Organic small molecules and nanoprobes in the near-infrared II region and their application in the detection of positive surgical margins in osteosarcoma

Abstract

The present invention provides an organic small molecule, BBTD14, capable of imaging in the near-infrared region II. BBTD14 is further combined with the chemiluminescent substrate CPPO to form a chemiluminescent nanoprobe capable of producing chemiluminescence in the presence of hydrogen peroxide. PD-L1 antibodies are also attached to the surface of the nanomicelles to enhance their targeting ability for osteosarcoma cells. Ultimately, after surgical resection of osteosarcoma, the resection margin is locally incubated and cleaned, and sprayed with hydrogen peroxide to enable instant imaging, revealing residual tumor tissue. The chemiluminescent nanoprobe constructed in this invention eliminates the need for an external excitation light source, reducing interference from autofluorescence and enabling surgeons to more intuitively determine positive osteosarcoma resection margins, thus overcoming the limitation of selective verification during intraoperative freezing of osteosarcoma. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a near-infrared II region organic small molecule and a nanoprobe thereof and their application in the positive detection of surgical margins of osteosarcoma, and specifically to the application of a near-infrared chemiluminescent probe in the field of imaging diagnosis of osteosarcoma. Background Art

[0002] Osteosarcoma is the most common type of malignant bone tumor. It develops from the mesenchymal cell lineage. The rapid growth of the tumor is due to the direct or indirect formation of tumor bone-like tissue and bone tissue after the cartilage stage. Typical osteosarcomas originate from within the bone. Another completely different type of osteosarcoma is the osteosarcoma that is parallel to the bone cortex and originates from the periosteum and nearby connective tissue. The current treatment method for osteosarcoma is mainly surgery. After the osteosarcoma is resected, the determination of whether the resection margin is positive depends on intraoperative frozen sections. However, this method can only selectively send part of the tissue for examination and cannot detect the entire resection margin. It also requires waiting for 20-30 minutes and is not real-time.

[0003] Currently, various organic small molecules based on benzothiadiazole structures operating in the near-infrared II region (NIR-II, 1000-1700 nm) (e.g., BTD540, BBTD700, IR-BGP6, BBTD-TP, etc.) have been developed for applications in bioimaging and therapy. These NIR-II organic small molecules are often used to construct NIR-II photoluminescent nanomaterials for imaging-guided tumor resection. These nanomaterials utilize the enrichment effect of tumors and, under external light excitation, differentiate between tumors and normal tissue based on the different signal-to-noise ratios between the two groups to determine whether the tumor has been cleared. However, these photoluminescent fluorescence navigation methods require an external excitation light source, and the body spontaneously fluoresces under external excitation light, making it difficult to precisely distinguish between tumor and normal tissue.

[0004] To achieve an emission wavelength reaching NIR-II, the structural design and synthesis of organic small molecule compounds are crucial. To avoid the problems of fluorescent nanomaterials, combining organic small molecule compounds with chemiluminescence is also a must-solve issue. To enable real-time observation of surgical margins, enhancing the tumor targeting of organic small molecule compounds is also a critical consideration. Therefore, there is an urgent need to develop a new organic small molecule in the near-infrared II region to address these issues. Summary of the Invention

[0005] In order to overcome at least one problem existing in the prior art, the present invention has developed and designed an organic molecule with an emission wavelength in the near-infrared II region, and the energy level difference between its excited state and normal state is similar to that of the chemiluminescent intermediate, which is conducive to the subsequent construction of chemiluminescent nanomaterials; using the principle of chemiluminescence, the above-mentioned organic molecule and the chemiluminescent substrate are constructed into a near-infrared II region chemiluminescent nanoprobe, avoiding the use of photoluminescence for external excitation light imaging; and the PD-L1 antibody can be modified on the chemiluminescent nanoprobe to enhance tumor targeting, which can be used locally on the entire resection margin after tumor resection to detect in real time whether there is tumor residue at the resection margin.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention is to provide a near-infrared II region organic small molecule, wherein the near-infrared II region organic small molecule is BBTD14, and its chemical structure is shown below:

[0008]

[0009] The second aspect of the present invention is to provide a method for synthesizing a near-infrared II region organic small molecule, wherein the near-infrared II region organic small molecule is BBTD14, and its synthesis route is:

[0010]

[0011] Step S1, dissolving compound 1, N,N-diphenyl-4-(tributyltin)aniline, and tetrakis(triphenylphosphine)palladium in toluene in a predetermined ratio and reacting at room temperature for 18 to 30 hours (preferably 20 to 28 hours, more preferably 24 hours) to obtain compound 2;

[0012] Step S2, dispersing compound 2 in a THF solution, adding an n-butyl lithium solution at -85 to -70°C (preferably -80 to -75°C, more preferably -78°C), reacting at -85 to -70°C (preferably -80 to -75°C, more preferably -78°C) for 0.5 to 2 hours (preferably 0.8 to 1.5 hours, more preferably 1 hour), adding tributyltin chloride (preferably stirring for 20 to 40 minutes, more preferably stirring for 30 minutes), and reacting at room temperature for 1 to 4 hours (preferably 1.5 to 3 hours, more preferably 2 hours) to obtain compound 3;

[0013] Step S3, dissolving compound 3, compound 4, tris(dibenzylacetone)dipalladium, and tris(o-methylphenyl)phosphine in toluene and mixing, degassing for 15 to 60 minutes (preferably 20 to 40 minutes, more preferably 30 minutes); reacting at 100 to 150° C. in a nitrogen atmosphere for 36 to 60 hours (preferably stirring at 120 to 140° C. for 42 to 54 hours, more preferably stirring at 130° C. for 48 hours) to obtain the organic small molecule BBTD14.

[0014] Furthermore, in step S1, the molar ratio of compound 1, N,N-diphenyl-4-(tributyltin)aniline, and tetrakis(triphenylphosphine)palladium is 10-15:8-12:0.05-0.4; the preferred molar ratio is 11.5-13.5:9-11:0.1-0.3; and the more preferred molar ratio is 12.6:10.2:0.17.

[0015] Furthermore, in step S1, the amount of toluene solvent used is preferably sufficient to completely dissolve and mix the reactants, and the amount used can be 20 to 80 mL, preferably 30 to 60 mL, and more preferably 40 mL.

[0016] Furthermore, in step S2, the molar ratio of compound 2, n-butyl lithium, and tributyltin chloride is 4-6:6-10:6-10; preferably, the molar ratio is 4.5-5.5:7-9:7-9; and more preferably, the molar ratio is 5.2:8.0:8.0. The n-butyl lithium solution is prepared by dispersing n-butyl lithium in hexane, and its concentration is adjustable, preferably 1.59 M.

[0017] Furthermore, in step S2, the amount of the solvent THF solution used is preferably such that it can completely dissolve and mix the reactants, and the amount used can be 4 to 20 mL, preferably 8 to 15 mL, and more preferably 10 mL.

[0018] Furthermore, in step S3, the molar ratio of compound 3, compound 4, tris(dibenzylacetone)dipalladium, and tris(o-methylphenyl)phosphine is 0.5-2:0.1-0.4:0.01-0.04:0.1-0.4; the preferred molar ratio is 0.8-1.5:0.2-0.3:0.02-0.03:0.15-0.3; and the more preferred molar ratio is 1:0.25:0.025:0.2.

[0019] Furthermore, in step S3, the amount of toluene solvent used is preferably sufficient to completely dissolve and mix the reactants, and the amount used can be 0.5 to 4 mL, preferably 0.8 to 2 mL, and more preferably 1.5 mL.

[0020] The third aspect of the present invention is to provide a nanoprobe prepared from any near-infrared II region organic small molecule described in the first aspect, or a near-infrared II region organic small molecule prepared by any synthesis method described in the second aspect.

[0021] Furthermore, the nanoprobe is a near-infrared II region chemiluminescent nanoprobe. It is understood that the above-mentioned near-infrared II region organic small molecules can also be prepared into near-infrared II region fluorescent nanoprobes.

[0022] Furthermore, the preparation method of the near-infrared II region chemiluminescent nanoprobe includes: dissolving carboxylated F127, organic small molecule BBTD14 and bisoxalate in tetrahydrofuran solution, fully mixing and evaporating the organic solvent, adding PBS buffer, and allowing them to self-assemble into nanomicelles to obtain the chemiluminescent nanoprobe.

[0023] Furthermore, the mass ratio of carboxylated F127, organic small molecule BBTD14 and bisoxalate is 80-120:0.1-0.8:20-60; the preferred mass ratio is 90-115:0.1-0.5:30-50; and the more preferred mass ratio is 100:0.3:40.

[0024] Furthermore, the amount of tetrahydrofuran solution used is preferably sufficient to completely dissolve and mix the reactants, and the amount used can be 2 to 20 mL, preferably 2 to 10 mL, and more preferably 5 mL.

[0025] Furthermore, the amount of PBS buffer used is suitable for successfully preparing nanomicelles, and the amount used can be 1 to 10 mL, preferably 1 to 5 mL, and more preferably 2.5 mL.

[0026] Furthermore, the nanomicelles prepared above need to be stored at 4° C. in the dark, and can be further diluted with PBS buffer, for example, to a concentration of 20, 30, 40, 50, 60 μM, etc.

[0027] Furthermore, the method for preparing the near-infrared II region chemiluminescent nanoprobe further includes the step of modifying the chemiluminescent nanoprobe (nanomicelle) with a PD-1 antibody; wherein the chemiluminescent nanoprobe modified with the PD-1 antibody is named CLNP. The modification is achieved by an amide reaction between amino groups on the antibody protein and the carboxylated F127 on the exterior of the micelle.

[0028] Furthermore, the preparation of the CLNP specifically includes the steps of: adding EDC solution and NHS solution to the chemiluminescent nanoprobe solution (nanomicellar solution, prepared by diluting PBS buffer), incubating in the dark at 4°C for 20 to 60 minutes (preferably 30 to 50 minutes, more preferably 40 minutes); ultrafiltration of the solution, centrifugation of the filtrate, removal of the supernatant, re-dissolution, adding PD-L1 antibody, and reacting at 4°C in the dark for 12 to 36 hours (preferably 18 to 30 hours, more preferably 24 hours) to obtain the CLNP.

[0029] Furthermore, the concentration of the chemiluminescent nanoprobe solution is 20-100 μM, and the volume is 400-600 μL; preferably, the concentration is 40-100 μM, and the volume is 180-540 μL; more preferably, the concentration is 50 μM, and the volume is 500 μL. The concentration of the chemiluminescent nanoprobe solution is based on the BBTD14 concentration.

[0030] Furthermore, the EDC solution is prepared by dissolving EDC at a concentration of 200 mM in a 10 mM aqueous solution of morpholineethanesulfonic acid, with an amount of 50 to 80 μL, preferably 56 to 70 μL, and more preferably 62.5 μL.

[0031] Furthermore, the NHS solution is prepared by dissolving sulo-NHS at a concentration of 200 mM in a 10 mM aqueous solution of morpholineethanesulfonic acid, with an amount of 60 to 90 μL, preferably 65 to 80 μL, and more preferably 75 μL.

[0032] Furthermore, the dosage of the PD-L1 antibody is 40 to 60 μg; preferably, the dosage is 45 to 55 μg; and more preferably, the dosage is 50 μg.

[0033] Furthermore, the ultrafiltration and centrifugation steps are as follows: using an ultrafiltration tube with a molecular weight cutoff of 100,000, the filtrate is centrifuged at 6,000 rpm for 5 minutes, and repeated twice.

[0034] It is understandable that the above-mentioned near-infrared II region organic small molecules and chemiluminescent nanoprobes can all be successfully prepared within the process operation parameter range listed above, and there are certain differences in product yield, performance of organic small molecules and nanoprobes, etc.; after optimization selection, the organic small molecules and nanoprobes prepared under the above-mentioned optimal conditions have the best performance.

[0035] The fourth aspect of the present invention is to provide an application of a near-infrared II region organic small molecule as described in any one of the first aspects, or a near-infrared II region organic small molecule obtained by the synthesis method described in any one of the second aspects, or a nanoprobe as described in any one of the third aspects; wherein the application is an application in biological imaging; and / or the application is an application in the preparation of a biological therapeutic agent.

[0036] Furthermore, the application is the use of chemiluminescent nanoprobe CLNP as a bioimaging agent in the detection of positive surgical margins of osteosarcoma.

[0037] Furthermore, during the bioimaging process, the minimum concentration of hydrogen peroxide (H2O2) used can be 0.5 mM, and the specific concentration used can be 50, 100, 200 mM, etc.; the concentration of CLNP used (in units of BBTD14 concentration) is 50-100 μM.

[0038] Furthermore, the specific operation of the bioimaging is as follows: after locally incubating CLNP (50 μM, 70 μL) with the tumor margin for 10 min, hydrogen peroxide (100 mM) is sprayed on the wound surface for chemiluminescence imaging.

[0039] Compared with the prior art, the present invention adopts the above technical solution to achieve the following beneficial effects:

[0040] This invention designed and synthesized for the first time a new organic molecule, BBTD14, capable of imaging in the near-infrared region II. BBTD14 and the chemiluminescent substrate CPPO were encapsulated in F127 to form chemiluminescent nanoprobes, which can produce chemiluminescence in the presence of hydrogen peroxide (H2O2). PD-L1 antibodies were then linked to the surface of nanomicelles to construct chemiluminescent nanoprobes (CLNPs) modified with PD-1 antibodies, enhancing their targeting ability for osteosarcoma cells. Ultimately, after surgical resection of osteosarcoma, the resection margins were locally incubated and cleaned, and sprayed with hydrogen peroxide to enable instant imaging, showing residual tumor tissue. Since no external excitation light source is required, the interference of autofluorescence is reduced; at the same time, the limitation of selective verification during intraoperative freezing is overcome.

[0041] The chemiluminescent nanoprobe CLNP modified with PD-1 antibodies prepared by the present invention can visualize residual tumor tissue after osteosarcoma resection through local incubation and hydrogen peroxide spraying. The above-mentioned chemiluminescence does not require an external excitation light source, so there is no interference from the body's autofluorescence and has a higher signal-to-noise ratio. In addition, during surgery, CLNP can be applied to the resection margin after tumor resection to detect whether there is any tumor residue, overcoming the time and space limitations of local pathological verification in clinical practice, and providing a new adjuvant treatment method for osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0043] Figure 1 This is the synthesis route of the infrared II region organic small molecule BBTD14 in one embodiment of the present invention;

[0044] Figure 2 This is the 1H NMR spectrum of the organic small molecule BBTD14 in the infrared II region in one embodiment of the present invention;

[0045] Figure 3 This is the mass spectrum of the organic small molecule BBTD14 in the infrared II region in one embodiment of the present invention;

[0046] Figure 4 Schematic diagram of the absorption and emission spectra of BBTD14 and CLNP (chemiluminescent nanoprobe modified with PD-L1 antibody) in one embodiment of the present invention; wherein, part a is the emission and absorption spectra of BBTD14 in THF; part b is the emission and absorption spectra of CLNP in PBS buffer; part c is the particle size distribution and transmission electron microscopy image of CLNP in PBS buffer; part d is the chemiluminescent emission spectrum of CLNP in hydrogen peroxide;

[0047] Figure 5 1 is an element distribution diagram of C, Cl, N, S, and O of the chemiluminescent nanoprobe CLNP in one embodiment of the present invention;

[0048] Figure 6 1 is a schematic diagram of the results of the chemiluminescent performance test of the chemiluminescent nanoprobe CLNP in one embodiment of the present invention; wherein part a is the statistical and imaging results of the luminescence intensity of CLNP at different BBTD14 concentrations, and the small figure is the corresponding chemiluminescence image detected 10 minutes after the addition of hydrogen peroxide; part b is the statistics of the chemiluminescence intensity of CLNP at different H2O2 concentrations; part c is the signal-to-noise ratio corresponding to the maximum intensity time point of each curve in part b; part d is the chemiluminescence imaging of CLNP after adding different concentrations of H2O2 at 37°C; part e is the curve of the change of chemiluminescence intensity of CLNP at different concentrations with time after adding 100mM H2O2; part f is the corresponding chemiluminescence imaging of part e;

[0049] Figure 7 This is a near-infrared confocal image of mouse (LM8) and human (U2OS) osteosarcoma cells, muscle tissue (C2C12), vascular epithelial cells (HUVEC), connective tissue (3T3), and human skin fibroblasts (HSF) labeled with the chemiluminescent nanoprobe CLNP in one embodiment of the present invention;

[0050] Figure 8This is a schematic diagram of the results of the cell targeting and imaging test of the chemiluminescent nanoprobe CLNP in one embodiment of the present invention; wherein, part a is a photograph of the mouse after most of the subcutaneous tumor was removed; part b is the bright field after most of the subcutaneous tumor was removed from the mouse; part c is the chemical imaging image after most of the subcutaneous tumor was removed from the mouse, the resection edge was incubated with CLNP, and H2O2 was sprayed; part d is the superposition of the chemical imaging and bright field in part c; part e is the chemical imaging image after the positive tissue was cut off according to the chemiluminescent signal; part f is the superposition of the chemical imaging and bright field in part e; part g is the chemical imaging image after the resection edge was incubated with CLNP again and H2O2 was sprayed, confirming that there is no tumor residue at the resection edge; part h is the use of HE staining to confirm the tumor tissue removed under chemiluminescence guidance. DETAILED DESCRIPTION

[0051] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. It is obvious that the described embodiments are only some of the embodiments of the present invention, and are not all of the embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally measured in accordance with national standards. The experimental materials in the following examples, for which the sources are not specified, are all commercially available raw materials. The equipment used in each step of the following examples is conventional equipment. If there are no corresponding national standards, the steps are carried out in accordance with general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise specified, all parts are by weight, and all percentages are by mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention.

[0052] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0053] In the following examples, all chemicals used in the synthesis were purchased from Sigma-Aldrich, Shenzhen Ruixun Optoelectronic Materials Technology Co., Ltd., and Shanghai Titan Technology Co., Ltd. without further purification. Phosphate buffer (1×PBS, pH 7.4), DMEM medium, and physiological saline were purchased from Sangon Biotech (Shanghai) Co., Ltd. F127 was purchased from Tanshtech. Bis[3,4,6-trichloro-2-(pentyloxycarbonyl)phenyl]oxalate (98%) and hydrogen peroxide (30%) were provided by Sinopharm Chemical Reagent Co., Ltd.; 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), sodium n-hydroxysulfosuccinimide (Sulfo-NHS), and 2-n-morpholinoethanesulfonic acid (MES) were obtained from Adamas. Simulated body fluid (SBF, MX0952-500ml) was purchased from Maokang Biotechnology Co., Ltd. PD-L1 (atezolizumab) monoclonal antibody (abs190358) was provided by Aibixin (Shanghai) Biotechnology Co., Ltd. Nude mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd. Cells were purchased from Shanghai Yaji Biotechnology Co., Ltd.

[0054] Example 1 - Synthesis of BBTD14 small organic molecule for near-infrared II imaging

[0055] This embodiment is a preferred method for synthesizing the BBTD14 organic small molecule, which specifically includes the following steps:

[0056] like Figure 1 As shown, N,N-diphenyl-4-(tributyltin)aniline (5.4 g, 10.2 mmol), compound 1 (5.2 g, 12.6 mmol) and tetrakis(triphenylphosphine)palladium (200 mg, 0.17 mmol) were dissolved in toluene (40 mL) and refluxed for 24 hours. The mixture was purified by SiO2 column chromatography (hexane / ethyl acetate = 50 / 1) to obtain compound 2 as a yellow oily substance (yield 61%).

[0057] Compound 2 (2.71 g, 5.2 mmol) was dispersed in a THF solution (10 mL), and a -78°C n-butyllithium solution (1.59 M, dispersed in hexane, 5.0 ml, 8.0 mmol) was added dropwise. After stirring at -78°C for 1 hour, tributyltin chloride (n-Bu3SnCl) (2.6 g, 8.0 mmol) was added and stirred for 30 minutes, followed by stirring at room temperature for 2 hours. The mixture was purified by Al2O3 column chromatography (hexane: 100%) to obtain compound 3 as a yellow oil (yield 62%). Both compounds 2 and 3 were synthesized by Suzhou Zhuoxinya Technology Co., Ltd.

[0058] Compound 3 (811.33 mg, 1 mmol) and compound 4 (bromobenzobisthiadiazole, 87.45 mg, 0.25 mmol), tris(dibenzylacetone)dipalladium (22.893 mg, 0.025 mmol), tris(o-methylphenyl)phosphine (63.92 mg, 0.21 mmol) and 1.5 mL of toluene were added to a 10 mL three-necked flask and degassed for 30 minutes. The mixture was heated to 130° C. and stirred for 48 hours under a nitrogen atmosphere to obtain a crude product. The product was purified by SiO2 column chromatography (petroleum ether / dichloroethylene = 5 / 1) to obtain a dark green solid BBTD14 molecule (yield 35%). The 1H NMR spectrum and mass spectrum of BBTD14 are shown in FIG. Figure 2-3 The NMR data are as follows: 1H NMR (400 MHz, Chloroform-d) δ 7.52–7.47 (m, 4H), 7.31–7.24 (m, 11H), 7.23 (d, J = 3.7 Hz, 2H), 7.16–7.11 (m, 8H), 7.06 (q, J = 7.9, 7.3 Hz, 8H), 2.56 (d, J = 7.1 Hz, 4H), 1.19–0.83 (m, 15H), 0.80 (tt, J = 10.1, 4.9 Hz, 2H), 0.71–0.60 (m, 6H), 0.47 (t, J = 7.3 Hz, 6H).

[0059] Example 2 - Synthesis of Chemiluminescent Nanoprobes with or without Modified PD-L1 Antibodies

[0060] This embodiment provides a method for synthesizing a preferred chemiluminescent nanoprobe (including chemiluminescent nanoparticles modified or unmodified with PD-L1 antibodies), which specifically includes the following steps:

[0061] (1) Synthesis of chemiluminescent nanoprobes;

[0062] Dissolve 100 mg of carboxylated F127, 0.3 mg of BBTD14 (25 nmol), and 40 mg of bis(oxalate) ester (CPPO) in 5 mL of tetrahydrofuran. Mix thoroughly and evaporate the organic solvent using a vacuum rotary evaporator. Add 2.5 mL of PBS buffer to allow micelles to self-assemble. Store at 4°C in the dark. Dilute to the desired concentration with PBS buffer before use.

[0063] To determine the optimal CLNP-dye coating ratio, the concentrations of CPPO and carboxylated F127 were kept constant while varying the amount of BBTD14. The ideal coating ratio of the material was evaluated based on the highest signal observed during 10 minutes of imaging, as detailed in Example 3.

[0064] (2) Synthesis of chemiluminescent nanoprobes (CLNPs) modified with PD-L1 antibodies;

[0065] The binding of PD-L1 antibody to micelles occurs through an amide reaction between the amino groups on the antibody protein and the carboxylated F127 on the outside of the micelles. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide sulfonic acid sodium salt (sulo-NHS) were dissolved in an aqueous solution (10 mM) of morpholineethanesulfonic acid (MES) at a concentration of 200 mM to obtain EDC solution and NHS solution. 62.5 μL of EDC solution and 75 μL of NHS solution were added to 500 μL of micelle solution (chemiluminescent nanoprobe solution prepared in step (1)) at a concentration of 50 μM, respectively, and incubated in the dark at 4°C for 40 minutes. Then, using a 100,000 molecular weight cut-off ultrafiltration tube, the filtrate was centrifuged at 6,000 rpm for 5 minutes, and repeated twice. After centrifugation to remove the supernatant, the solution was redissolved in 1 mL of PBS solution, 50 μg of PD-L1 antibody was added, and the solution was shaken at 4°C for 24 hours. The whole process was completely protected from light.

[0066] Example 3 - Performance Verification of Near-Infrared II Region Organic Small Molecule BBTD14 and Chemiluminescent Nanoprobe CLNP This example verifies the optical performance of BBTD14 prepared in Example 1 and CLNP prepared in Example 2, including:

[0067] (1) Absorption and emission spectra of BBTD14 and CLNPs: First, 100 nmol of BBTD14 was dissolved in 1 mL of tetrahydrofuran (THF), and its absorption spectrum was detected using a UV spectrometer, and its emission spectrum was detected under 808 nm excitation light; secondly, the prepared CLNPs were dissolved in PBS buffer (50 μM), and their absorption and emission spectra were detected; finally, hydrogen peroxide (100 mM) was added to CLNPs (100 μM), and its chemical generation spectrum was detected under no excitation light.

[0068] The above experimental results are as follows Figure 4 As shown in the figure, specifically: the absorption spectrum of BBTD14 in the organic phase shows a near-infrared absorption peak at 688 nm, and the maximum absorption extends to 900 nm; the emission wavelength of BBTD14 is in the NIR-II region, with the main peak appearing at 1026 nm and the tail extending to 1400 nm ( Figure 4 The absorption and emission spectra of CLNP are similar to those of BBTD14 ( Figure 4 Part b of the figure shows that it can be excited by near-infrared light; the characterization of CLNPs was further evaluated using transmission electron microscopy (DLS) and dynamic light scattering (DLS). The results showed that CLNPs were spherical nanoparticles with a diameter of about 21 nm ( Figure 4The chemiluminescence emission spectrum of CLNPs activated by hydrogen peroxide confirmed the successful occurrence of the CRET (ortho-triggered chemiluminescence resonance energy transfer) process ( Figure 4 The d part of the CLNP is the longest emission wavelength reported so far for CRET systems. In addition, the CLNPs were detected by element scanning, and the results showed that the C, Cl, N, S and O elements were uniformly distributed in the CLNPs (e.g. Figure 5 shown).

[0069] (2) Chemiluminescence performance test of CLNP;

[0070] 1) Determination of CLNP CL Intensity at Different H2O2 Concentrations: All in vitro chemiluminescence characterization of CLNPs was performed in 96-well plates. Since the micelle synthesis ratio was fixed, the micelle concentration was defined by the BBTD14 concentration in the solution. The total reaction volume was 200 μL. With a fixed CLNP concentration of 60 μM, the hydrogen peroxide concentration was varied (10, 50, 100, 200, 400, 600, 800, and 1000 mM) to achieve the desired experimental conditions. The effects of different hydrogen peroxide concentrations on the experiment were quantitatively evaluated and the minimum detectable hydrogen peroxide concentration was determined. After the reactants were thoroughly mixed, the plate was immediately placed in a real-time imager and images were recorded from 0 to 60 minutes with an exposure time of 2 seconds. Subsequent CL intensity statistics were measured using ImageJ software.

[0071] The above experimental results are as follows Figure 6 Specifically, the following steps are performed: first, the contents of F127 and CPPO used for micelle coating are fixed, and the amount of BBTD14 added (5, 10, 15, 20, 25, 30, 35, 40 nmol) is adjusted to form micelles. After activation by hydrogen peroxide, the optimal coating amount of BBTD14 in the micelles is determined according to the CL intensity ( Figure 6 The results show that the 25 nmol BBTD14 encapsulation group exhibits the most outstanding performance after 10 min of hydrogen peroxide addition. Secondly, at a fixed micelle concentration, the CL signal under different hydrogen peroxide concentrations was detected ( Figure 6 (Part b) It was observed that each group could continue to emit light for more than 1 hour. The change of CL intensity with time at 50, 100, and 200 mM hydrogen peroxide concentrations showed an initial increase and then a gradual decrease, with corresponding half-lives of 83, 77, and 64 minutes, respectively. Furthermore, the signal-to-noise ratio at the time point of the strongest signal was calculated for each curve, and it was observed that its maximum value reached 137.6 ( Figure 6 It is worth noting that under NIR-II imaging, the CL signal of CLNPs is still clearly visible at a hydrogen peroxide concentration as low as 0.5 mM ( Figure 6 (d)

[0072] 2) CL Intensity Measurement at Different CLNP Concentrations: As with the CL assay described above, the total reaction volume was set at 200 μL, the hydrogen peroxide concentration was fixed at 100 mM, and the CLNP concentration was adjusted accordingly (1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM). After the reactants were thoroughly mixed, the plate was immediately placed in a real-time imager, and images were recorded from 0 to 60 minutes with an exposure time of 2 seconds.

[0073] The above experimental results are as follows Figure 6 Specifically, in order to ensure long-term, stable and strong CL luminescence, the change of CL intensity over time under the action of different concentrations of CLNP (in units of BBTD14 concentration) was observed ( Figure 6 (Fig. e and f). When the CLNP concentration increased from 1 μM to 30 μM, the CL intensity showed a continuous downward trend, indicating an excess of hydrogen peroxide. In the 40–100 μM range, the chloride ion intensity initially increased and then decreased, with a half-life exceeding 60 minutes at concentrations above 50 μM. This suggests that CLNPs in the 50–100 μM range could provide the necessary time and space for intraoperative guidance.

[0074] Example 4 - Cell Targeting and Imaging Capabilities of Chemiluminescent Nanoprobe CLNP

[0075] This example verifies the cell targeting and imaging capabilities of the chemiluminescent nanoprobe CLNP in vitro and in vivo in mice, specifically including:

[0076] (1) Confocal imaging was used to evaluate the affinity of CLNP for osteosarcoma cells in vitro: LM8, U2OS, C2C12, HUVEC, 3T3, and HSF cells (2×10 5 Each cell type was seeded in a confocal dish and allowed to adhere overnight (DMEM high-glucose medium, 37°C, 5% CO2). Each cell type was co-cultured with CLNPs (5 μM) for 10 minutes and then washed three times with PBS. Fresh DMEM medium was then added to replace the original medium, and near-infrared imaging was performed under 808 nm excitation light.

[0077] The above experimental results are as follows Figure 7 As shown in the figure, specifically: CLNP modified with PD-LI has a strong affinity for osteosarcoma cells. In mouse osteosarcoma cells LM8 and human osteosarcoma cells U2OS, stronger fluorescence signals were observed by confocal microscopy, while there was no significant fluorescence signal in other cell lines, confirming that CLNP has good targeting specificity at the cellular level.

[0078] (2) Using mice with subcutaneous tumors, the imaging effect of residual tumors was evaluated by incubating CLNPs locally after tumor resection and spraying hydrogen peroxide after clearing: female balb / c nude mice (4-6 weeks old, purchased from Shanghai Slake Animal Laboratory Co., Ltd.) were injected subcutaneously with U2OS osteosarcoma cells (2×10 6 When the tumor volume reached 200 mm 3 The experiment was conducted around 1:10 pm. Under gas anesthesia, the mouse tumor was largely resected. CLNPs (50 μM, 70 μL) were then incubated locally at the tumor margin for 10 minutes. The wound was then washed three times with saline to remove nonspecifically bound nanoprobes. Finally, hydrogen peroxide (100 mM) was sprayed onto the wound surface for chemiluminescence imaging, and near-infrared imaging was used to capture the CL signal. Once a signal was detected, the tissue within the signaling area was immediately excised, and the above process was repeated to confirm a negative margin.

[0079] The above experimental results are as follows Figure 8 Specifically, after the mouse was anesthetized, most of the tumor was removed. The photos after removal are as follows: Figure 8 As shown in part a of the figure, there is no obvious tumor tissue remaining under the naked eye, and it is recorded under the bright field of the near-infrared imager ( Figure 8 Secondly, after local incubation, washing and spraying of hydrogen peroxide on CLNP, the chemiluminescence signal was detected by the near-infrared imager, and after fusion with the bright field image, the signal was found to be located at the resection margin of the tumor ( Figure 8 c and d). Under chemiluminescence guidance, secondary resection of the tumor was performed, and it was observed that the tissue with chemiluminescence signal was removed ( Figure 8 Finally, repeat the incubation-washing-hydrogen peroxide spraying process again, and complete the operation after confirming that there is no chemiluminescence signal ( Figure 8 HE staining was used to confirm the tumor tissue ( Figure 8 The h section of the tissue is fixed, dehydrated, transparent, embedded, and then sectioned. The tissue is then dewaxed and hydrated, and finally stained with hematoxylin and eosin. After staining, the tissue is dehydrated, transparent, and mounted. Microscopic examination of tissue with significant cellular atypia, an increased nuclear-cytoplasmic ratio, increased mitotic activity, and structurally disorganized tissue indicates residual tumor tissue.

[0080] From the above examples, it can be seen that (1) the chemiluminescent probe CLNP of the present invention can replace intraoperative freezing to confirm the negative resection margin in osteosarcoma resection surgery. Currently, after osteosarcoma resection, in order to confirm that there is no tumor residual at the surgical resection margin, part of the suspicious tissue can only be cut off and immediately sent for intraoperative freezing. This method requires waiting for the intraoperative freezing result on the operating table and cannot detect the entire surgical resection margin. The method of the present invention can observe the surgical resection margin in real time to see if there is any residual tumor signal; (2) In the past, image-guided surgery was mainly based on fluorescence imaging under photoluminescence, which relied on normal The fluorescent probes accumulated in tissue and tumor tissue are different and have different signal-to-noise ratios, so as to judge whether it is tumor tissue. Since tissue has spontaneous fluorescence under photoluminescence, the distinction between the two will be blurred. However, the chemiluminescence in the present invention does not require external excitation light, avoiding the influence of spontaneous fluorescence, allowing the surgeon to more intuitively judge the positive resection margin; (3) The wavelength of chemiluminescence in the past was mainly below 900nm, while the chemiluminescence wavelength of CLNP in the present invention can reach above 1400nm. The longer wavelength can have a better penetration depth to achieve deep imaging, and has greater application prospects in the future.

[0081] While the specific embodiments of the present invention have been described in detail above, these are intended to be exemplary only, and the present invention is not limited thereto. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

Claims

1. A near-infrared II region organic small molecule, characterized in that: The near-infrared II region organic small molecule is BBTD14, and the chemical structure of BBTD14 is shown below:

2. A method for synthesizing organic small molecules in the near-infrared II region, characterized in that: The near-infrared II region organic small molecule is BBTD14, and the synthesis route of BBTD14 is: Step S1, dissolving compound 1, N,N-diphenyl-4-(tributyltin)aniline, and tetrakis(triphenylphosphine)palladium in toluene in a predetermined ratio, and reacting at room temperature for 18 to 30 hours to obtain compound 2; Step S2, dispersing compound 2 in a THF solution, adding a -85 to -70°C n-butyl lithium solution, reacting at -85 to -70°C for 0.5 to 2 hours, adding tributyltin chloride, and reacting at room temperature for 1 to 4 hours to obtain compound 3; Step S3: dissolve compound 3, compound 4, tris(dibenzylacetone)dipalladium, and tris(o-methylphenyl)phosphine in toluene, mix, and degas for 15 to 60 minutes; react at 100 to 150° C. in a nitrogen atmosphere for 36 to 60 hours to obtain the organic small molecule BBTD14.

3. The synthesis method according to claim 2, characterized in that In step S1, the molar ratio of compound 1, N,N-diphenyl-4-(tributyltin)aniline, and tetrakis(triphenylphosphine)palladium is 10-15:8-12:0.05-0.4; And / or, in step S2, the molar ratio of compound 2, n-butyl lithium, and tributyltin chloride is 4-6:6-10:6-10; and / or, in step S3, the molar ratio of compound 3, compound 4, tris(dibenzylacetone)dipalladium, and tris(o-methylphenyl)phosphine is 0.5-2:0.1-0.4:0.01-0.04:0.1-0.

4.

4. A nanoprobe CNLP, characterized in that The preparation method of the nanoprobe CNLP includes: dissolving carboxylated F127, an organic small molecule BBTD14, and a bisoxalate in a tetrahydrofuran solution, fully mixing and evaporating the organic solvent, adding a PBS buffer solution, and self-assembling them into nanomicelles to obtain a chemiluminescent nanoprobe; adding an EDC solution and an NHS solution to the chemiluminescent nanoprobe solution, respectively, and incubating in the dark at 4°C for 20 to 60 minutes; ultrafiltration of the solution, centrifugation of the filtrate, removal of the supernatant, redissolving, adding a PD-L1 antibody, and reacting at 4°C in the dark for 12 to 36 hours to obtain the nanoprobe CLNP; The chemical structure of the organic small molecule BBTD14 is shown below: The mass ratio of the carboxylated F127, the organic small molecule BBTD14 and the bisoxalate is 80-120:0.1-0.8:20-60; In addition, the concentration of the chemiluminescent nanoprobe solution is 20-100 μM, and the volume is 400-600 μL; and, the EDC solution is prepared by dissolving EDC at a concentration of 200 mM in a 10 mM aqueous solution of morpholineethanesulfonic acid, and the amount used is 50-80 μL; and, the NHS solution is prepared by dissolving sulo-NHS at a concentration of 200 mM in a 10 mM aqueous solution of morpholineethanesulfonic acid, and the amount used is 60-90 μL; and, the amount of the PD-L1 antibody is 40-60 μg; and, the steps of ultrafiltration and centrifugation are: using a 100,000 molecular weight cut-off ultrafiltration tube, centrifuging the filtrate at 6000 rpm for 5 minutes, and repeating twice.

5. Use of the nanoprobe CNLP as claimed in claim 4 in the preparation of a biological imaging agent.

6. Use of the nanoprobe CNLP according to claim 4 in preparing a reagent for detecting positive surgical margins of osteosarcoma.

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