Preparation and application of fluorescent nanoprobe for rapidly detecting in-vitro tissue in tumor resection

Abstract

The invention relates to the technical field of medical treatment, in particular to preparation of a fluorescent nanoprobe and a method for applying the fluorescent nanoprobe to detecting whether tumor tissue remains in an in-vitro tissue in tumor resection. The invention provides a fluorescent molecule with aggregation-induced emission characteristics for tumor imaging. The structural general formula of the fluorescent molecule is as follows: # imgabs0 #. The invention also provides a method for preparing nanoparticles NPs by using the fluorescent molecule and a method for obtaining a fluorescent probe by modifying an antibody / polypeptide by using the nanoparticles NPs. The in-vitro tissue biopsy device can realize accurate biopsy of in-vitro tissues in tumor resection.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and particularly to a method for preparing a fluorescent nanoprobe and its application in detecting whether residual tumor tissue remains in excised tissue during tumor resection surgery. Background Art

[0002] How to specifically cure malignant tumors remains a worldwide problem. The clinical treatment methods for malignant tumors mainly include surgical methods, chemotherapy, radiotherapy, etc. The surgical method aims to completely remove tumor tissue while minimizing collateral damage to normal tissue; chemotherapy uses chemical drugs to kill tumor cells, which can extend the survival period of patients, but is accompanied by severe adverse reactions and may even cause irreversible physical damage; the core principle of radiotherapy is to skillfully use high-energy radiation to kill tumor tissue, and radiotherapy is accompanied by varying degrees of side effects, such as skin damage in the irradiated field, nausea and vomiting, fever, decrease in peripheral blood picture, fatigue, etc. Accurately defining the boundary between the tumor and normal tissue during the operation is a key factor in reducing postoperative recurrence and secondary surgery. Frozen section is a common pathological technique for rapid diagnosis of tissue samples during surgery. It rapidly cools the tissue to a certain hardness under low-temperature conditions and then slices it. Although this technique has the advantages of simplicity, rapidity, and preservation of tissue components and has been widely used in various fields, it also has certain limitations. During the production process, it requires rapid freezing and cutting, and ice crystals are easily generated in the tissue block during the freezing process, which affects the morphological structure of cells and the localization of antigenic substances, and the quality of the section may be affected to a certain extent. In addition, the frozen section technique has high technical requirements for operators, who need to proficiently master skills such as freezing, cutting, and observation, and a pathologist is required to determine whether there is residual tumor tissue through tissue morphology. These relative defects increase the recurrence rate of tumors.

[0003] In order to improve the defects of the current frozen section technique and inspired by the fluorescence surgical navigation technique, we propose a new detection method - fluorescence section technique, that is, to rapidly determine whether there is tumor residue in the excised tissue during tumor surgery through fluorescence imaging. During the operation, we co-incubate the freshly excised tissue with a fluorescent probe with targeting ability and use a fluorescence imaging system to accurately visualize the location and boundary of the tumor. This method greatly reduces the technical requirements for personnel, and since multiple tissues can be co-incubated with the fluorescent probe simultaneously by this method, it improves the biopsy efficiency of the excised tissue, effectively reduces the operation duration, and reduces the probability of postoperative recurrence and secondary surgery.

[0004] Traditional fluorescent probes such as fluorescein, rhodamine, and boron-dipyrromethene (BODIPY) exhibit good luminescence properties in dilute solution. However, once the concentration increases or aggregation occurs, the fluorescence intensity will significantly decrease or even disappear due to intermolecular π-π interactions. This phenomenon is called the Aggregation-Caused Quenching (ACQ) effect. In the study of fluorescence slicing technology, when such fluorescent probes are applied to bioimaging, they aggregate in tumor cells due to targeting, resulting in the ACQ effect and a decrease in fluorescence intensity in cells. When the expression of patient-specific targets is low, false-negative results are likely to occur. Different from traditional ACQ molecules, Aggregation-Induced Emission (AIE) materials dissipate the excited-state energy mainly through non-radiative decay due to active molecular motion in dilute solution or single-molecule state. In the aggregated state, due to restricted molecular motion, the non-radiative energy transition path is inhibited, leading to a significant enhancement of fluorescence. This feature enables them to still have strong fluorescence inside cells, reducing the probability of false negatives in patients. To endow AIE fluorescent probes with better biocompatibility, optical stability, and tumor targeting ability, they are encapsulated into fluorescent nanoparticles and polypeptides / antibodies are modified on the particle surface.

[0005] In summary, developing a convenient and fast new intraoperative biopsy method for the discrimination of positive margins during tumor resection and designing a fluorescent molecular probe with excellent luminescence intensity, good optical / chemical stability, reverse quenching in the aggregated state, and precise targeting ability for this application scenario have clear industrialization prospects and practical significance.

[0006] The invention of CN116425739B, "Compounds for Tumor Targeted Imaging and Photodynamic Therapy, Their Synthesis Methods and Applications", discloses that the structural formula of the fluorescent molecule is

[0007] In the formula, R1 is a straight-chain or branched-chain alkyl with 1-20 carbon atoms, R2 is one of hydrogen, a straight-chain or branched-chain alkyl with 1-20 carbon atoms, and a straight-chain or branched-chain alkoxy with 1-20 carbon atoms, and R3 is one of hydrogen, arylvinyl, a straight-chain or branched-chain alkyl with 1-20 carbon atoms, a straight-chain or branched-chain alkoxy with 1-20 carbon atoms, and a straight-chain carboxyl with 1-20 carbon atoms. The fluorescence wavelength of this fluorescent molecule is in the first near-infrared region (around 815 nm), which is more suitable for fluorescence imaging in the human body, and it has a certain ability to generate ROS. It mainly serves as a navigation imaging agent during tumor resection surgery and a photosensitizer for tumor photodynamic therapy. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a fluorescent nanosphere probe that can be used for in vitro tissue biopsy during tumor resection surgery, and its preparation method and use.

[0009] To solve the above problems, the present invention provides a fluorescent molecule with aggregation-induced emission characteristics for tumor imaging, and the structural general formula of the fluorescent molecule is as follows:

[0010]

[0011] In the structural general formula, R1 is a straight chain with 0 to 20 carbon atoms, and R is any one of the following: triphenylamine, 4,4'-dimethyltriphenylamine, 4,4'-diphenyltriphenylamine, tetraphenylethylene, 4-methyltetraphenylethylene, 4-methoxytetraphenylethylene group.

[0012] As an improvement of the fluorescent molecule of the present invention, it is any one of the following:

[0013]

[0014] Eco-A1110, R1 is a straight-chain hexyl group, and R is triphenylamine; Eco-A4410, R1 is a straight-chain dodecyl group, and R is 4,4'-dimethyltriphenylamine; Eco-A9510, R1 is a straight-chain butyl group, and R is 4,4'-diphenyltriphenylamine; Eco-A2110, R1 is a straight-chain hexyl group, and R is tetraphenylethylene; Eco-A6410, R1 is a straight-chain dodecyl group, and R is 4-methoxytetraphenylethylene.

[0015] The fluorescent molecule synthesized by the present invention that can be used for infrared region I fluorescence imaging can effectively reduce the ACQ effect in the aggregated state, laying a foundation for the next step of preparing nanoparticles that can be used for in vitro tissue biopsy during nanoparticle tumor resection.

[0016] The present invention also simultaneously provides a preparation method for the above fluorescent molecule, including the following steps:

[0017]

[0018] 1), Prepare precursor 2:

[0019] React precursor 1 with 2-bromo-3-(R1)thiophene under the action of the basic environment formed by tetrakis(triphenylphosphine)palladium catalysis and potassium carbonate and solvent (1,4-dioxane) to carry out a suzuki coupling reaction. The suzuki coupling reaction is carried out at 100 ± 10 °C for 12 to 24 h; after purification of the reaction product, precursor 2 is obtained;

[0020] The molar ratio of precursor 1:2-bromo-3-(R1)thiophene:tetrakis(triphenylphosphine)palladium:potassium carbonate = 1 to 2:1:0.03 to 0.1:10 to 20;

[0021] The precursor 1 is The precursor 2 is

[0022] 2), Preparation of precursor 3:

[0023] React precursor 2 with n-butyllithium in a solvent (tetrahydrofuran) at -78 ± 10 °C for 1 - 2 h, and then add tributyltin chloride and transfer to room temperature for reaction for 4 - 6 h; the resulting crude product named precursor 3 (i.e., obtain the reaction product containing precursor 3);

[0024] The molar ratio of precursor 2: n-butyllithium: tributyltin chloride = 1: 1.1 - 1.3: 1.2 - 1.5;

[0025] The precursor 3 is

[0026] Note: The reaction product of this step does not need to be purified and can be directly used in the next step;

[0027] 3), Carry out a Stille coupling reaction on the crude product of precursor 3 and 4,7-dibromo-2,1,3-benzothiadiazole under the catalysis of tetrakis(triphenylphosphine)palladium in a solvent (toluene), and the Stille coupling reaction is carried out at 100 ± 10 °C for 12 - 24 h;

[0028] The molar ratio of precursor 3: 4,7-dibromo-2,1,3-benzothiadiazole: tetrakis(triphenylphosphine)palladium = 3 - 5: 1: 0.03 - 0.1;

[0029] After the reaction product is purified, a fluorescent molecule for imaging is obtained; the fluorescent molecule is

[0030] Among precursor 1, precursor 2, precursor 3, and the fluorescent molecule:

[0031] R1 is a straight chain with 0 - 20 carbon atoms, and R is any one of the following: triphenylamine, 4,4'-dimethyltriphenylamine, 4,4'-biphenyltriphenylamine, tetraphenylethylene, 4-methyltetraphenylethylene, 4-methoxytetraphenylethylene groups.

[0032] As an improvement to the preparation method of the fluorescent molecule of the present invention: The preparation of the precursor 1 is:

[0033] Carry out a Suzuki coupling reaction on the tetraphenylethylene compound and bis(pinacolato)diboron under the catalysis of palladium(II) dichloride DPPF (i.e., [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium) and the action of the alkaline environment formed by potassium acetate and a solvent (1,4-dioxane), and the Suzuki coupling reaction is carried out at 100 ± 10 °C for 12 - 24 h,

[0034] The molar ratio of tetraphenylethylene compound: pinacol borane: dichlorobis(triphenylphosphine)palladium(II): potassium acetate is 1: 1-2: 0.03-0.1: 10-20;

[0035] When the tetraphenylethylene compound is 1-(4-bromophenyl)-1,2,2-triphenylethylene, the obtained precursor 1 is 4,4,5,5-tetramethyl-2-(4-(1,2,2-triphenylethylenyl)phenyl)-1,3,2-dioxaborolane;

[0036] When the tetraphenylethylene compound is [1,2-diphenyl-1-(4-bromophenyl)-2-(4-methoxyphenyl)]ethylene, the obtained precursor 1 is (E)-2-(4-(2-(4-methoxyphenyl)-1,2-diphenylethylenyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane.

[0037] The present invention also provides a method for preparing nanoparticles NPs using the above-mentioned fluorescent molecules, including the following steps:

[0038] Dissolve the fluorescent molecule and DSPE-PEG2000-Mal (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-maleimide) in tetrahydrofuran to form a mixed solution, where the mass ratio of the fluorescent molecule: DSPE-PEG2000-Mal is 1: 5.

[0039] According to the volume ratio of tetrahydrofuran: ultrapure water = 1: 9, add the mixed solution to ultrapure water (rapidly add) under ultrasonic conditions, and then continue to ultrasonicate for 10 ± 4 min to promote uniform dispersion of the system. Then, bubble nitrogen to remove tetrahydrofuran to obtain nanoparticles NPs.

[0040] The present invention also provides a method for obtaining a fluorescent probe by modifying nanoparticles NPs with an antibody / polypeptide, including the following steps:

[0041] Dissolve nanoparticles NPs in tetrahydrofuran to prepare an NPs solution with a concentration of 2 mg / mL;

[0042] Dissolve the antibody / polypeptide in PBS buffer to prepare an antibody solution with a concentration of 10 μg / mL or a polypeptide solution with a concentration of 1 mg / mL;

[0043] According to the volume ratio of tetrahydrofuran: PBS buffer = 1: 2, mix the NPs solution with the antibody solution / polypeptide solution and react overnight (12-14 h) at room temperature in the dark; then dialyze (the cut-off molecular weight is 8000-14000) to remove the excess antibody / polypeptide to obtain the fluorescent nanoprobe NPs-AB / NPs-peptide.

[0044] Improvement of the method for modifying antibodies / polypeptides with nanoparticles (NPs) of the present invention to obtain fluorescent probes:

[0045] The antibody is EphA2 antibody (i.e., Anti-Eph receptor A2 antibody [EPR17660-120]), and the fluorescent probe NPs-EphA2-AB is obtained;

[0046] The antibody is Anti-EGFR antibody [EGFR1], and the fluorescent probe NPs-EGFR-AB is obtained;

[0047] The polypeptide is CFFGYSAYPDSVPMMS, and the fluorescent probe NPs-YSA-peptide is obtained,

[0048] The polypeptide is CFFGYHWYGYTPENVI, and the fluorescent probe NPs-GE11-peptide is obtained.

[0049] The structural formula of the polypeptide CFFGYSAYPDSVPMMS is:

[0050]

[0051] The structural formula of the polypeptide CFFGYHWYGYTPENVI is:

[0052]

[0053] Among them, the antibody Anti-Eph receptor A2 antibody [EPR17660-120] and the -CFFGYSAYPDSVPMMS polypeptide target is EphA2; the Anti-EGFR antibody [EGFR1] and the -CFFGYHWYGYTPENVI target is EGFR.

[0054] Taking the fluorescent molecule Eco-A1110 as an example, the obtained nanoparticles (NPs) modified antibodies are Eco-A1110 NPs-EphA2-Ab, Eco-A1110 NPs-EGFR-Ab, and the obtained nanoparticles (NPs) modified polypeptides are Eco-A1110 NPs-YSA-peptide, Eco-A1110

[0055] NPs-GE11-peptide.

[0056] The present invention also simultaneously provides a method for precise biopsy of ex vivo tissues during tumor resection, including the following steps:

[0057] 1), According to the characteristics of the tumor / cancer, select a fluorescent probe with good corresponding targeting;

[0058] 2) Cut a part from the edge of the ex vivo tumor tissue, and then put the excised tissue into Krebs-Hensleit's Solution containing 5% fetal bovine serum for blocking. The blocking temperature is 4 - 8 °C, and the soaking time is 2 - 5 min (multiple positions can be excised and soaked simultaneously);

[0059] 3) Transfer the blocked excised tissue obtained in step 2) to phosphate buffered saline containing 30 - 100 uM fluorescent probe (KCl: 2.67 mM; NaCl: 137.07 mM; Na2HPO3: 10 mM; KH2PO4: 1.84 mM; pH is 7.4 ± 0.2), and incubate for 10 - 20 min; after incubation, rinse with phosphate saline containing PEG-400 (mass concentration 30%) for 1 - 3 times, 1 - 2 min each time; then image the tissue with a fluorescence imaging system and observe the fluorescence intensity.

[0060] 4) Image the excised tissue obtained by the treatment in step 3) with a fluorescence imaging system and observe the fluorescence intensity; thereby determine the tumor boundary.

[0061] The imaging boundary of the ex vivo tissue resection margin of the present invention is clear and easy to distinguish.

[0062] As an improvement to the method for precise biopsy of ex vivo tissue during tumor resection of the present invention:

[0063] When it is breast cancer (4T1), colon cancer (MC-38), select fluorescent probes NPs-EphA2-Ab and fluorescent probe NPs-YSA-peptide,

[0064] When it is oral squamous cell carcinoma, select fluorescent probe NPs-GE11-peptide and fluorescent probe NPs-EGFR-Ab.

[0065] As a further improvement to the method for precise biopsy of ex vivo tissue during tumor resection of the present invention, as a preference:

[0066] The soaking time in step 2) is 3 min;

[0067] In step 3): the concentration of the fluorescent nanoprobe is 50 uM, the incubation time is 10 - 15 min, the number of rinsing times is 2 - 3 times, and the rinsing time each time is 1 min.

[0068] That is, the primary object of the present invention is to provide a fluorescent nanosensor for ex vivo tissue biopsy during tumor resection surgery. Another important object of the present invention is to provide a method for preparing a fluorescent molecule for ex vivo tissue biopsy during tumor resection surgery. Another object of the present invention is to provide a method for preparing nanoparticles from fluorescent molecules and modifying them with polypeptides / antibodies. Another important object of the present invention is to provide a new method for ex vivo tissue biopsy during tumor resection surgery.

[0069] The Eco-Axx10 series of fluorescent nanosensors of the present invention have the following advantages and beneficial effects compared with the prior art:

[0070] (1) A series of fluorescent molecules in the present invention use benzothiadiazole as the core, triarylamine as the electron donor, and 3-alkyl-substituted thiophene as the π-bridge to construct a D-π-A-π-D molecular structure, effectively improving the fluorescence intensity.

[0071] (2) The fluorescent molecules prepared in the present invention have better stability compared with traditional cyanine fluorescent molecules (such as ICG).

[0072] (3) The series of fluorescent molecules of the present invention have a twisted molecular configuration, which can effectively alleviate the ACQ effect caused by molecular stacking, so that they still have strong fluorescence after being prepared into nanoparticles, and the imaging boundary of the ex vivo tissue resection margin during the operation is clear and easy to distinguish.

[0073] (4) The nanoparticles prepared in the present invention can be conjugated with multiple antibodies / polypeptides, with excellent targeting and a low false positive probability.

[0074] The immersion intraoperative fluorescence biopsy method adopted in the present invention has the following advantages and beneficial effects compared with the prior art:

[0075] (1) The traditional frozen section method requires professional pathologists to determine whether there is tumor residue through tissue morphology, which requires high experience of doctors; the immersion intraoperative fluorescence biopsy method determines the resection margin sample through fluorescence brightness, and the discrimination standard is simple, greatly reducing the requirements for doctors.

[0076] (2) The traditional frozen section method is limited by time, operation, and personnel, and the number of detection points for a single sample submission is small; the new intraoperative method can achieve multi-site simultaneous biopsy, with a large number of detectable sites, simple operation, and can be independently completed by one doctor in a single operation, effectively improving the complete tumor resection rate, shortening the operation duration, reducing the possibility of secondary surgery and the risk of postoperative lesion metastasis.

[0077] In summary, a series of fluorescent nanosensors in the present invention are mainly applied to ex vivo tissue biopsy during tumor resection surgery, providing a new method for determining the tumor boundary during tumor resection surgery, preventing secondary surgery and postoperative lesion metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The following further elaborates on the specific implementation manners of the present invention in conjunction with the accompanying drawings.

[0079] Figure 1 Flow chart of a new method for ex vivo tissue biopsy during tumor resection surgery - fluorescence sectioning technique;

[0080] Figure 2 1H NMR spectrum of Eco-A1110 prepared in Example 1-1;

[0081] Figure 3 1H NMR spectrum of Eco-A4410 prepared in Example 1-2;

[0082] Figure 4 1H NMR spectrum of Eco-A9510 prepared in Example 1-3;

[0083] Figure 5 1H NMR spectrum of Eco-A2110 prepared in Example 2-1;

[0084] Figure 6 1H NMR spectrum of Eco-A6410 prepared in Example 2-2;

[0085] Figure 7 High-resolution mass spectrum of Eco-A1110 prepared in Example 1-1;

[0086] Figure 8 High-resolution mass spectrum of Eco-A4410 prepared in Example 1-2;

[0087] Figure 9 High-resolution mass spectrum of Eco-A9510 prepared in Example 1-3;

[0088] Figure 10 High-resolution mass spectrum of Eco-A2110 prepared in Example 2-1;

[0089] Figure 11 High-resolution mass spectrum of Eco-A6410 prepared in Example 2-2;

[0090] Figure 12 UV absorption and fluorescence emission spectra of Eco-A1110 prepared in Example 1-1;

[0091] Figure 13 UV absorption and fluorescence emission spectra of Eco-A4410 prepared in Example 1-2;

[0092] Figure 14 UV absorption and fluorescence emission spectra of Eco-A9510 prepared in Example 1-3;

[0093] Figure 15 UV absorption and fluorescence emission spectra of Eco-A2110 prepared in Example 2-1;

[0094] Figure 16 UV absorption and fluorescence emission spectra of Eco-A6410 prepared in Example 2-2;

[0095] Figure 17 Normalized fluorescence emission spectra (left figure) of Eco-A1110 prepared in Example 1-1 in THF / H2O mixed solutions with different volume ratios, and aggregation-induced emission curve (right figure);

[0096] Figure 18 Normalized fluorescence emission spectra (left figure) of Eco-A4410 prepared in Example 1-2 in THF / H2O mixed solutions with different volume ratios, and aggregation-induced emission curve (right figure);

[0097] Figure 19 Normalized fluorescence emission spectra (left figure) of Eco-A9510 prepared in Example 1-3 in THF / H2O mixed solutions with different volume ratios, and aggregation-induced emission curve (right figure);

[0098] Figure 20 Normalized fluorescence emission spectra (left figure) of Eco-A2110 prepared in Example 2-1 in THF / H2O mixed solutions with different volume ratios, and aggregation-induced emission curve (right figure);

[0099] Figure 21 Normalized fluorescence emission spectra (left figure) of Eco-A6410 prepared in Example 2-2 in THF / H2O mixed solutions with different volume ratios, and aggregation-induced emission curve (right figure);

[0100] Figure 22 Normalized fluorescence emission spectra (left figure) of commercially available indocyanine green (ICG) in MeOH / THF mixed solutions with different volume ratios, and aggregation-induced emission curve (right figure);

[0101] Figure 23 Liquid phase diagram of the stability experiment of Eco-A1110 prepared in Example 1-1;

[0102] Figure 24 Liquid phase diagram of the stability experiment of indocyanine green (ICG);

[0103] Figure 25For the transplanted mouse breast cancer cells (4T1) subcutaneous tumors in mice, the fluorescence images of the excised tumor tissues were obtained by the IVIS Lumina II imaging system; the left figure is the fluorescence image of the tumor tissue under the IVIS Lumina II imaging system, and the right figure is the tumor tissue under normal white light;

[0104] Figure 26 For the transplanted mouse breast cancer cells (4T1) subcutaneous tumors in mice, the fluorescence images of the excised tumor tissues were obtained by the IVIS Lumina II imaging system; the left figure is the fluorescence image of the tumor tissue under the IVIS Lumina II imaging system, and the right figure is the tumor tissue under normal white light;

[0105] Figure 27 For the transplanted human tongue squamous carcinoma cells (HN6) in mice, the fluorescence images of the excised tumor tissues were obtained by the IVIS Lumina II imaging system; the left figure is the fluorescence image of the tumor tissue under the IVIS Lumina II imaging system, and the right figure is the tumor tissue under normal white light;

[0106] Figure 28 For the transplanted human tongue squamous carcinoma cells (HN6) in mice, the fluorescence images of the excised tumor tissues were obtained by the IVIS Lumina II imaging system; the left figure is the fluorescence image of the tumor tissue under the IVIS Lumina II imaging system, and the right figure is the tumor tissue under normal white light;

[0107] Figure 29 For the transplanted mouse breast cancer cells (4T1) subcutaneous tumors in mice, the fluorescence images of the excised tumor tissues were obtained by the IVIS Lumina II imaging system;

[0108] Figure 30 For the transplanted human tongue squamous carcinoma cells (HN6) in mice, the fluorescence images of the excised tumor tissues were obtained by the IVIS Lumina II imaging system;

[0109] Figure 31 For the HE staining map of the excised human oral squamous cell carcinoma tissues used in Experiment 7, the left figure is the tumor tissue under normal white light, and the right figure is the HE staining map of the tumor tissue after paraffin section;

[0110] Figure 32To obtain the fluorescence image of Eco-A1110 NPs-GE11-peptide in ex vivo human oral squamous cell carcinoma tissues using the IVIS Lumina II imaging system;

[0111] Figure 33 It is the HE staining image of the ex vivo human oral squamous cell carcinoma tissues used in Experiment 8. The left figure is the tumor tissue under normal white light, and the right figure is the HE staining image of the tumor tissue after paraffin sectioning.

[0112] Figure 34 To obtain the fluorescence image of Eco-A1110 NPs-EGFR-Ab in ex vivo human oral squamous cell carcinoma tissues using the IVIS Lumina II imaging system. Detailed implementation manners

[0113] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0114] The boiling point of the petroleum ether selected in the present invention is 60-90°C;

[0115] The PBS solution is a PBS buffer (KCl: 2.67 mM; NaCl: 137.07 mM; Na2HPO3: 10 mM; KH2PO4: 1.84 mM; pH is 7.4 ± 0.2).

[0116] Example 1-1: Preparation of Eco-A1110 fluorescent molecules

[0117]

[0118] Step 1: Synthesis of compound 1 (as precursor 2):

[0119] Put 4-(diphenylamino)phenylboronic acid pinacol ester (1851 mg, 5.0 mmol), 2-bromo-3-(hexyl)thiophene (823 mg, 3.3 mmol), and tetrakis(triphenylphosphine)palladium (269.6 mg, 0.23 mmol) into a reaction flask. Under a nitrogen atmosphere, add 100 mL of 1,4-dioxane to dissolve completely, and then add 25 mL of 2M potassium carbonate solution (V 二氧六环 :V 碱溶液 = 4:1), and heat to 100°C for reaction for 24 h. Extract the reaction solution (500 mL of water, 800 mL of dichloromethane), collect the organic phase, dry it with 8 g of anhydrous magnesium sulfate, filter it, remove the solvent (dichloromethane) under reduced pressure, and then perform column chromatography separation (using about 50 g of silica gel with 200-300 meshes), and elute with 300 mL of eluent. The ratio of the eluent is V PE :V DCM= 20:1 (PE is petroleum ether, DCM is dichloromethane), the eluate was collected, and after removing the solvent under reduced pressure, 1.2 g of compound 1 (colorless liquid) was obtained with a yield of 89%.

[0120] Step 2: Synthesis of compound 2 (as precursor 3):

[0121] Place compound 1 (730 mg, 1.77 mmol) in a reaction flask, dissolve it in 20 ml of tetrahydrofuran under a nitrogen atmosphere, stir it at -78 °C for 15 min, add n-butyllithium (2.4 mL, 2.12 mmol) and keep the reaction at -78 °C for 2 h, add tributyltin chloride (748.6 mg, 2.3 mmol), warm up to room temperature and react for 5 h, remove the solvent (tetrahydrofuran) under reduced pressure, and the obtained crude product of compound 2 (1.18 g) was obtained with a yield of 95%.

[0122] Step 3: Synthesis of compound Eco-A1110:

[0123] Place the crude product of compound 2 (1.24 g, 1.77 mmol), 4,7-dibromo-2,1,3-benzothiadiazole (130 mg, 0.44 mmol), and tetrakis(triphenylphosphine)palladium (35 mg, 0.031 mmol) in a reaction flask, add 33 mL of toluene under a nitrogen atmosphere, warm up to 110 °C and stir for 18 h, remove the solvent (toluene) under reduced pressure and then extract (400 mL of water, 600 mL of dichloromethane), collect the organic phase and dry and filter it with 8 g of anhydrous magnesium sulfate, remove the solvent (dichloromethane) under reduced pressure and then perform column chromatography separation (using about 50 g of silica gel with a mesh size of 200 - 300), and elute with 500 mL of eluent, and the ratio of the eluent is V PE :V DCM = 10:1 (PE is petroleum ether, DCM is dichloromethane), collect the eluate, and finally obtain 348 mg of red solid compound Eco-A1110 after removing the solvent under reduced pressure, with a yield of 83%.

[0124] Example 1-2: Preparation of Eco-A4410 fluorescent molecule

[0125] Compared with Example 1-1, the following changes were made: change 4-(diphenylamino)phenylboronic acid pinacol ester in Step 1 to 4,4-dimethyltriphenylamine-4-boronic acid pinacol ester, and keep the molar amount unchanged; change 2-bromo-3-(hexyl)thiophene to 2-bromo-3-(dodecyl)thiophene, and keep the molar amount unchanged;

[0126] The rest is the same as Example 1-1; compound Eco-A4410 was obtained.

[0127] Example 1-3: Preparation of Eco-A9510 fluorescent molecule

[0128] Compared with Example 1-1, the following changes are made: change the raw material 4-(diphenylamino)phenylboronic acid pinacol ester in Step 1 to N,N-bis(4-biphenylyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, and keep the molar amount unchanged; change 2-bromo-3-(hexyl)thiophene to 2-bromo-3-(butyl)thiophene, and keep the molar amount unchanged;

[0129] The rest is the same as Example 1-1; Compound Eco-A9510 is obtained.

[0130]

[0131] The 1H NMR spectrum of Eco-A1110 is as shown in Figure 2 the figure, the 1H NMR spectrum of Eco-A4410 is as shown in Figure 3 the figure, and the 1H NMR spectrum of Eco-A9510 is as shown in Figure 4 the figure; the high-resolution mass spectrum of Eco-A1110 is as shown in Figure 7 the figure; the high-resolution mass spectrum of Eco-A4410 is as shown in Figure 8 the figure; the high-resolution mass spectrum of Eco-A9510 is as shown in Figure 9 the figure.

[0132] Example 2-1: Preparation of Eco-A2110 Fluorescent Molecule

[0133]

[0134] Step 1: Synthesis of Compound 3 (as Precursor 1):

[0135] Put 1-(4-bromophenyl)-1,2,2-triphenylethylene (4110 mg, 10.0 mmol), bis(pinacolato)diboron (3046 mg, 12.0 mmol), palladium(II) dichloride bis(diphenylphosphinoferrocene) (593.2 mg, 0.46 mmol), and potassium acetate (2940 mg, 30.0 mmol) into a reaction flask. Add 50 mL of 1,4-dioxane under a nitrogen atmosphere, and heat the mixture to 100 °C and react for 24 h. Extract the reaction solution (500 mL of water, 800 mL of dichloromethane), remove the solvent (dichloromethane), and then perform column chromatography separation (using about 50 g of silica gel with a mesh size of 200-300). Elute with 500 mL of an eluent, and the ratio of the eluent is V PE : V DCM = 20:1 (PE is petroleum ether, DCM is dichloromethane). Collect the obtained eluate, rotary evaporate it under reduced pressure to obtain 4.17 g of a colorless liquid, named Compound 3, with a yield of 91%.

[0136] Step 2: Synthesis of Compound 4 (as Precursor 2):

[0137] Place Compound 3 (2292 mg, 5.0 mmol), 2-bromo-3-(hexyl)thiophene (823 mg, 3.3 mmol), and tetrakis(triphenylphosphine)palladium (269.6 mg, 0.23 mmol) in a reaction flask. Under a nitrogen atmosphere, add 100 mL of 1,4-dioxane and 25 mL of 2 M potassium carbonate solution (V 二氧六环 :V 碱溶液 = 4:1), and heat the mixture to 100 °C and react for 24 h. Extract the reaction solution (450 mL of water, 700 mL of dichloromethane), collect the organic phase, dry it over 8 g of anhydrous magnesium sulfate, filter it, remove the solvent (dichloromethane), and then perform column chromatography separation (using approximately 50 g of silica gel with a mesh size of 200 - 300). Elute with 300 mL of an eluent, and the ratio of the eluent is V PE :V DCM = 20:1 (PE is petroleum ether, DCM is dichloromethane). Collect the obtained eluate, rotary evaporate it under reduced pressure to obtain 1.43 g of a colorless liquid, named Compound 4, with a yield of 87%.

[0138] Step 3: Synthesis of Compound 5 (as Precursor 3):

[0139] Place Compound 4 (882.6 mg, 1.77 mmol) in a reaction flask. Under a nitrogen atmosphere, dissolve it in 20 ml of tetrahydrofuran, place it at -78 °C and stir for 15 min, add n-butyllithium (2.4 mL, 2.12 mmol) and react for 2 h, then add tributyltin chloride (748.6 mg, 2.3 mmol), and raise the temperature to room temperature and react for 5 h. After the reaction is completed, remove the solvent (tetrahydrofuran) under reduced pressure to obtain a crude product (1.3 g) named Compound 5, with a yield of 94%.

[0140] Step 4: Synthesis of Compound Eco-A2110:

[0141] Place the crude product of Compound 5 (1.4 g, 1.77 mmol), 4,7-dibromo-2,1,3-benzothiadiazole (130 mg, 0.44 mmol), and tetrakis(triphenylphosphine)palladium (35 mg, 0.031 mmol) in a reaction flask. Under a nitrogen atmosphere, add 33 mL of toluene, heat the mixture to 110 °C and stir and react for 18 h. After removing toluene under reduced pressure, extract (400 mL of water, 600 mL of dichloromethane), collect the organic phase, dry it over 8 g of anhydrous magnesium sulfate, filter it, remove the solvent (dichloromethane) under reduced pressure, and then perform column chromatography separation (using approximately 50 g of silica gel with a mesh size of 200 - 300). Elute with 500 mL of an eluent, and the ratio of the eluent is V PE :V DCM= 10:1 (PE is petroleum ether, DCM is dichloromethane). The obtained eluate was collected, and after rotary evaporation under reduced pressure, 437 mg of red solid Eco-A2110 was obtained with a yield of 88%.

[0142] Example 2-2. Preparation of Eco-A6410 Fluorescent Molecule

[0143] Compared with Example 2-1, the following changes were made: In step one, the raw material 1-(4-bromophenyl)-1,2,2-triphenylethylene was changed to (E)-(1-(4-bromophenyl)-2-(4-methoxyphenyl)ethene-1,2-diyl)dibenzene, and in step two, 2-bromo-3-(hexyl)thiophene was changed to 2-bromo-3-(dodecyl)thiophene, and the rest was the same as Example 2-1; then the compound Eco-A6410 could be obtained.

[0144]

[0145] The 1H NMR spectrum of Eco-A2110 is as Figure 5 shown, and the 1H NMR spectrum of Eco-A6410 is as Figure 6 shown. The high-resolution mass spectrum of Eco-A2110 is as Figure 10 shown, and the high-resolution mass spectrum of Eco-A6410 is as Figure 11 shown.

[0146] The Eco-A1110, Eco-A4410, Eco-A9510, Eco-A2110, and Eco-A6410 obtained in the above examples were used as test substances for experiments.

[0147] Experiment 1: Optical Property Test

[0148] I. UV determination: The instrument used was a Shimadzu UV-1900i ultraviolet spectrometer. The concentration of the test substance was 50 μM, and the absorption wavelength scanning range was 900 nm to 500 nm; the recording range was 0 Abs to 2.5 Abs; the scanning speed was high; the data interval was 0.2 nm; the recording method was covering. The determination results are as Figures 12 - 16 shown.

[0149] II. Fluorescence determination: The instrument used was a Shimadzu RF-6000 fluorescence spectrometer. The concentration of the test substance was 10 μM, the excitation wavelength was set to the maximum absorption wavelength of the test substance, and the emission spectrum wavelength range was 500 nm to 900 nm; the slit width (nm): Ex = 5, Em = 5; the sensitivity was Low; the data interval was 1.0 nm; the scanning speed was 2000 nm / min; the response time was automatic. The determination results are as Figures 12 - 16 shown.

[0150] III. Study on the ACQ effect of the test articles: In the present invention, the degree of torsion of the molecular structure is increased by connecting alkylthiophene in the middle to prevent its stacking, effectively alleviating the aggregation-caused quenching (ACQ) effect.

[0151] 1. Methods and conditions

[0152] Since the molecules to be tested are all soluble in THF (tetrahydrofuran) but insoluble in H2O (water), when the proportion of H2O in the THF / H2O mixed solution of the molecules to be tested continuously increases, the molecules to be tested in the solvent system will gradually aggregate to form aggregates. Prepare THF / H2O mixed solutions with different ratios (the proportion of water is 0 - 90%) of each molecule to be tested (keeping the concentration of the molecules to be tested at 10 μM), and use a Shimadzu RF-6000 fluorescence spectrometer to measure the maximum fluorescence emission intensity of the molecules in the THF / water mixed solution at different volume ratios with a specific excitation wavelength.

[0153] ICG (indocyanine green), as a control group, is soluble in MeOH (methanol) but insoluble in THF (tetrahydrofuran). When the proportion of THF in the MeOH / THF mixed solution of ICG continuously increases, the molecules to be tested in the solvent system will gradually aggregate to form aggregates. Prepare MeOH / THF mixed solutions with different ratios (the proportion of THF is 0 - 90%) of ICG (keeping the concentration of the molecules to be tested at 10 μM), and use a Shimadzu RF-6000 fluorescence spectrometer to measure the maximum fluorescence emission intensity of the molecules in the MeOH / THF mixed solution at different volume ratios with a specific excitation wavelength.

[0154] 2. Results and analysis

[0155] As Figures 17 - 21 shown, when measuring Eco-A1110, Eco-A4410, Eco-A9510, Eco-A2110, and Eco-A6410 in THF / H2O mixed solutions with different volume ratios, as the volume continuously increases, the fluorescence intensities of the Eco-A1110, Eco-A4410, Eco-A9510, Eco-A2110, and Eco-A6410 fluorescent probes show a trend of first decreasing and then increasing. When measuring ICG in MeOH / THF mixed solutions with different volume ratios, as the volume continuously increases, the fluorescence intensity of ICG gradually decreases until quenching, indicating that the Eco-A1110, Eco-A4410, Eco-A9510, Eco-A2110, and Eco-A6410 fluorescent probes alleviate the ACQ effect to a certain extent.

[0156] Experiment II. Molecular stability test

[0157] 1. Materials and instruments

[0158] The test molecules are five fluorescent probe molecules, namely Eco-A1110, Eco-A4410, Eco-A9510, Eco-A2110, and Eco-A6410, and the control sample is indocyanine green (ICG). Characterization was performed using LCMS-2020 (Shimadzu system).

[0159] 2. Methods and Conditions

[0160] Taking Eco-A1110 as an example, 10 mg (0.01 mmol) of Eco-A1110 was added to the reaction flask in the experimental group. After dissolving it with 5 mL of N,N-dimethylformamide, 0.56 mL (12.8 mmol) of triethylamine was added, and the mixture was stirred at 60 °C without light shielding for 12 h. In the control group reaction flask, 7.75 mg (0.01 mmol) of ICG was added, dissolved with 5 mL of N,N-dimethylformamide, 0.56 mL (12.8 mmol) of triethylamine was added, and the mixture was stirred at 60 °C without light shielding for 12 h. Subsequently, approximately 1 mL of the reaction solutions from the experimental group and the control group were taken respectively, and characterization was performed using LCMS-2020 (Shimadzu system).

[0161] 3. Results and Analysis

[0162] As Figure 23 and Figure 24 are the liquid chromatography spectra of the stability test of Eco-A1110 and the liquid chromatography spectrum of the control ICG respectively. As Figure 23 can be seen, under the set alkaline, heating, and light conditions, Eco-A1110 is basically not significantly damaged and no impurity peaks appear; as Figure 24 for ICG, there is more damage under the same conditions and obvious impurity peaks appear.

[0163] The test results of the other four light molecules (Eco-A4410, Eco-A9510, Eco-A2110, Eco-A6410) are similar to those of Eco-A1110, all proving that the molecules shown in the present invention have good stability (alkaline, heating, light).

[0164] In summary, Eco-A1110, which has a relatively large emission wavelength and still has strong fluorescence intensity, has certain advantages as a fluorescent molecular probe compared to the other four molecules.

[0165] Experiment 3: Preparation of Nanoparticles (NPs) of Each Molecule

[0166] Taking Eco-A1110 as an example: 1 mg of Eco-A1110 as a lipophilic photosensitizer and 5 mg of DSPE-PEG2000-Mal (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000-maleimide) as an amphiphilic polymer were dissolved in 1.0 mL of tetrahydrofuran until clear. The mixture was quickly added to 9 mL of ultrapure water under ultrasonic conditions, and then ultrasonic treatment was continued for 10 min to promote uniform dispersion of the system. Thereafter, nitrogen gas was bubbled into the sample to remove tetrahydrofuran, and finally, Eco-A1110 NPs were obtained and stored refrigerated for later use.

[0167] Similarly, Eco-A4410 NPs, Eco-A9510 NPs, Eco-A2110-NPs, and Eco-A6410 NPs were obtained accordingly.

[0168] Experiment 4: Modification of nanoparticles (NPs) with antibodies / polypeptides

[0169] Eco-A1110 NPs were dissolved in tetrahydrofuran to prepare a 2 mg / mL Eco-A1110 NPs solution. 500 μL of the Eco-A1110 NPs solution was mixed with 1 mL of a PBS solution of EphA2 antibody or polypeptide-CFFGYSAYPDSVPMMS (antibody concentration: 10 μg / mL PBS, polypeptide concentration: 1 mg / mL PBS) in the dark overnight. Dialysis (the molecular weight cut-off of the dialysis bag used was 8000 - 14000) was performed to remove the excess antibody / polypeptide, and the products Eco-A1110 NPs-EphA2-Ab and Eco-A1110 NPs-YSA-peptide were obtained accordingly.

[0170] Similar to the above steps, by replacing the antibody with Anti-EGFR antibody [EGFR1] or replacing the polypeptide sequence with CFFGYHWYGYTPENVI, the products Eco-A1110 NPs-EGFR-Ab and Eco-A1110 NPs-GE11-peptide could be obtained.

[0171] Experiment 1: Fluorescence imaging of the excised tumor tissues of mice with transplanted subcutaneous tumors of mouse breast cancer cells (4T1) using the Eco-A1110 NPs-EphA2-Ab fluorescent nanoprobe

[0172] 1. Materials and instruments

[0173] Mouse breast cancer cells (4T1) were purchased from the American Type Culture Collection (ATCC). The cells were grown in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humid environment at 37 °C with 5% CO2, and mycoplasma contamination was regularly checked.

[0174] 2. Preoperative preparation:

[0175] To establish a 4T1 tumor-bearing mouse model, healthy female Balb / C mice (18 - 22 g) were used in the experiment. After depilation of the right axilla of the mice, 50 mL of 4T1 cell suspension (1X10 7 cells) was injected. When the tumor volume reached approximately 93 - 119 mm 2 after 15 days, intraoperative biopsy experiments were started.

[0176] 3. Intraoperative procedures:

[0177] S1: The tumor tissue excised after surgery was first washed with PBS solution to remove excess blood, grease, etc. Subsequently, the immersion surface was determined by the naked eye, and the immersion surface mainly contained tumor tissue. The tissue was completely immersed in the blocking solution (PBS solution containing 5% FBS), and the original height of the solution in the immersion dish was ≥5 mm, and blocked at 4°C for 3 min.

[0178] S2: The tissue sample was taken out from the blocking solution and completely immersed in the fluorescent nanoprobe solution, and the original height of the solution in the immersion dish was ≥5 mm. The fluorescent nanoprobe solution was a PBS solution containing 50 μM Eco-A1110 NPs-EphA2-Ab, and incubated at room temperature for 15 min. Subsequently, the tissue sample was washed 3 times with PBS, 1 min each time.

[0179] S3: After washing, fluorescence images were obtained through the IVIS Lumina II imaging system to determine whether there was residual tumor tissue at the tissue margin.

[0180] 4. Experimental results

[0181] As Figure 25 shown, the fluorescence of tumor tissue in ex vivo tissues was obvious, and the boundary with the surrounding normal tissues was clear, and the tumor margin could be clearly identified, indicating that the ex vivo tissues were margin positive.

[0182] Experiment 2: Fluorescence imaging of ex vivo tumor tissues of mice transplanted with subcutaneous tumors of breast cancer cells (4T1) using Eco-A1110 NPs-YSA-peptide fluorescent nanoprobes.

[0183] The following changes were made compared with Experiment 1: In step S2 of Experiment 1, the "fluorescent nanoprobe solution" was changed from "PBS solution containing 50 μM Eco-A1110 NPs-EphA2-Ab" to "PBS solution containing 50 μM Eco-A1110 NPs-YSA-peptide"; the rest was the same as Experiment 1.

[0184] The experimental results were:

[0185] AsFigure 26 As shown, the fluorescence of the tumor tissue in the ex vivo tissue was obvious, and the boundary with the surrounding normal tissue was clear. The tumor margin could be clearly identified, indicating that the ex vivo tissue was positive for the resection margin.

[0186] Experiment 3: Fluorescence imaging of the ex vivo tumor tissue of mice transplanted with subcutaneous tumors of human tongue squamous carcinoma cells (HN6) using the Eco-A1110 NPs-EGFR-Ab fluorescent nanoprobe.

[0187] The following changes were made compared to Experiment 1: In the "Preoperative Preparation" of Experiment 1, "injecting 50 mL of 4T1 cell suspension (1X10 7 cells)" was changed to "injecting 50 mL of human tongue squamous carcinoma cell (HN6) suspension (1X10 7 cells)" to establish an HN6 tumor-bearing mouse model; the human tongue squamous carcinoma cells (HN6) were purchased from the American Type Culture Collection (ATCC); and "PBS solution containing 50 μM Eco-A1110

[0188] NPs-EphA2-Ab" was changed to "PBS solution containing 50 μM Eco-A1110 NPs-EGFR-Ab"; the rest was the same as Experiment 1.

[0189] The experimental results were as follows:

[0190] As Figure 27 shown, the fluorescence of the tumor tissue in the ex vivo tissue was obvious, and the boundary with the surrounding normal tissue was clear. The tumor margin could be clearly identified, indicating that the ex vivo tissue was positive for the resection margin.

[0191] Experiment 4: Fluorescence imaging of the ex vivo tumor tissue of mice transplanted with subcutaneous tumors of human tongue squamous carcinoma cells (HN6) using the Eco-A1110 NPs-GE11-peptide.

[0192] The following changes were made compared to Experiment 3: The "fluorescent nanoprobe solution" in Experiment 3 was changed from "PBS solution containing 50 μM Eco-A1110 NPs-EGFR-Ab" to "PBS solution containing 50 μM Eco-A1110 NPs-GE11-peptide"; the rest was the same as Experiment 3.

[0193] The experimental results were as follows:

[0194] As Figure 28 shown, the fluorescence of the tumor tissue in the ex vivo tissue was obvious, and the boundary with the surrounding normal tissue was clear. The tumor margin could be clearly identified, indicating that the ex vivo tissue was positive for the resection margin.

[0195] Since the other four fluorescent molecules, Eco-A4410, Eco-A9510, Eco-A2110, and Eco-A6410, are not as excellent as this molecule in terms of emission wavelength and fluorescence intensity, although their nanoparticles modified with polypeptides / antibodies still have certain effects in fluorescence section experiments, they are all inferior to the fluorescent nanoprobe based on the Eco-A1110 fluorescent molecule, and will not be elaborated here.

[0196] In order to study the advantages and disadvantages of nanomaterials and non-nanomaterials in fluorescence section technology, the present invention synthesized the fluorescent molecule Eco-Ab110.

[0197] The synthesis method of the fluorescent molecule Eco-Ab110 refers to Patent CN116425739A. That is, 4,9-dibromonaphthothiadiazole used in CN116425739A was changed to 4,7-dibromo-2,1,3-benzothiadiazole, and the remaining steps were the same as those in Example 5 thereof, thereby obtaining the fluorescent molecule Eco-Ab110. The structural formula of Eco-Ab110 is:

[0198]

[0199] React Eco-Ab110, N-(2-aminoethyl) maleimide hydrochloride, HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), and TEA (triethylamine) in a nitrogen atmosphere for 5 h (the molar ratio of Eco-Ab110:N-(2-aminoethyl) maleimide hydrochloride:HBTU:TEA is 1:2:2:4, and 5 mL of DMF (N,N-dimethylformamide) is added per 100 mg of Eco-Ab110). Subsequently, directly add the polypeptide-CFFGYSAYPDSVPMMS to the reaction solution and react overnight. Then, the reaction solution is purified by high-performance liquid chromatography (HPLC) to obtain the product Eco-Ab110-YSA-peptide;

[0200] The purification by high-performance liquid chromatography (HPLC) is specifically as follows: Use methanol (containing 0.05% trifluoroacetic acid) and water (containing 0.05% trifluoroacetic acid) as the eluent, and the elution gradient is successively 70% water + 30% methanol for 10 min; 40% water + 60% methanol for 5 min; 100% methanol for 4 min; 70% water + 30% methanol for 4 min. The flow rate is 8 mL / min. Collect the eluent with a retention time of 16.7 min, put it into the refrigerator to freeze and ice, and then put it into a freeze dryer for vacuum sublimation (-55 °C, 92 Pa pressure) to obtain the Eco-Ab110-YSA-peptide product.

[0201] The structural formula of Eco-Ab110-YSA-peptide is as follows:

[0202]

[0203] Following the above experimental steps, change the polypeptide - CFFGYSAYPDSVPMMS to - CFFGYHWYGYTPENVI, and the product Eco - Ab110 - GE11 - peptide can be obtained.

[0204]

[0205] Experiment 5: Fluorescence imaging of the excised tumor tissues of mice with subcutaneous tumors of transplanted breast cancer cells (4T1) using non - nanomaterial Eco - Ab110 - YSA - peptide.

[0206] Make the following changes compared to Experiment 1: change the "fluorescent nanoprobe solution" in step S2 of Experiment 1 from "PBS solution containing 50 μM Eco - A1110NPs - EphA2 - Ab" to "PBS solution containing 50 μM Eco - Ab110 - YSA - peptide"; the rest is the same as Experiment 1.

[0207] The experimental results are as follows:

[0208] As Figure 29 shown, both tumor tissues and normal tissues in the excised tissues have certain fluorescence responses. This is because when rinsing the residual fluorescent probes, the excess fluorescent probes were not removed, which increased the false - positive probability of fluorescence sections and affected the doctor's judgment; this result indicates that preparing fluorescent molecules into nanoparticles can facilitate the removal of excess fluorescent probes remaining in the tissues and reduce the false - positive probability of fluorescence sections.

[0209] Experiment 6: Fluorescence imaging of the excised tumor tissues of mice with subcutaneous tumors of transplanted human tongue squamous carcinoma cells (HN6) using non - nanomaterial Eco - Ab110 - GE11 - peptide.

[0210] Make the following changes compared to Experiment 5: change the Eco - Ab110 - YSA - peptide probe used in Experiment 5 to an Eco - Ab110 - GE11 - peptid probe, and change "inject 50 mL of 4T1 cell suspension (1X10 7 cells)" in the "pre - operative preparation" to "inject 50 mL of human tongue squamous carcinoma cell (HN6) suspension (1X10 7 cells)" to establish a subcutaneous tumor model bearing HN6; the rest is the same as Experiment 1.

[0211] The experimental results:

[0212] As Figure 30As shown, both tumor tissues and normal tissues in ex vivo tissues have a certain fluorescence response. This is due to the failure to remove the excess fluorescent probes during the washing of the residual fluorescent probes, which increases the false positive probability of fluorescence sections and affects the doctor's judgment. This result indicates that preparing fluorescent molecules into nanoparticles can facilitate the removal of the excess fluorescent probes remaining in the tissues and reduce the false positive probability of fluorescence sections.

[0213] To further study the application of fluorescence section technology in actual tumor resection, the present invention selected freshly resected human oral squamous cell carcinoma tissues with high expression of EGFR as tumor sections for fluorescence section technology experiments.

[0214] Experiment 7: Fluorescence imaging of tumor tissues in human oral squamous cell carcinoma with Eco-A1110 NPs-GE11-peptide.

[0215] 1. Materials and instruments:

[0216] Human oral squamous cell carcinoma tumor tissues were obtained from Tianjin Stomatological Hospital.

[0217] 2. Imaging operation:

[0218] S1: Wash the human oral squamous cell carcinoma tumor tissues with PBS solution to remove excess blood, grease, etc. Subsequently, determine the immersion surface by naked eye, and the immersion surface mainly contains tumor tissues. Immerse the tissues completely in the blocking solution (PBS solution containing 5% FBS), and the original height of the solution in the immersion dish is ≥5 mm. Block at 4°C for 3 min.

[0219] S2: Take out the tissue samples from the blocking solution and immerse the tissues completely in the fluorescent nanoprobe solution, and the original height of the solution in the immersion dish is ≥5 mm. The fluorescent nanoprobe solution is a PBS solution containing 50 μM Eco-A1110 NPs-GE11-peptide. Incubate at room temperature for 15 min. Subsequently, wash the tissue samples 3 times with PBS, 1 min each time.

[0220] S3: After washing, obtain fluorescence images through the IVIS Lumina II imaging system to judge whether there is residual tumor tissue at the tissue margin.

[0221] 3. Experimental results:

[0222] As Figure 31 shown, the human oral squamous cell carcinoma tumor tissues were paraffin-sectioned and stained with HE to prove the presence of residual tumor tissues; as Figure 32 shown, the fluorescence at the tumor sites in the ex vivo tissues after the immersion procedure was obvious, and the boundary with the surrounding normal tissues was clear. The tumor margin could be clearly identified without false positives.

[0223] Experiment 8: Tumor tissue fluorescence imaging of Eco-A1110 NPs-EGFR-Ab in human oral squamous cell carcinoma.

[0224] Make the following changes compared to Experiment 7: Change the "fluorescent nanoprobe solution" in Step S2 from "PBS solution containing 50 μM Eco-A1110 NPs-GE11-peptide" to "PBS solution containing 50 μM Eco-A1110 NPs-EGFR-Ab", and the rest is the same as Experiment 7.

[0225] Experimental results:

[0226] As Figure 33 shown, after paraffin sectioning of human oral squamous cell carcinoma tumor tissue, HE staining was used to prove the presence of residual tumor tissue; as Figure 34 shown, the fluorescence at the tumor site in the ex vivo tissue after the soaking procedure was obvious, and the boundary with the surrounding normal tissue was clear. The tumor margin could be clearly identified, and there was no false positive.

[0227] It can be seen from the above two experiments that there is no obvious difference between Eco-A1110 NPs-GE11-peptide and Eco-A1110 NPs-EGFR-Ab in terms of experimental results. However, since Eco-A1110 NPs-EGFR-Ab is a product of modifying Eco-A1110 NPs with EGFR antibody, its cost is relatively higher than that of Eco-A1110 NPs-GE11-peptide.

[0228] Comparison: Among the currently existing fluorescent molecules, the existing fluorescent molecule closest to the molecular structural formula of the present invention is the fluorescent molecule in Patent CN116425739B

[0229] "Compound for tumor-targeted imaging and photodynamic therapy, its synthesis method and application", and its structural formula is;

[0230]

[0231] According to the "optical property test" and "molecular stability test", the obtained results are as follows:

[0232] The absorption wavelength of this molecule is 546 nm, the emission wavelength is 815 nm, the fluorescence intensity is 4.6 w, and the molecule is stable under alkaline, heating, and light conditions.

[0233] According to the above experiment, the results of the experiment on breast cancer (4T1) mouse subcutaneous tumor, human tongue squamous cell carcinoma (NH6) mouse subcutaneous tumor, and human oral squamous cell carcinoma are: the molecule has a large absorption wavelength and emission wavelength, and a relatively low fluorescence intensity (the absorption wavelength of Eco-A1110 in the present invention is 511nm, the emission wavelength is 680nm, and the fluorescence intensity is 74w. The other molecules of the present invention are similar to this molecule, see for details Figures 12 - 16 ), which can easily lead to false negative results when performing fluorescent sectioning, making it difficult for doctors to judge whether there is any tumor residue in the tumor resection tissue.

[0234] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A fluorescent molecule with aggregation-induced emission properties for tumor imaging, characterized in that The general structural formula of the fluorescent molecule is as follows: In the general structural formula, R1 is a straight chain with 0 to 20 carbon atoms, and R is any one of the following: triphenylamine, 4,4'-dimethyltriphenylamine, 4,4'-diphenyltriphenylamine, tetraphenylethylene, 4-methyltetraphenylethylene, 4-methoxytetraphenylethylene group.

2. The fluorescent molecule according to claim 1, characterized in that is any one of the following:

3. The preparation method of the fluorescent molecule according to claim 1 or 2, characterized in that Comprises the following steps: 1), Prepare precursor 2: Precursor 1 and 2-bromo-3-(R1)thiophene are subjected to a suzuki coupling reaction under the catalysis of tetrakis(triphenylphosphine)palladium and the action of a basic environment formed by potassium carbonate and a solvent. The suzuki coupling reaction is carried out at 100±10°C for 12 to 24 h; after purification of the reaction product, precursor 2 is obtained; The molar ratio of precursor 1:2-bromo-3-(R1)thiophene:tetrakis(triphenylphosphine)palladium:potassium carbonate = 1 to 2:1:0.03 to 0.1:10 to 20; The precursor 1 is The precursor 2 is 2), Prepare precursor 3: Precursor 2 and n-butyllithium are reacted in a solvent at -78±10°C for 1 to 2 h, and then tributyltin chloride is added and the reaction is transferred to room temperature for 4 to 6 h; the resulting crude product named precursor 3; The molar ratio of precursor 2:n-butyllithium:tributyltin chloride = 1:1.1 to 1.3:1.2 to 1.5; The precursor 3 is 3), The crude product of precursor 3 and 4,7-dibromo-2,1,3-benzothiadiazole are subjected to a Stille coupling reaction in a solvent under the catalysis of tetrakis(triphenylphosphine)palladium. The Stille coupling reaction is carried out at 100±10°C for 12 to 24 h; The molar ratio of precursor 3:4,7-dibromo-2,1,3-benzothiadiazole:tetrakis(triphenylphosphine)palladium = 3 to 5:1:0.03 to 0.1; After purification of the reaction product, a fluorescent molecule for imaging is obtained; the fluorescent molecule is Among precursor 1, precursor 2, precursor 3, and the fluorescent molecule: R1 is a straight chain with 0 to 20 carbon atoms, and R is any one of the following: triphenylamine, 4,4'-dimethyltriphenylamine, 4,4'-diphenyltriphenylamine, tetraphenylethylene, 4-methyltetraphenylethylene, 4-methoxytetraphenylethylene group.

4. The method for preparing a fluorescent molecule according to claim 3, wherein: The preparation of precursor 1 is as follows: A tetraphenylethylene compound and bis(pinacolato)diboron are subjected to a suzuki coupling reaction under the catalysis of palladium(II) dichloride DPPF and the action of a basic environment formed by potassium acetate and a solvent. The suzuki coupling reaction is carried out at 100±10°C for 12 to 24 h; The molar ratio of the tetraphenylethylene compound:bis(pinacolato)diboron:palladium(II) dichloride DPPF:potassium acetate is 1:1 to 2:0.03 to 0.1:10 to 20; When the tetraphenylethylene compound is 1-(4-bromophenyl)-1,2,2-triphenylethylene, the resulting precursor 1 is 4,4,5,5-tetramethyl-2-(4-(1,2,2-triphenylethylenyl)phenyl)-1,3,2-dioxaborolane; When the tetraphenylethylene compound is [1,2-diphenyl-1-(4-bromophenyl)-2-(4-methoxyphenyl)]ethylene, the resulting precursor 1 is (E)-2-(4-(2-(4-methoxyphenyl)-1,2-diphenylethynyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane.

5. A method for preparing nanoparticles NPs using the fluorescent molecule according to claim 1 or 2, characterized in that Comprising the following steps: Dissolve the fluorescent molecule and DSPE-PEG2000-Mal in tetrahydrofuran to form a mixed solution, where the mass ratio of the fluorescent molecule to DSPE-PEG2000-Mal is 1:

5. According to the volume ratio of tetrahydrofuran to ultrapure water of 1:9, add the mixed solution to ultrapure water under ultrasonic conditions, and then continue to ultrasonicate for 10 ± 4 min to promote uniform dispersion of the system. Then, purge with nitrogen to remove tetrahydrofuran, obtaining nanoparticles NPs.

6. Method for obtaining a fluorescent probe by modifying an antibody / polypeptide with nanoparticles NPs, characterized in that Comprising the following steps: Dissolve the nanoparticles NPs in tetrahydrofuran to prepare an NPs solution with a concentration of 2 mg / mL. Dissolve the antibody / polypeptide in PBS buffer to prepare an antibody solution with a concentration of 10 μg / mL or a polypeptide solution with a concentration of 1 mg / mL. According to the volume ratio of tetrahydrofuran to PBS buffer of 1:2, mix the NPs solution with the antibody solution / polypeptide solution and react overnight at room temperature in the dark. Then, dialyze to remove the excess antibody / polypeptide to obtain the fluorescent nanoprobe NPs-AB / NPs-peptide.

7. The method for modifying an antibody / polypeptide with nanoparticles to obtain a fluorescent probe according to claim 6, characterized in that: The antibody is the EphA2 antibody, obtaining the fluorescent probe NPs-EphA2-AB. The antibody is the Anti-EGFR antibody [EGFR1], obtaining the fluorescent probe NPs-EGFR-AB. The polypeptide is CFFGYSAYPDSVPMMS, obtaining the fluorescent probe NPs-YSA-peptide. The polypeptide is CFFGYHWYGYTPENVI, obtaining the fluorescent probe NPs-GE11-peptide. The structural formula of the polypeptide CFFGYSAYPDSVPMMS is: The structural formula of the polypeptide CFFGYHWYGYTPENVI is: Among them, the antibody Anti-Eph receptor A2 antibody [EPR17660-120] and the -CFFGYSAYPDSVPMMS polypeptide target is EphA2; the Anti-EGFR antibody [EGFR1] and the -CFFGYHWYGYTPENVI target is EGFR.

8. Method for precise biopsy of ex vivo tissues during tumor resection, characterized in that Comprising the following steps: 1), According to the characteristics of tumors / cancers, select a fluorescent probe with good corresponding targeting. 2), Cut a part from the edge of the ex vivo tumor tissue, and then place the excised tissue in Krebs-Hensleit's Solution containing 5% fetal bovine serum for blocking. The blocking temperature is 4 - 8 °C, and the soaking time is 2 - 5 min. 3), Transfer the resected tissue after step 2) to phosphate buffered saline containing 30 - 100 uM fluorescent probe, and incubate for 10 - 20 min; After incubation, rinse with phosphate saline containing PEG - 400 for 1 - 3 times, with each rinse for 1 - 2 min; 4), Image the resected tissue obtained from step 3) with a fluorescence imaging system to observe the fluorescence intensity; thereby determining the tumor boundary.

9. The method for precise biopsy of ex vivo tissue during tumor resection according to claim 8, wherein: When it is breast cancer or colon cancer, select fluorescent probe NPs - EphA2 - Ab and fluorescent probe NPs - YSA - peptide, When it is oral squamous cell carcinoma, select fluorescent probe NPs - GE11 - peptide and fluorescent probe NPs - EGFR - Ab.

10. The method for precise biopsy of ex vivo tissue during tumor resection according to claim 8 or 9, wherein: The soaking time in step 2) is 3 min; In step 3): the concentration of the fluorescent nanoprobe is 50 uM, the incubation time is 10 - 15 min, the number of rinses is 2 - 3 times, and the time for each rinse is 1 min.

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