Near-infrared two-region fluorescent probe for double-target characterization of tumor immunosuppression microenvironment as well as preparation method and application of near-infrared two-region fluorescent probe

Abstract

The invention provides a near-infrared two-region fluorescent probe for double-target characterization of tumor immunosuppression microenvironment and a preparation method and application thereof.The near-infrared two-region fluorescent probe comprises a fluorescent probe system composed of two NIR-II quantum dots with different emission spectrums and used for double-target characterization of tumor immunosuppression microenvironment, the NIR-II quantum dots with different emission spectrums are IIa QD and IIb QD, pbS (lead sulfide) is taken as a core of the probe, CdS (cadmium sulfide) is wrapped outside the probe, the surface of the probe is rich in carboxyl polyethylene glycol PEG (polyethylene glycol) chain segments through chemical modification, PD-L1 and VEGF monoclonal antibodies are respectively coupled to form NIR-II non-overlapping emission fluorescent molecular probes IIaQD at alpha VEGF and IIb QD at alpha PD-L1, and finally, the probe is mixed to form a fluorescent marker for representing highly specific dissimilation by double targets. The near-infrared two-region fluorescent mixed probe disclosed by the invention can be used for independently and clearly imaging two target spots at the same time, monitoring dynamic changes of VEGF and PD-L1 biomarkers in vivo in real time, avoiding the problem of imaging interference possibly existing in a single probe, and also has good stability, biocompatibility and safety.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and particularly relates to a near-infrared second near-infrared fluorescent probe for dual-target characterization of tumor immunosuppressive microenvironment, and a preparation method and application thereof.

Background Art

[0002] At present, traditional treatment methods for malignant tumors (such as surgical resection, radiotherapy, chemotherapy, and targeted therapy, etc.) have problems such as strong toxic side effects, incomplete treatment, poor prognosis of patients, and easy generation of drug resistance, resulting in poor tumor treatment effects. In recent years, immunotherapy has become a forefront hotspot in tumor treatment methods. It enables the body to produce anti-tumor specific immune responses actively or passively to achieve the ability of T cells to recognize and eliminate tumors. It has the advantages of small side effects, high specificity and adaptability, and the ability to produce immune memory. However, at present, only a small number of patients benefit from tumor immunotherapy, and there are risks such as accelerating tumor progression.

[0003] With the development of modern medicine, especially the progress of oncology, personalized treatment methods based on specific molecular markers have received increasing attention. However, most existing diagnostic tools and technologies still have limited understanding of the complex immunosuppressive microenvironment inside tumors, especially in identifying and quantifying key regulatory factors such as programmed death ligand 1 (PD-L1) and vascular endothelial growth factor (VEGF). These molecules not only participate in the process of cancer cells escaping immune surveillance, but are also important targets of various anti-tumor drugs. Therefore, developing new methods that can efficiently locate and monitor these two markers has become one of the current research focuses.

Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a near-infrared second near-infrared fluorescent probe for dual-target characterization of tumor immunosuppressive microenvironment, and a preparation method and application thereof. This near-infrared second near-infrared fluorescent hybrid probe can independently and clearly image two targets at the same time, and can monitor the dynamic changes of two key biomarkers, VEGF and PD-L1, in vivo in real time, avoiding the imaging interference problem that may exist in a single probe. At the same time, it also has good stability, biocompatibility and biosafety.

[0005] The present invention is implemented as follows:

[0006] A near-infrared II region fluorescent probe for dual-target characterization of the tumor immunosuppressive microenvironment, comprising a fluorescent probe system for dual-target characterization of the tumor immunosuppressive microenvironment composed of two NIR-II quantum dots with different emission spectra. Among them, the NIR-II quantum dots with different emission spectra are type IIa QD and type IIb QD. Both are based on lead sulfide (PbS) as the core, wrapped with a layer of cadmium sulfide (CdS) on the outside, and chemically modified to make their surfaces rich in carboxyl polyethylene glycol (PEG) segments. Then, they are respectively conjugated with two monoclonal antibodies, PD-L1 and VEGF, to form NIR-II non-overlapping emission fluorescent molecular probes, namely IIa QD@αVEGF probe and IIb QD@αPD-L1 probe. Finally, the mixed probes form a highly specific fluorescent marker for dual-target characterization.

[0007] Furthermore, the emission spectrum of the IIa QD is 950 nm - 1100 nm, and the emission spectrum of the IIb QD is 1500 nm - 1600 nm;

[0008] The particle size of the IIa QD is 4.69 ± 1.18 nm, and the particle size of the IIb QD is 6.70 ± 0.84 nm.

[0009] Furthermore, in the mixed fluorescent probe system, the dosage ratio of the IIa QD@αVEGF probe to the IIb QD@αPD-L1 probe is 1:1.

[0010] Furthermore, a preparation method of a near-infrared II region fluorescent probe for dual-target characterization of the tumor immunosuppressive microenvironment, the method steps are as follows:

[0011] Step 1: Using the hot injection method, PbS quantum dots (PbS QD), including IIa PbS QD and IIb PbS QD, are generated by using a sulfur source precursor solution and a lead source precursor solution; then, a CdS shell layer is coated on the surface of the PbS quantum dots by the cation exchange method to obtain a lipophilic PbS / CdS core-shell structure quantum dot (PbS@CdS QD, abbreviated as QD), including lipophilic IIa QD and IIb QD;

[0012] Step 2: DSPE-PEG 2000 and DSPE-PEG-COOH 2000 are ultrasonically dissolved and mixed evenly in tetrahydrofuran (THF). QD is also ultrasonically dissolved alone in THF. Then, the mixture of DSPE-PEG 2000 and DSPE-PEG-COOH 2000 is mixed with QD again and ultrasonically treated. Then, the above-mentioned mixed solution of DSPE-PEG 2000, DSPE-PEG-COOH 2000 and QD is injected into the ultrasonic ddH2O at one time and ultrasonically treated for a period of time; subsequently, THF is removed by overnight evaporation to obtain water-soluble QD, including water-soluble IIa QD and IIb QD;

[0013] Step 3: Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in ddH2O, add water-soluble QD, and react at room temperature for 30 ± 5 min. After the reaction, mix 4-Arm-PEG-NH2 and QD in a mass ratio of 5:1 in the solution for amide condensation; after the reaction, ultrafilter to remove excess PEG and reaction by-products to obtain QD coated with 4-Arm-PEG-NH2 on the surface;

[0014] Step 4: Dissolve EDC and NHS in ddH2O, add monoclonal murine antibody mAb-PD-L1 or mAb-VEGF, and co-incubate at room temperature for more than 3 hours. After the incubation, add the QD solution coated with 4-Arm-PEG-NH2 on the surface for amide condensation, and ultrafilter to remove by-products to obtain near-infrared second-region fluorescence probe Ⅱa QD@αVEGF or Ⅱb QD@αPD-L1 for in vivo imaging of PD-L1 or VEGF;

[0015] Finally, mix the separately prepared near-infrared second-region fluorescence probes Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 in a ratio of 1:1 to obtain a mixed probe solution.

[0016] Further, the sulfur source is sulfur powder, and the lead source is lead chloride.

[0017] Further, the mass ratio of DSPE-PEG 2000 to DSPE-PEG-COOH 2000 is 1:1; the mass ratio of the mixture of DSPE-PEG 2000 and DSPE-PEG-COOH 2000 to QD is 5:1; the mass ratio of 4-Arm-PEG-NH2 to QD is 5:1; the mass ratio of QD coated with 4-Arm-PEG-NH2 on the surface to the monoclonal antibody is 25:3.

[0018] Further, the application of the near-infrared second-region fluorescence probe for dual-target characterization of tumor immunosuppressive microenvironment in the preparation of tumor living cell imaging detection reagents.

[0019] The present invention has the following advantages:

[0020] (1) The near-infrared second-region fluorescence mixed probes (Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1) in the present invention have good stability, biocompatibility and biosafety;

[0021] (2) The near-infrared second near-infrared fluorescence hybrid probe in the present invention has excellent near-infrared second near-infrared fluorescence emission interval and tumor targeting property, and can realize in-situ dual-color simultaneous fluorescence imaging of VEGF and PD-L1 in the tumor immunosuppressive microenvironment, thereby indicating the levels of VEGF and PD-L1 in the tumor. Compared with the existing immunohistochemical detection method for ex vivo tumor tissues, the near-infrared second near-infrared fluorescence probe in the present invention can realize in-vivo, rapid, and non-invasive visualization imaging of VEGF and PD-L1. Compared with the existing single-probe imaging, the near-infrared second near-infrared fluorescence hybrid probe in the present invention can independently and clearly image two targets at the same time, and can monitor the dynamic changes of two key biomarkers, VEGF and PD-L1, in the body in real time, avoiding the imaging interference problem that may exist in a single probe.

Description of the Drawings

[0022] The present invention will be further described below with reference to the accompanying drawings in conjunction with embodiments.

[0023] Figure 1 It is a schematic diagram of the characterization of Ⅱa QD and Ⅱb QD quantum dots in the embodiment of the present invention, where (a) Transmission electron microscope images and particle size frequency distribution diagrams of Ⅱa QD and Ⅱb QD dispersed in n-hexane solution respectively; (b) Transmission electron microscope images of Ⅱa QD and Ⅱb QD dispersed in aqueous solution respectively; (c) Fluorescence spectra of Ⅱa QD and Ⅱb QD in aqueous solution.

[0024] Figure 2 It is a schematic diagram of the synthesis process of the near-infrared second near-infrared fluorescence probes Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 in Example 2 of the present invention.

[0025] Figure 3 It is a schematic diagram of the characterization of the probes Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 in Example 3 of the present invention, where (a) Particle size distribution diagrams of Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1; (b) Zeta potential characterization of Ⅱa QD, Ⅱa QD@αVEGF, Ⅱb QD and Ⅱb QD@αPD-L1; (c) NIR-II fluorescence images of Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1.

[0026] Figure 4 It is a non-invasive in-vivo dual-color fluorescence imaging diagram of a mouse bearing H22 subcutaneous tumor in the NIR-II window in Example 4 of the present invention.

[0027] Figure 5 It is the cytotoxicity results of the Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 hybrid probes in hepatoma cells (Hepa1-6, H22) and normal mouse hepatocytes in Example 5 of the present invention.

[0028] Figure 6 For the stability evaluation of the Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 probes in Example 6 of the present invention, where (a, b) are the particle size changes of the Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 probes during storage at 4°C; (c) are the fluorescence intensity changes of the Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 probes during storage at 4°C.

[0029] Figure 7 For the in vivo fluorescence imaging of H22 tumor-bearing mice and the immunohistochemical images of their tumor tissues in Example 7 of the present invention (scale bar: 100 μm).

Specific Embodiments

[0030] The following will combine the attached Figure 1-7 drawings and specific embodiments to clearly and completely describe the technical solutions of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained by purchasing in the market.

[0031] Example 1: Prepare Ⅱa QD and Ⅱb QD respectively

[0032] S1: Preparation of Ⅱa QD

[0033] S1-1: Weigh 0.08 g of sulfur powder into a two-necked flask, add 7.5 mL of oleylamine, evacuate, and then stir at 120°C under an argon atmosphere for 30 min to fully dissolve the sulfur powder, and cool to room temperature to obtain a sulfur source precursor solution;

[0034] S1-2: Weigh 0.834 g of lead chloride (PbCl2) into a three-necked flask, add 7.5 mL of oleylamine, evacuate, heat to 100°C under an argon atmosphere, evacuate again, and then quickly inject 2.5 mL of oleic acid under an argon atmosphere, heat up to 120°C and continue to react for 30 min. It can be observed that the reaction system changes from milky white turbidity to light yellow clear transparency to obtain a lead source precursor solution;

[0035] S1-3: Cool the temperature of the lead source precursor solution obtained in step S1-2 to 45 °C, and inject 2.25 mL of the sulfur source precursor solution obtained in step S1-1 at one time. React for 30 min in an argon environment at 45 °C. After adding 10 mL of n-hexane and 20 mL of absolute ethanol, quickly transfer the reaction device to an ice-water mixture and cool it to room temperature to quench the reaction. Collect the reaction solution and centrifuge it at 10000 rpm for 10 min. Discard the supernatant, resuspend the precipitate with 5 - 10 mL of n-hexane, add oleic acid with at least twice the volume of n-hexane, shake it at room temperature for 30 min, and centrifuge it again at 10000 rpm for 10 min to remove the excess sulfur in the product; repeat this step 2 - 3 times until the supernatant is colorless and transparent to obtain the precipitate of type IIa PbS quantum dots, and resuspend the precipitate in 10 mL of 1-octadecene (ODE);

[0036] S1-4: Weigh 0.6 g of cadmium oxide into a three-necked flask, add 4 mL of oleic acid and 15 mL of ODE. After evacuating the vacuum, heat it to 200 °C under argon protection and react for 30 min. Then cool it down to 100 °C to prepare the cadmium (Cd) source solution;

[0037] S1-5: Inject 5 mL of the type IIa PbS quantum dot solution obtained in step S1-3 into the Cd source solution obtained in step S1-4. After continuously reacting at 100 °C under argon for 100 min, quickly add 5 mL of pre-cooled n-hexane, transfer the reaction device to an ice-water mixture to quench the reaction. Collect the reaction solution and centrifuge it at 10000 rpm for 10 min. Wash the centrifuged precipitate with absolute ethanol three times to obtain the precipitate of lipophilic type IIa PbS / CdS core-shell structure quantum dots (abbreviated as type IIa QD). Resuspend the precipitate with 10 mL of n-hexane to obtain a lipophilic type IIa QD solution, and store it in a refrigerator at 4 °C for standby.

[0038] S2: Preparation of type IIb QD

[0039] S2-1: Heat the lead source precursor solution obtained in step S1-2 to 160 °C, and inject 2.25 mL of the sulfur source precursor solution obtained in step S1-1 at one time. React for 30 min in an argon environment at 160 °C. Add 10 mL of pre-cooled n-hexane and 20 mL of absolute ethanol, and quickly transfer the reaction device to an ice-water mixture to quench the reaction. Collect the reaction solution, and centrifuge and wash it according to the method described in step S1-3 to obtain the precipitate of type IIb PbS quantum dots, and resuspend the precipitate in 10 mL of ODE;

[0040] S2-2: Weigh 1.20 g of cadmium oxide into a three-necked flask, add 8 mL of oleic acid and 20 mL of ODE. After evacuating the vacuum, heat it to 200 °C under argon protection and react for 90 min; then cool it down to 100 °C to prepare the Cd source solution;

[0041] S2-3: Inject 5 mL of the type IIb PbS quantum dot solution obtained in step S2-1 into the Cd source solution prepared in step S2-2, and continuously react under argon at 100 °C for 60 min. Subsequently, quickly add 5 mL of n-hexane to quench the reaction; collect the reaction solution and centrifuge it at 10,000 rpm for 10 min. Discard the supernatant, wash the precipitate with anhydrous ethanol three times to obtain a precipitate of lipophilic type IIb PbS / CdS core-shell structure quantum dots (abbreviated as type IIb QD). Redisperse the obtained precipitate in 10 mL of n-hexane to obtain a lipophilic type IIb QD solution, and store it in a refrigerator at 4 °C for later use.

[0042] S2-4: Mix DSPE-PEG 2000 and DSPE-PEG-COOH 2000 in a mass ratio of 1:1, add tetrahydrofuran (THF), and ultrasonically dissolve for 20 min. Type IIa QD (or type IIb QD) is also ultrasonically dissolved in THF for 20 min; mix the DSPE-PEG 2000 and DSPE-PEG-COOH 2000 mixture with type IIa QD (or type IIb QD) and ultrasonicate; then inject it into ddH2O under ultrasonic treatment and continue to ultrasonicate for 20 min; let the ultrasonically treated solution stand overnight at room temperature to allow THF to volatilize naturally to obtain water-soluble type IIa QD (or type IIb QD).

[0043] Characterize type IIa QD and type IIb QD quantum dots respectively, and the results are as Figure 1 shown, Figure 1 a Transmission electron microscopy images show that in n-hexane, type IIa QD and type IIb QD are evenly dispersed, but there are slight differences in their particle sizes, which are approximately 4.69 ± 1.18 nm and 6.70 ± 0.84 nm respectively. This difference can lead to different fluorescence emission wavelengths of type IIa QD and type IIb QD. After being modified with DSPE-PEG 2000 and DSPE-PEG-COOH 2000, both type IIa QD and type IIb QD show excellent dispersibility in aqueous solution, which is more conducive to subsequent biological applications ( Figure 1 b). As Figure 1 seen from the fluorescence spectra in c, type IIa QD has an obvious fluorescence emission peak near 950 nm, and the peak shape is symmetrically distributed without tailing; while the fluorescence emission peak of type IIb QD is near 1500 nm, and there is no obvious overlap with the emission peak of type IIa QD, indicating that there is a significant fluorescence emission interval in the second near-infrared region (NIR-II) between the two, which can effectively avoid spectral crosstalk artifacts in fluorescence imaging and contribute to their dual-color imaging in the tumor immune microenvironment.

[0044] Example 2: Preparation of type IIa QD@αVEGF and type IIb QD@αPD-L1 probes

[0045] S1: Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in ddH2O, add water-soluble type IIa QD (or type IIb QD), and react at room temperature for 30 min. After the reaction, mix type IIa QD (or type IIb QD) with 4-Arm-PEG-NH2 at a mass ratio of 1:5 for amide condensation. After the condensation is completed, ultrafilter to remove excess PEG and reaction by-products to obtain type IIa QD (or type IIb QD) coated with 4-Arm-PEG-NH2 on the surface.

[0046] S2: Dissolve EDC and NHS in ddH2O, add monoclonal murine antibody (mAb-PD-L1 or mAb-VEGF), and co-incubate at room temperature for more than 3 hours. After the incubation, add the type IIa QD (or type IIb QD) solution prepared in step S1 for amide condensation, and ultrafilter to remove by-products to obtain a near-infrared second-region fluorescence probe (IIa QD@αVEGF or IIb QD@αPD-L1) for in vivo imaging of VEGF or PD-L1. The specific synthesis process is as Figure 2 shown.

[0047] Example 3: Characterization of IIa QD@αVEGF and IIb QD@αPD-L1 probes

[0048] Measure the particle size distribution of the quantum dots after antibody conjugation by dynamic light scattering (DLS). The results are as Figure 3 shown in a. The hydrodynamic particle size distributions of IIa QD@αVEGF and IIb QD@αPD-L1 are uniform, and the average particle size is significantly increased compared with the quantum dots without antibody conjugation. Figure 3 The Zeta potential diagram in b shows that after antibody conjugation, more positive charges accumulate on the surface of the quantum dots, resulting in a significant decrease in the charge of IIa QD@αVEGF and IIb QD@αPD-L1. These changes prove the successful synthesis of the antibody-conjugated quantum dot probes. Then, investigate the fluorescence imaging ability of the probes by a second-region imager. The results are as Figure 3 shown in c. After antibody conjugation, the fluorescence spectra of the two quantum dot probes are also basically completely separated, and the fluorescence crosstalk between them is negligible, still showing excellent dual-color near-infrared second-region fluorescence imaging ability.

[0049] Example 4: In vivo fluorescence imaging of the tumor immunosuppressive microenvironment

[0050] First, prepare the NIR-II fluorescence probes IIa QD@αVEGF and IIb QD@αPD-L1 according to the method provided in Example 2, and mix them in a mass ratio of 1:1 to obtain a mixed probe solution.

[0051] H22 tumor-bearing mice were injected via the tail vein with a mixture of Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 probes (1 mg / mouse, i.e., 500 μg Ⅱa QD@αVEGF + 500 μg Ⅱb QD@αPD-L1 / mouse). At different time points after injection (1 h, 3 h, 6 h, 9 h, and 12 h), near-infrared fluorescence in vivo imaging system was used (for NIR-Ⅱa channel, 808 nm excitation and 900 nm LP emission filter plus 1200 nm SP emission filter were used; for NIR-Ⅱb channel, 808 nm excitation and 1500 nm LP emission filter were used) to collect the near-infrared second-region fluorescence signals at the subcutaneous tumor sites of the mice. The results are as Figure 4 shown. Within 1 hour after injection, the NIR-Ⅱa channel signal representing Ⅱa QD@αVEGF and the NIR-Ⅱb channel signal representing Ⅱb QD@αPD-L1 rapidly accumulated at the tumor sites, and obvious fluorescence co-localization occurred between the two. This phenomenon could last until the 12th hour after injection, indicating that the mixed probe of Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 had excellent in vivo tumor targeting ability and outstanding long-term dual-color NIR-Ⅱ imaging ability, realizing the visualization imaging of VEGF and PD-L1 in the tumor microenvironment.

[0052] Example 5: In vitro cytotoxicity of Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 quantum dot probes

[0053] The CCK8 kit was used to detect the cytotoxicity of the mixed probe of Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 (ⅡaQD@αVEGF:ⅡbQD@αPD-L1 = 1:1) against hepatoma cells Hepa1-6, H22, and mouse embryonic hepatocytes CL2. The results are as Figure 5 shown. When the mixed probe was co-incubated with the three types of cells Hepa1-6, H22, and CL2 for 12 hours respectively, the proliferation of the three types of cells was hardly affected, indicating that the mixed probe had good biosafety.

[0054] Example 6: Stability of Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 probes

[0055] The storage stability of Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 was detected by dynamic light scattering and fluorescence spectroscopy. The results are as Figure 6 shown. When the Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 probes were stored in an environment at 4℃ for seven days, neither the particle size nor the fluorescence intensity of the probes changed significantly, indicating that the Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 probes had good storage stability.

[0056] Example 7: In Vivo Prediction Performance of Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 Quantum Dot Probes

[0057] H22 liver cancer cells were inoculated subcutaneously in mice to establish a subcutaneous liver cancer tumor model. A mixture of quantum dot probes (Ⅱa QD@αVEGF:Ⅱb QD@αPD-L1 = 1:1) was injected into the tumor-bearing mice via the tail vein. One hour later, in vivo fluorescence imaging was performed on the mice. According to the fluorescence signal intensity at the tumor site, mice with different fluorescence intensities were selected. The tumor tissues of each mouse were surgically removed, and the protein expressions of VEGF and PD-L1 in the excised tissues were detected by immunohistochemistry. The results are as follows Figure 7 shown. The fluorescence signal attenuation of the two probes, Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1, was positively correlated with the positive rates of VEGF and PD-L1 immunohistochemistry in the tumor tissues. This result indicates that the Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 quantum dot probes can effectively reflect the protein expression levels of VEGF and PD-L1 in the tumor microenvironment through the changes in their fluorescence intensities, achieving the in vivo visualization of VEGF and PD-L1.

[0058] In summary, in the present invention, the reaction temperature and time were controlled by the hot injection method, and the nucleation and growth of quantum dots were controlled at different reaction stages to synthesize NIR-Ⅱ quantum dots PbS@CdS QD (abbreviated as QD) with different particle sizes and different emission spectra, namely Ⅱa QD (emission spectrum 950 nm) and Ⅱb QD (emission spectrum 1500 nm). Both QDs have lead sulfide quantum dots (PbS QD) as the core and cadmium sulfide quantum dots (CdS QD) as the shell. Then, carboxyl PEG and 4-Arm-PEG were sequentially coated on the surfaces of the two QDs through hydrophobic interaction and amide condensation to increase biocompatibility and the probability of coupling antibodies. Finally, two monoclonal antibodies, PD-L1 and VEGF, were respectively coupled to form NIR-Ⅱ non-overlapping emission fluorescence molecular probes, namely Ⅱa QD@αVEGF probe and Ⅱb QD@αPD-L1 probe. This probe can achieve the visualization and quantitative imaging detection of the key targets PD-L1 and VEGF in tumor immunotherapy. That is, the present invention aims at the key targets PD-L1 and VEGF of the "T+A" combination therapy, adopts the NIR-Ⅱ region non-overlapping emission fluorescence imaging technology, constructs a molecular probe to visualize VEGF and PD-L1 in vivo, and improves clinical treatment decision-making and efficacy prediction.

[0059] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we described are illustrative rather than used to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered by the scope protected by the claims of the present invention.

Claims

1. A near-infrared second-zone fluorescent probe for dual-target characterization of tumor immunosuppressive microenvironment, characterized by: The system includes two NIR-II quantum dots with different emission spectra, each of which is composed of a dual-target fluorescent probe for characterizing the tumor immunosuppressive microenvironment. The NIR-II quantum dots with different emission spectra are Ⅱa QD and Ⅱb QD. Both of them have lead sulfide PbS as the core, are wrapped with a layer of cadmium sulfide CdS on the outside, and are chemically modified to enrich their surfaces with carboxyl polyethylene glycol PEG segments. They are then coupled with two monoclonal antibodies, PD-L1 and VEGF, to form NIR-Ⅱ non-overlapping emission fluorescent molecular probes, namely ⅡaQD@αVEGF probe and Ⅱb QD@αPD-L1 probe, respectively. Finally, the probes are mixed to form a dual-target fluorescent marker for characterizing highly specific tumors.

2. A near-infrared second-region fluorescent probe for characterizing tumor immunosuppressive microenvironment with dual targets according to claim 1, characterized in that: The emission spectrum of the IIa QD is 950nm-1100nm, and the emission spectrum of the IIb QD is 1500nm-1600nm; The particle size of the IIa QD is 4.69±1.18 nm, and the particle size of the IIb QD is 6.70±0.84 nm.

3. A near-infrared second-region fluorescent probe for characterizing tumor immunosuppressive microenvironment with dual targets according to claim 1, characterized in that: The dosage ratio of Ⅱa QD@αVEGF probe and Ⅱb QD@αPD-L1 probe in the mixed fluorescent probe system is 1:

1.

4. A method for preparing a near-infrared second-zone fluorescent probe for dual-target characterization of tumor immunosuppressive microenvironment, characterized by: The method steps are as follows: Step 1: using a hot injection method, using a sulfur source precursor solution and a lead source precursor solution to generate PbS quantum dots PbS QD, including Ⅱa PbS QD and Ⅱb PbS QD; then using a cation exchange method to coat a CdS shell layer on the surface of the PbS quantum dots to obtain fat-soluble PbS / CdS core-shell structure quantum dots PbS@CdS QD, referred to as QD, including fat-soluble Ⅱa QD and Ⅱb QD; Step 2, DSPE-PEG 2000 and DSPE-PEG-COOH 2000 are ultrasonically dissolved and mixed in tetrahydrofuran THF, and QD is also ultrasonically dissolved in THF alone, and then the mixed solution of DSPE-PEG 2000 and DSPE-PEG-COOH 2000 is mixed with QD again and ultrasonicated, and then the mixed solution of DSPE-PEG 2000, DSPE-PEG-COOH 2000 and QD is injected into the ultrasonic ddH2O at one time, and the ultrasonication is continued for a period of time; Subsequently, THF was removed by evaporation overnight to obtain water-soluble QDs, including water-soluble IIa QDs and IIb QDs; Step 3, dissolving 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS in ddH2O, adding water-soluble QD, reacting at room temperature for 30±5min, after the reaction, mixing 4-Arm-PEG-NH2 and QD in a mass ratio of 5:1 in the solution for amide condensation; after the reaction, removing excess PEG and reaction by-products by ultrafiltration to obtain QD with 4-Arm-PEG-NH2 coated on the surface; Step 4, dissolving EDC and NHS in ddH2O, adding monoclonal mouse antibody mAb-PD-L1 or mAb-VEGF and incubating at room temperature for more than 3 hours, after the incubation, adding QD solution coated with 4-Arm-PEG-NH2 on the surface for amide condensation; after the reaction, ultrafiltration was performed to remove byproducts to obtain near-infrared second-zone fluorescent probe ⅡaQD@αVEGF or Ⅱb QD@αPD-L1 for in vivo imaging of PD-L1 or VEGF; Finally, the prepared near-infrared second-zone fluorescent probes Ⅱa QD@αVEGF and Ⅱb QD@αPD-L1 were mixed in a 1:1 ratio to obtain a mixed probe solution.

5. The method for preparing a near-infrared second-region fluorescent probe for characterizing tumor immunosuppressive microenvironment with dual targets according to claim 4, characterized in that: The sulfur source is sulfur powder, and the lead source is lead chloride.

6. The method for preparing a near-infrared second-region fluorescent probe for characterizing tumor immunosuppressive microenvironment with dual targets according to claim 4, characterized in that: The mass ratio of the DSPE-PEG 2000 and DSPE-PEG-COOH 2000 is 1:1; the mass ratio of the mixed solution of DSPE-PEG 2000 and DSPE-PEG-COOH 2000 to QD is 5:1; the mass ratio of the 4-Arm-PEG-NH2 to QD is 5:1; the mass ratio of the QD coated with 4-Arm-PEG-NH2 to the monoclonal antibody is 25:

3.

7. The use of a near-infrared second-region fluorescent probe for characterizing tumor immunosuppressive microenvironment with dual targets according to any one of claims 1 to 3, characterized in that: Application in the preparation of tumor living cell imaging detection reagents.

Images