic acid, stirring at room temperature, and centrifuging to obtain a covalent organic framework-coated core.

[0015] Preferably, the thickness of the covalent organic framework is 1–5 nm;

[0016] Preferably, the particle size of the PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation biological probe is 5–20 nm.

[0017] As a preferred option, the molar ratio of superparamagnetic nanoparticles, covalent organic frameworks and PD-L1 antibody is (250-310):(100-150):(50-70).

[0018] Preferably, the core also includes Raman signal molecules, which are attached to the surface of the superparamagnetic nanoparticles.

[0019] Another objective of this invention is achieved through the following technical solution:

[0020] A method for preparing a PD-L1-guided bioprobe for specific targeting of CTCs and tumor boundary differentiation includes the following steps:

[0021] (1) Prepare superparamagnetic nanoparticles and attach Raman signal molecules to the surface of the superparamagnetic nanoparticles;

[0022] (2) Add the product of step (1), TAPB and BTCA to acetonitrile, then add acetic acid, stir at room temperature for 12 to 48 hours, and centrifuge to obtain the covalent organic framework-coated core.

[0023] (3) Add PD-L1 antibody to the covalent organic framework-coated core, stir, centrifuge, and resuspend in phosphate buffer to obtain a PD-L1-guided biological probe for specific targeting of CTC detection and tumor boundary differentiation.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The PD-L1-guided specific targeted CTC detection combined with the tumor boundary differentiation biological probe provided by the present invention utilizes the high concentration of the specific antibody component PD-L1 in the target area, and utilizes the Raman signal enhancement properties of oil-phase superparamagnetic nanoparticles to form a specific Raman signal image in the target tissue, thereby distinguishing the tumor boundary at a microscopic level. The oil-phase superparamagnetic nanoparticles have magnetization properties, which can magnetically separate PD-L1-positive circulating tumor cells, and perform Raman detection on the separated CTCs.

[0026] 2. The PD-L1-guided, oil-phase superparamagnetic nanoparticle-based specific targeted CTC detection combined with tumor boundary differentiation bioprobe of this invention can be transported across the membrane into the body, successfully entering PD-L1-positive malignant tumor lesions for Raman imaging. This eliminates the need for puncture biopsy, thus avoiding serious complications such as bleeding, infection, and even massive hemorrhage and sepsis. It also allows for dynamic evaluation of breast lesion progression and treatment efficacy. This non-invasive, sensitive, and patient-acceptable evaluation method can dynamically monitor breast pathological changes. Using Raman dynamic monitoring, the PD-L1-guided, oil-phase superparamagnetic nanoparticle-based specific targeted CTC detection combined with tumor boundary differentiation bioprobe directly reflects and quantifies the deposition status of PD-L1 in triple-negative breast cancer, colorectal cancer, and lung cancer, monitoring the lesion status of malignant tumors.

[0027] 3. The oil-phase superparamagnetic Fe3O4 nanoparticles of the PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation biological probe of the present invention have a particle size that is preferably ≤15nm. The ultra-small particle size is more conducive to transmembrane transport.

[0028] 4. The superparamagnetic nanoparticles in the core of this invention are oil-phase superparamagnetic Fe3O4 nanoparticles. The aggregation effect of the oil-phase Fe3O4 particles enhances the signal of the surface-connected SERS signal molecules, making SERS detection more stable.

[0029] 5. The covalent organic framework coated on the core surface of this invention is TAPB-BTCA COF, which is prepared by monomers including TAPB and BTCA. The covalent organic framework has good solubility and is coated on the core surface. The benzene ring structure of COF can covalently bind to the specific antibody component, thereby stably binding the core and the specific antibody component together, improving the stability and safety of the PD-L1 guided biological probe for specific targeting of CTC detection and tumor boundary differentiation using oil-phase superparamagnetic nanoparticles as carriers.

[0030] 6. The present invention employs a method for preparing a PD-L1-guided biological probe for the specific targeting of CTCs and the differentiation of tumor boundaries using oil-phase superparamagnetic nanoparticles as carriers. The resulting biological probe is stable in properties, uniform in size, with a particle size of 5–20 nm, and has excellent penetrating power, enabling it to successfully enter PD-L1-positive tumor lesions. Furthermore, the preparation process is simple and the cost of large-scale production is low. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the preparation process of the PD-L1-guided biological probe for specific targeting of CTC detection and tumor boundary differentiation using oil-phase superparamagnetic nanoparticles as carriers, as described in Example 1 of the present invention.

[0032] Figure 2 TEM image of the oil-phase superparamagnetic nanoparticles prepared in Example 1;

[0033] Figure 3 The image shows a comparison of HRTEM images of the PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation biological probe needles in Example 1 and Comparative Example 1 of the present invention.

[0034] Figure 4 The Raman spectra of the PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation biological probes of Example 1 and Comparative Example 1 of the present invention are compared.

[0035] Figure 5 This is a fluorescence confocal image of the PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation biological probe of Example 1 of the present invention;

[0036] Figure 6Raman spectra of different concentrations of MDA-MB-231 in rabbit blood were measured using a PD-L1-guided specific targeted CTC detection combined with a biological probe for tumor boundary differentiation in Example 1 of this invention.

[0037] Figure 7 Raman spectra of blood samples from breast cancer patients and healthy individuals obtained by combining PD-L1-guided specific targeted CTC detection with biological probes for tumor boundary differentiation in Embodiment 1 of the present invention.

[0038] Figure 8 This invention provides an example of a PD-L1-guided bioprobe for the detection of specific CTCs using oil-phase superparamagnetic nanoparticles as a carrier, combined with tumor boundary differentiation, used for T2 imaging of triple-negative breast cancer in Balb / c female nude mice.

[0039] Figure 9 The PD-L1-guided bioprobe for the detection of specific targeted CTCs using oil-phase superparamagnetic nanoparticles as carriers, combined with tumor boundary differentiation, is used in Balb / c female nude mice for the differentiation of triple-negative breast cancer tumor boundaries.

[0040] Figure 10 for Figure 9 Raman spectra measured at selected boundary points. Detailed Implementation

[0041] In the following description, embodiments of the PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation biological probe and its preparation method of the present invention will be described in detail. However, these embodiments are exemplary and the disclosure of the present invention is not limited thereto.

[0042] The following details the PD-L1-guided biological probe for specific targeting of CTCs combined with tumor boundary differentiation.

[0043] In some embodiments of the present invention, the PD-L1-guided specific targeting CTC detection combined with tumor boundary differentiation biological probe includes a core, a covalent organic framework (COF), and a PD-L1 antibody. The core includes superparamagnetic nanoparticles, the covalent organic framework is coated on the surface of the core, and the PD-L1 antibody is bound to the surface of the covalent organic framework.

[0044] This invention selects programmed cell death ligand 1 (PCD1), which is highly expressed in triple-negative breast cancer lesions but not in normal breast tissue and low in other breast cancers, as an imaging target. A PD-L1-guided bioprobe, using superparamagnetic nanoparticles as a carrier, was synthesized for the specific targeting of CTCs and tumor boundary differentiation. This bioprobe utilizes the high concentration of the specific antibody component PD-L1 in the target area and leverages the Raman signal properties of the superparamagnetic nanoparticles to create a specific Raman signal image in the target tissue, enabling microscopic differentiation of tumor boundaries. Furthermore, the superparamagnetic nanoparticles possess magnetization properties, allowing for magnetic separation of CTCs for Raman detection.

[0045] The superparamagnetic nanoparticles refer to nanoscale particles with magnetic responsiveness, preferably one or more of superparamagnetic Fe2O3 nanoparticles and superparamagnetic Fe3O4 nanoparticles.

[0046] The particle size of the superparamagnetic nanoparticles is preferably ≤15nm, and can be any value or a range between any two values ​​from 2, 5, 9, 10, 11, 12, 13, 14, to 15nm, specifically 7–15nm. Superparamagnetic nanoparticles with a particle size ≤15nm are more easily transported across the membrane.

[0047] Preferably, the superparamagnetic nanoparticles are oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm. Oil-phase superparamagnetic nanoparticles are more likely to aggregate, and the Raman spectral signal generated by the PD-L1-guided specific targeted CTC detection using oil-phase superparamagnetic nanoparticles as carriers combined with the tumor boundary differentiation biological probe of this invention is stronger and more stable.

[0048] Preferably, the preparation method of the oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm includes the following steps:

[0049] A polar solvent, cis-9-octadecenoic acid, and an inorganic base were mixed to obtain a mixture. Then, an aqueous solution of ferrous ammonium sulfate was added to the mixture, and the mixture was heated at 100–500 °C for 1–20 h. The supernatant was removed, and the precipitate was dispersed in a non-polar solvent. After washing, the precipitate was stored in a non-polar solvent to obtain oil-phase superparamagnetic Fe3O4 nanoparticles.

[0050] In the above method for preparing oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm, the polar solvent includes one or more of anhydrous ethanol, methanol, acetonitrile, acetone, etc.

[0051] In the above method for preparing oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm, the inorganic base includes one or more of sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, and copper hydroxide.

[0052] In the above method for preparing oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm, the nonpolar solvent includes one or more of cyclohexane, hexane, benzene, carbon tetrachloride, dichloroethane, and dichloromethane.

[0053] In the above method for preparing oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm, a polar solvent such as anhydrous ethanol is used to clean the precipitate.

[0054] In the above method for preparing oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm, the volume ratio of polar solvent to cis-9-octadecenoic acid is 0.8~1.2:1; the mass ratio of inorganic base to ferrous ammonium sulfate is 1:0.4~1.

[0055] The covalent organic framework coats the core surface, with a preferred thickness of 1–5 nm. According to this preferred scheme, the biological probe that specifically targets CTCs and distinguishes tumor boundaries has strong penetrating power, enabling it to successfully reach tumors with high PD-L1 expression for CTC detection and Raman imaging.

[0056] The covalent organic framework is TAPB-BTCA COF, which comprises TAPB (1,3,5-tris(4-aminophenyl)benzene, with the chemical formula C... 24 H 21 The sample was prepared from monomers of N3 and BTCA (1,3,5-benzaldehyde, chemical formula C9H6O3). According to this preferred scheme, the covalent organic framework (COF) is highly soluble and coats the core surface. The benzene ring structure of the COF can covalently bind to the specific antibody PD-L1, thereby stably binding the core to the specific antibody PD-L1 and improving the stability and safety of the PD-L1-guided specific targeted CTC detection bioprobe combined with tumor boundary differentiation.

[0057] The steps for coating a core surface with a covalent organic framework can include: adding the core, TAPB, and BTCA to acetonitrile, followed by the addition of acetic acid, stirring at room temperature, and centrifuging to obtain a covalent organic framework-coated core. The stirring time at room temperature can range from 12 to 48 hours.

[0058] The molar ratio of TAPB to BTCA can be listed as 0.9 to 1.2:1.

[0059] The PD-L1 antibody is preferably a monoclonal PD-L1 antibody. The advantages of using a monoclonal PD-L1 antibody are high specificity, high uniformity and high reproducibility, which is beneficial for Raman imaging and CTC detection using biological probes that distinguish tumor boundaries and specifically target CTCs with PD-L1 antibodies.

[0060] The preferred molar ratio of superparamagnetic nanoparticles, covalent organic framework, and PD-L1 antibody is (250–310):(100–150):(50–70). In this text, the values ​​in parentheses include endpoint values. The PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation bioprobe formed according to the above molar ratio exhibits superior stability. The covalent organic framework effectively binds the core and specific antibody components together. Furthermore, the PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation bioprobe using oil-phase superparamagnetic nanoparticles as a carrier enables precise localization, accurate qualitative analysis, and efficacy detection of diseased cells.

[0061] The preferred particle size of the PD-L1-guided specific targeted CTC detection and tumor boundary differentiation bioprobe is 5–20 nm. According to this preferred scheme, the PD-L1-guided specific targeted CTC detection and tumor boundary differentiation bioprobe particles have a moderate particle size and excellent penetrability, enabling transmembrane transport and successful entry into PD-L1-highly expressed malignant tumor lesions for Raman imaging.

[0062] In some embodiments of the present invention, the core further includes Raman signal molecules attached to the surface of superparamagnetic nanoparticles. The Raman signal molecules are organic compounds exhibiting conjugated vibrations in the Raman spectrum, including but not limited to one or more of alizarin red (AR), mercaptopyridine, 4-mercaptoaniline, mercaptonaphthalene, p-fluorothiophenol, rhodamine, crystal violet, 4-mercaptobenzoic acid (4-MBA), and Nell blue.

[0063] The following details the preparation method of the PD-L1-guided specific targeted CTC detection and tumor boundary differentiation biological probe of the present invention.

[0064] In some embodiments of the present invention, the preparation method of the PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation biological probe includes the following steps:

[0065] (1) Prepare superparamagnetic nanoparticles and attach Raman signal molecules to the surface of the superparamagnetic nanoparticles;

[0066] (2) Add the product of step (1), TAPB and BTCA to acetonitrile, then add acetic acid, stir at room temperature for 12 to 48 hours, and centrifuge to obtain the covalent organic framework-coated core.

[0067] (3) Add PD-L1 antibody to the covalent organic framework-coated core, stir for 3 to 30 hours, centrifuge and resuspend in phosphate buffer to obtain a PD-L1-guided specific target CTC detection and tumor boundary differentiation biological probe.

[0068] The superparamagnetic nanoparticles in step (1) are preferably oil-phase superparamagnetic Fe3O4 nanoparticles, and more preferably oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm. The preparation method of oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15nm is preferably including the following steps: a polar solvent, cis-9-octadecenoic acid and an inorganic base are mixed to obtain a mixed solution, and then an aqueous solution of ferrous ammonium sulfate (Fe(NH4)2(SO4)2·6H2O) is added to the above mixed solution. The mixture is heated at 100-500℃ for 1-20h, the supernatant is removed, the precipitate is dispersed with a non-polar solvent, washed and stored in a non-polar solvent to obtain oil-phase superparamagnetic Fe3O4 nanoparticles.

[0069] Step (1) involves attaching Raman signal molecules to the surface of superparamagnetic nanoparticles, which includes: adding the superparamagnetic nanoparticles to a Raman signal molecule solution and stirring the reaction. The Raman signal molecule solution is formed by dissolving the Raman signal molecules in a solvent; any solvent capable of dissolving Raman signal molecules is acceptable, such as water, ethanol, tetrahydrofuran, etc., and the concentration of the Raman signal molecule solution is not limited. The stirring reaction is carried out at a temperature of 5–40°C, preferably at room temperature, and the stirring reaction time is 5–60 min.

[0070] In step (3), the pH of the phosphate buffer solution is 7.0 to 7.5.

[0071] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the specific scope of the present invention. Furthermore, the accompanying drawings used herein are merely for better illustrating the content disclosed in the present invention and do not limit the scope of protection. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0072] Example 1

[0073] The schematic diagram of the preparation process of the PD-L1-guided bioprobe for specific targeting of CTCs using oil-phase superparamagnetic nanoparticles as carriers, combined with tumor boundary differentiation, in this embodiment is shown below. Figure 1 As shown, the specific steps include:

[0074] (1) Preparation of oil-phase superparamagnetic Fe3O4 nanoparticles with SERS function

[0075] Take 10 mL of anhydrous ethanol and cis-9-octadecenoic acid and add them to a 50 mL beaker. Add 1 g of NaOH and stir at 600 rpm until dissolved to obtain a mixture. Then take 0.78 g of Fe(NH4)2(SO4)2·6H2O and dissolve it in 20 mL of water. Slowly add the solution to the mixture using a dropper. After it is completely dissolved, transfer it to a reaction vessel and heat it at 230 °C for 10 h. Then, take the supernatant, disperse the precipitate with cyclohexane, wash it three times with ethanol, and store it in 10 mL of cyclohexane.

[0076] (2) Preparation of oil-phase Fe3O4 (Fe3O4@AR) linked with Raman signal molecules

[0077] Alizarin Red was dissolved in water to obtain 1*10 -3 Take 10 mL of Alizarin Red (AR) solution M and add it to 10 mL of oil phase Fe3O4. Stir for 16 h, centrifuge and wash three times at 10000 rpm and 15 min to obtain oil phase Fe3O4@AR nanoparticles.

[0078] (3) Encapsulation of covalent organic framework materials

[0079] Take 0.0299 mmol of the oil phase Fe3O4@AR obtained in step (2) (calculated based on iron content), add 17 mL of acetonitrile, 0.006 mmol of BTCA and 0.006 mmol of TAPB, add 2 mL of acetic acid, stir at room temperature for 24 h, centrifuge at 12000 rpm for 20 min, take the precipitate and resuspend it in 1 mL of Tris-HCl buffer to obtain the oil phase Fe3O4@AR@COF.

[0080] (7) Conjugation of surface antibodies

[0081] Add 10 μL of 1.095 mg / mL monoclonal PD-L1 antibody to the product of step (3), stir for 12 hours, centrifuge 3 times at 10000 rpm and 10 min, and resuspend the precipitate in pH 7.4 PBS buffer to obtain the final PD-L1-guided biological probe for the detection of CTCs and the differentiation of tumor boundaries using oil-phase superparamagnetic nanoparticles as carriers.

[0082] Comparative Example 1

[0083] The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, step (1) involves the preparation of aqueous superparamagnetic Fe3O4 nanoparticles. The specific steps of step (1) are as follows:

[0084] Take 10 mL of anhydrous ethanol and cis-9-octadecenoic acid and add them to a 50 mL beaker. Add 1 g of NaOH and stir at 600 rpm until dissolved to obtain a mixture. Then, dissolve 0.78 g of Fe(NH4)2(SO4)2·6H2O in 20 mL of water and slowly add it to the mixture using a dropper. After complete dissolution, transfer the mixture to a reaction vessel and heat at 230 °C for 10 h. Then, collect the supernatant, disperse the precipitate with cyclohexane, wash three times with ethanol, and store it in 10 mL of cyclohexane. Then, convert the oil phase Fe3O4 to the aqueous phase. Add the above solution to a long, narrow glass bottle, then add 0.2 g of citric acid, 5 mL of DMF, and 5 mL of chloroform. Seal the bottle with a sealing film and sonicate for 6 h. Then wash three times with ethanol. Finally, dissolve Fe3O4 in 10 mL of deionized water. The subsequent steps are the same as in Example 1, to prepare a PD-L1-guided biological probe for the specific targeting of CTC detection and tumor boundary differentiation using aqueous superparamagnetic nanoparticles as carriers.

[0085] Figure 2 The image shows a TEM image of the oil-phase superparamagnetic nanoparticles prepared in Example 1, with a particle size of approximately 8–12 nm. Figure 3 (1) is an HRTEM image of the PD-L1-guided bioprobe for specific CTC detection and tumor boundary differentiation using oil-phase superparamagnetic nanoparticles as a carrier, as described in Example 1. As can be seen from the image, the particle size of the PD-L1-guided bioprobe for specific CTC detection and tumor boundary differentiation using oil-phase superparamagnetic nanoparticles as a carrier is approximately 10–18 nm. Figure 3 (2) HRTEM images of the PD-L1-guided bioprobe for the detection of specific CTCs using aqueous superparamagnetic nanoparticles as a carrier, combined with tumor boundary differentiation, as shown in Comparative Example 1. (Comparison) Figure 3 (1) and Figure 3 (2) It can be seen that oil-phase superparamagnetic nanoparticles are more likely to aggregate together. Figure 4 (1) is the Raman spectrum of the PD-L1-guided biological probe for the detection of specific CTCs using oil-phase superparamagnetic nanoparticles as a carrier, combined with tumor boundary differentiation, as described in Example 1. Figure 4 (2) The Raman spectrum of the PD-L1-guided bioprobe for the detection of specific CTCs using aqueous superparamagnetic nanoparticles as a carrier, combined with tumor boundary differentiation, is shown in Comparative Example 1. (By comparison...) Figure 4 (1) and Figure 4(2) It can be seen that the Raman spectral signal of the PD-L1-guided CTC detection with oil-phase superparamagnetic nanoparticles as carrier combined with the tumor boundary differentiation biological probe is stronger and more stable. Figure 5 (1) A fluorescence confocal image of the biological probe for PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation in Example 1 (wherein, the left image is the cell nucleus of MDA-MB-231 breast cancer cells, the middle image is the biological probe for PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation, and the right image is the merge map of the cell nucleus of MDA-MB-231 breast cancer cells combined with the biological probe for PD-L1-guided specific targeted CTC detection combined with tumor boundary differentiation); combined with Figure 5 (2) (where the left figure is the nucleus of MCF-7 breast cancer cells, the middle figure is the biological probe for PD-L1-guided specific targeting CTC detection combined with tumor boundary differentiation, and the right figure is the PD-L1-guided specific targeting CTC detection combined with tumor boundary differentiation biological probe combined with the nucleus Merge diagram of MCF-7 breast cancer cells) It can be seen that the uptake of the probe by MDA-MB-231 with high PD-L1 expression is higher than that by MCF-7 with low PD-L1 expression.

[0086] Application Example 1

[0087] Rabbit blood CTC detection

[0088] The PD-L1-guided bioprobe for the detection of specific CTCs using oil-phase superparamagnetic nanoparticles as carriers, prepared in Example 1, was prepared into a 300 μg / mL solution using pH 7.4 phosphate buffer.

[0089] Seven mL of blood was collected from the marginal ear vein of a healthy rabbit and placed in a vacuum anticoagulant tube, then shaken for 3 minutes to prevent clotting. One mL of MDA-MB-231 breast cancer cells at different concentrations (500 Cells / mL, 200 Cells / mL, 100 Cells / mL, 50 Cells / mL, 10 Cells / mL) dispersed in PBS was taken separately and mixed with one mL of fresh rabbit blood. The mixture was then slowly added dropwise to one mL of lymphocyte separation medium. The sample separated into two layers: the upper layer was rabbit blood containing tumor cells, and the lower layer was lymphocyte separation medium. Centrifugation at 1500 rpm for 25 min resulted in four layers based on cell density: an upper layer of plasma, an upper-middle layer of lymphocytes and monocytes, a lower-middle layer of clear lymphocyte separation medium, and a lower layer of erythrocytes. Circulating tumor cells were located in the lymphocyte layer. The lymphocyte layer was removed using a pipette, mixed with 2 mL of PBS, and centrifuged at 1500 rpm for 5 min, followed by washing three times. Finally, the sample was dispersed in 200 μL of LMEM complete culture medium (10% FBS, 1% PS), and 200 μL of 300 μg / mL nanoprobe was added. The mixture was incubated at 37°C for 30 min. After incubation, the sample was centrifuged and washed three times at 1000 rpm for 5 min to remove excess probe that had not bound to cancer cells. The washed sample was then placed on a clean glass slide for Raman spectroscopy. Detection of different concentrations of circulating tumor cells (CTCs) was performed as follows: Figure 6 As shown, the nanoprobe of the present invention can bind to CTC and effectively detect CTC.

[0090] CTC testing in patients with triple-negative breast cancer (TNBC)

[0091] Blood samples of 1 mL were collected from two triple-negative breast cancer patients and two healthy individuals and placed in vacuum anticoagulant tubes, shaken for 3 minutes to prevent clotting. The blood was then slowly added dropwise to 1 mL of lymphocyte separation medium. At this point, the sample separated into two layers: the upper layer was human blood, and the lower layer was lymphocyte separation medium. Centrifugation was performed at 1500 rpm for 25 min, and the sample separated into four layers based on cell density (upper layer: plasma; upper middle layer: lymphocytes and monocytes; lower middle layer: clear lymphocyte separation medium; lower layer: erythrocytes). Circulating tumor cells were located in the lymphocyte layer. The lymphocyte layer was removed using a pipette, mixed with 2 mL of PBS, and centrifuged at 1500 rpm for 5 min, washing three times. Finally, the sample was dispersed in 200 μL of DMEM complete culture medium (10% FBS, 1% PS), and 200 μL of 300 μg / mL nanoprobes were added. The mixture was incubated at 37°C for 30 min, followed by centrifugation at 1000 rpm for 5 min, washing three times to remove excess probes that had not bound to cancer cells. After centrifugation and washing, the sample was dropped onto a clean glass slide for Raman spectroscopy. The CTC detection results from the blood of triple-negative breast cancer patients and healthy individuals are presented as Raman spectra. Figure 7 As shown.

[0092] Boundary differentiation in a BALB / c female nude mouse triple-negative breast cancer model

[0093] The PD-L1-guided bioprobe for the detection of specific CTCs using oil-phase superparamagnetic nanoparticles as carriers, prepared in Example 1, was prepared into a 300 μg / mL solution using pH 7.4 phosphate buffer and then injected into the test subject, typically at a dose of 60 μg / kg.

[0094] Before performing Raman imaging, it is necessary to explore the drug accumulation time in mice. Since the core of this probe is superparamagnetic iron oxide, MR imaging was performed to determine the time point. The BALB / c female nude mouse breast cancer model was established using the internationally accepted in situ injection method. The modeling process was as follows: MDA-MB-231 breast cancer cells were injected subcutaneously into the anterior upper epithelium of the right leg of BALB / c female nude mice. The model was successfully established on day 7; subsequently, the model was used for in vivo MR imaging. The MR imaging system was a German SEMENS 3.0TMR, with a horizontal scanning gantry inner diameter of 16 cm and a 38 mm rat head coil. After anesthesia with 4% isoflurane, the mice were placed in an acrylic scanning bed, and the brains were fixed using dental braces and ear rods. Anesthesia was maintained with a 1.5% isoflurane:air mixture, and heart rate and respiration were monitored. The scanning range covered the subcutaneous tumors of the mouse breast cancer. The scanning employed a prone T2-PD dual-echo MSME (mμlti slices mμlti echo) spin-echo sequence with the following parameters: TR 4000ms, TE 90ms; slice thickness 2mm, interslice spacing 0, number of slices 25, FOV 60mm×48mm, acquisition times 3, matrix 256×256, minimum spatial resolution 998μm×98μm, and scanning time approximately 30min. Mice were injected via tail vein with a PD-L1-guided bioprobe buffer for detecting and distinguishing tumor boundaries using oil-phase superparamagnetic nanoparticles, prepared in Example 1. The injection dose was 60μg / kg body weight. In vivo imaging results showed that 2 hours after injection of the nanoprobe carrying the PD-L1-targeting antibody, the T2 signal of mouse breast cancer lesions was significantly reduced. Figure 8 As shown. Therefore, after euthanizing the mouse spine at the time of peak probe aggregation (2 hours), the tumor was dissected for Raman mapping imaging, as shown. Figure 9 The two sets of images shown above and below are Raman bright field and mapping images of breast cancer MDA-MB-231 and colorectal cancer HT-29 tumors and normal mouse tissues, respectively, with the dashed lines separating their boundaries. Figure 10 for Figure 9 Raman spectra measured at selected boundary points.

[0095] Example 2

[0096] The difference between Example 2 and Example 1 is that the Raman signaling molecule in Example 1 is replaced with 4-MBA, while the remaining steps are the same as in Example 1. This allows for the preparation of a PD-L1-guided biological probe for specific targeting of CTCs, combining tumor boundary differentiation.

[0097] Example 3

[0098] The difference between Example 3 and Example 1 is that the Raman signaling molecule in Example 1 is replaced with rhodamine, while the remaining steps are the same as in Example 1. This allows for the preparation of a PD-L1-guided biological probe for specific targeting of CTCs, combining tumor boundary differentiation.

[0099] Example 4

[0100] The difference between Example 4 and Example 1 is that in Example 4, step (1) involves the preparation of oil-phase superparamagnetic Fe3O4 nanoparticles with SERS function as follows:

[0101] Take 15 mL of anhydrous ethanol and cis-9-octadecenoic acid and add them to a 100 mL beaker. Add 2 g of NaOH and stir at 800 rpm until dissolved to obtain a mixture. Then, take 1.25 g of Fe(NH4)2(SO4)2·6H2O and dissolve it in 30 mL of water. Slowly add the Fe(NH4)2(SO4)2·6H2O to the mixture using a dropper. After complete dissolution, transfer the mixture to a reaction vessel and heat at 200 °C for 14 h. Then, collect the supernatant, disperse the precipitate with cyclohexane, wash three times with ethanol, and store it in 10 mL of cyclohexane. The remaining steps are the same as in Example 1. A PD-L1-guided specific targeted CTC detection and tumor boundary differentiation biological probe can be prepared.

[0102] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0103] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0104] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A PD-L1-guided biological probe for specific targeting of CTCs combined with tumor boundary differentiation, characterized in that, It includes a core, a covalent organic framework, and a PD-L1 antibody, wherein the covalent organic framework is coated on the surface of the core, and the PD-L1 antibody is bound to the surface of the covalent organic framework. The core comprises superparamagnetic nanoparticles and Raman signal molecules, wherein the Raman signal molecules are attached to the surface of the superparamagnetic nanoparticles. The superparamagnetic nanoparticles are oil-phase superparamagnetic Fe3O4 nanoparticles with a particle size ≤15 nm, and the preparation method includes the following steps: A polar solvent, cis-9-octadecenoic acid and an inorganic base were mixed to obtain a mixture. Then, an aqueous solution of ferrous ammonium sulfate was added to the mixture and heated at 100~500 °C for 1~20 h. The supernatant was removed, the precipitate was dispersed with a non-polar solvent, washed and stored in a non-polar solvent to obtain oil-phase superparamagnetic Fe3O4 nanoparticles. The covalent organic framework is TAPB-BTCA COF, which is prepared from monomers including TAPB and BTCA; The biological probe has a particle size of 5~20 nm.

2. The biological probe for PD-L1-guided specific targeting of CTCs detection combined with tumor boundary differentiation according to claim 1, characterized in that, Polar solvents include one or more of anhydrous ethanol, methanol, acetonitrile, and acetone.

3. The biological probe for PD-L1-guided specific targeting of CTCs detection combined with tumor boundary differentiation according to claim 1, characterized in that, Inorganic bases include one or more of sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, and copper hydroxide.

4. The biological probe for PD-L1-guided specific targeting of CTCs detection combined with tumor boundary differentiation according to claim 1, characterized in that, Nonpolar solvents include one or more of cyclohexane, hexane, benzene, carbon tetrachloride, dichloroethane, and dichloromethane.

5. The biological probe for PD-L1-guided specific targeting of CTCs detection combined with tumor boundary differentiation according to claim 1, characterized in that, The steps for coating a core surface with a covalent organic framework include: adding the core, TAPB and BTCA to acetonitrile, followed by adding acetic acid, stirring at room temperature, and centrifuging to obtain a covalent organic framework-coated core.

6. The biological probe for PD-L1-guided specific targeting of CTCs detection combined with tumor boundary differentiation according to claim 1, characterized in that, The thickness of the covalent organic framework is 1~5 nm.

7. The biological probe for PD-L1-guided specific targeting of CTCs detection combined with tumor boundary differentiation according to claim 1, characterized in that, The molar ratio of superparamagnetic nanoparticles, covalent organic frameworks and PD-L1 antibody is (250~310):(100~150):(50~70).

8. The method for preparing a PD-L1-guided specific targeted CTC detection and tumor boundary differentiation biological probe as described in claim 1, characterized in that, Includes the following steps: (1) Prepare superparamagnetic nanoparticles and attach Raman signal molecules to the surface of the superparamagnetic nanoparticles; (2) Add the product of step (1), TAPB and BTCA to acetonitrile, then add acetic acid, stir at room temperature for 12-48 h, and centrifuge to obtain the covalent organic framework-coated core. (3) Add PD-L1 antibody to the covalent organic framework-coated core, stir, centrifuge, and resuspend in phosphate buffer to obtain a PD-L1-guided specific target CTC detection and tumor boundary differentiation biological probe.

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