High-specificity fluorescent tracer for metastatic lymph nodes

Abstract

The invention discloses a metastatic lymph node high-specificity fluorescent tracer and a preparation method and application thereof, and belongs to the field of biotechnology and medicine. The tracer takes ZIF-8 nanoparticles as a carrier, encapsulates indocyanine green (ICG), is modified by polyethylene glycol (PEG), and then is inserted into a vascular endothelial growth factor C (VEGFC) antibody to form an ICG (ZIF) PEG-VEGFC (ZIPV) compound. The particle size of the tracer is controllable (30-150 nm), the tracer emits light in a near-infrared I region (the emission wavelength is 825 nm), metastatic lymph nodes highly expressed by VEGFC can be specifically targeted, and the imaging sensitivity and specificity are remarkably improved. In-vivo and in-vitro experiments show that the ZIPV has good biocompatibility (the hemolysis rate is less than 5%), can accurately distinguish metastatic and non-metastatic lymph nodes, and has no liver and kidney toxicity. The preparation process is simple, the cost is low, and a novel tool is provided for diagnosis of gastric cancer lymph node metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and medicine, specifically relating to the preparation and application of a highly specific fluorescent tracer for metastatic lymph nodes. Background Technology

[0002] In tumor metastasis research, the pre-metastatic niche (PMN) has become one of the core theoretical frameworks for understanding cancer progression. Lymph nodes are one of the most common early metastatic sites for solid tumors. Studies have shown that certain tumors secrete specific factors that alter the lymph node microenvironment, thereby providing favorable conditions for the formation of the pre-metastatic niche. Vascular endothelial growth factor C (VEGFC) plays a crucial role in this process as a secreted protein. VEGFC is mainly secreted by tumor cells, fibroblasts, and immune cells. It promotes lymphangiogenesis and dilation by binding to the receptor VEGFR-3. Research has found that VEGFC accelerates tumor cell entry into the lymphatic system and increases the risk of distant metastasis by enhancing lymphatic drainage, altering the lymph node microenvironment, and recruiting immunomodulatory cells. Furthermore, VEGFC can indirectly promote angiogenesis, thereby accelerating tumor growth and spread.

[0003] In the progression of tumors, lymph node imaging technology plays a vital clinical role in the early detection of metastases, assessment of disease development, and guidance of treatment plans. Common lymph node imaging techniques include magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and contrast-enhanced ultrasound (CEUS). Furthermore, with the development of novel molecular imaging techniques such as near-infrared fluorescence imaging (NIRF) and super-resolution microscopy, the accurate detection of tumor-associated lymph nodes has become more sensitive and specific. However, commonly used tracers such as indocyanine green (ICG) cannot distinguish between metastatic and non-metastatic lymph nodes. Indocyanine green, a water-soluble fluorescent dye, is widely used in medical imaging and blood flow monitoring. Despite its significant clinical advantages, its short half-life, precise injection timing, and low specificity, coupled with susceptibility to non-specific uptake, may interfere with diagnostic results. Therefore, its limitations must be carefully weighed when using indocyanine green to optimize its application.

[0004] Research on pre-metastatic niches has provided a new perspective for understanding the mechanisms of tumor metastasis, while the development of lymph node imaging technology has provided an important tool for early diagnosis and precision treatment. Future research will further explore the formation mechanisms of pre-metastatic niches and optimize lymph node imaging techniques to better distinguish between metastatic and non-metastatic lymph nodes. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for preparing a fluorescent tracer for metastatic lymph nodes with good biocompatibility and targeting properties, and to apply it to the imaging of metastatic lymph nodes.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] This invention discloses a highly specific fluorescent tracer for metastatic lymph nodes, characterized in that it comprises the following components: a. ZIF-8 nanoparticles; b. Indocyanine green (ICG) encapsulated within ZIF-8 nanoparticles; c. Surface-modified polyethylene glycol (PEG); d. VEGFC antibodies inserted into PEG; Furthermore, the tracer has a particle size of 30-150 nm and a near-infrared fluorescence emission wavelength of 800-840 nm.

[0008] This invention also discloses a method for preparing the above-described fluorescent tracer, characterized in that the method includes the following steps: Step 1, Preparation of ZI: Dissolve 2-MIM in methanol, then add a mixed solution of ICG and Zn(NO3)2•6H2O, protect from light, stir at 750 r / min for 40 min, centrifuge at 7000 r / min for 10 min, discard the supernatant; vacuum dry at 120℃ to obtain powdered ZI; Step 2, ZIP preparation: (1) Measure 25 mg of ZI obtained in step one and dissolve it in 8 mL of methanol. Shake it on a vortex shaker to completely dissolve it and obtain a ZI solution. (2) Dissolve DSPE-PEG-COOH powder in methanol, and slowly and uniformly add it to the ZI solution obtained in (1). Protect from light, stir at 660 r / min for 12 h, and then centrifuge at 3000 r / min for 10 min to obtain a series of ZIP nanoparticles. Step 3, Preparation of ZIPV: Take the ZIP precipitate obtained in Step 2 after centrifugation, dissolve it in water, then add 6 μL of VEGFC antibody stock solution with a concentration of 1 mg / mL, and place it in an ultrasonic cleaner for 30 min of sonication to obtain ZIPV nanotracer.

[0009] Furthermore, in the synthesis system described in step one, the final concentration of 2-MIM is 32.5 mg / mL, the final concentration of ICG is 0.15 mg / mL, and the final concentration of Zn(NO3)2•6H2O is 5~15 mg / mL.

[0010] Furthermore, in step two, the final concentration of ZI in the synthesis system is 2.5 mg / mL, and the final concentration of DSPE-PEG-COOH is 0.5 mg / mL.

[0011] This invention also discloses the application of the above-described fluorescent tracer in the preparation of diagnostic reagents for metastatic lymph nodes, characterized in that the application includes: a. Delivery of tracers to the target area via subcutaneous injection; b. Use near-infrared fluorescence imaging to differentiate between metastatic and non-metastatic lymph nodes.

[0012] This invention also discloses the application of VEGFC antibody in the preparation of a highly specific fluorescent tracer for metastatic lymph nodes.

[0013] Furthermore, the tracer includes a ZIF-ICG-VEGFC antibody.

[0014] Furthermore, the VEGFC antibody specifically recognizes metastatic lymph nodes to achieve metastatic lymph node imaging.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] 1. Improved Lymphatic System Targeting: In existing technologies, many nano-tracers exhibit significant deviations in size control, affecting the effective uptake by the lymphatic system. This invention optimizes the feed ratio to stably control the nano-tracer particle size within 30-150 nm, ensuring more efficient lymphatic system uptake and retention, and improving imaging results.

[0017] 2. Precise Metastatic Lymph Node Imaging Based on High VEGFC Expression: Current lymph node imaging techniques typically rely on non-specific tracers, which may lead to high background noise or false positives. This invention utilizes the significantly high expression of VEGFCs in metastatic lymph nodes, combined with the ZIF-ICG-antibody complex system, to achieve highly specific and sensitive metastatic lymph node imaging. This improves the accuracy of lymph node metastasis detection and provides more reliable imaging support for gastric cancer staging.

[0018] 3. Enhanced fluorescence imaging capability and improved tissue penetration: Traditional ICG in aqueous solution typically emits fluorescence at around 800 nm, but is prone to quenching, resulting in limited skin penetration. This invention encapsulates ICG with ZIF, causing a redshift (shifting to a longer wavelength) of its fluorescence signal, enhancing near-infrared imaging capability and increasing tissue penetration depth, making deeper lymph node imaging possible.

[0019] 4. PEG modification improves stability, and antibody modification enhances specificity: Some existing nanoparticle formulations lack stable surface modifications, making them easily cleared or non-specifically absorbed in vivo. This invention improves biocompatibility and reduces immune system clearance by modifying the surface of nanoparticles with PEG, while simultaneously inserting specific antibodies to enhance the recognition and targeting ability of metastatic lymph nodes in gastric cancer.

[0020] 5. Good biocompatibility and high safety: Some existing nanomaterials may cause hemolysis or toxic reactions at high concentrations, limiting their clinical application. This invention exhibits a hemolysis rate of less than 5% at concentrations of 100 μg / mL and below, meeting biosafety standards and demonstrating good biocompatibility, thus improving the feasibility of clinical application.

[0021] 6. Simple preparation process, low cost, and low equipment requirements: Existing nano-tracer preparation processes are complex and require sophisticated equipment, limiting large-scale applications. This invention uses low-cost raw materials, has simple preparation steps, and low equipment requirements, which is conducive to large-scale production and widespread application. Attached Figure Description

[0022] Figure 1 Immunohistochemical staining for lymph node metastasis-related factors in gastric cancer patient tissues.

[0023] Figure 2 Diagrams illustrating the preparation methods for ZI, ZIP, and ZIPV series particles.

[0024] Figure 3 SEM images of ZI, ZIP, and ZIPV series nanoparticles.

[0025] Figure 4 TEM images of ZI, ZIP, and ZIPV nanoparticles.

[0026] Figure 5 The zeta potential is for ZI, ZIP, and ZIPV nanoparticles.

[0027] Figure 6 These are the UV-Vis absorption and fluorescence spectra of ZI, where A: UV spectrum of ZI series particles; B: fluorescence spectrum of ZI series particles.

[0028] Figure 7 Fluorescence confocal imaging of GA-0518 cells after incubation with ZIPV-1 for 1 h.

[0029] Figure 8 For the biocompatibility of ZIPV particles, where A: hemolysis rate of ZIPV-1 particles; B: hemolysis rate of ZIPV-2 particles; and C: hemolysis rate of ZIPV-3 particles.

[0030] Figure 9Luciferase chemiluminescence imaging of nude mice 1-9 weeks after tumor implantation.

[0031] Figure 10 Luciferase fluorescent staining of tumor tissue and bilateral popliteal lymph nodes in nude mice 9 weeks after tumor implantation.

[0032] Figure 11 In vivo fluorescence imaging of ZIPV nanotracers: Nude mice (A) after injection of ZIPV-1; Nude mice (B) after injection of ZIPV-2; Nude mice (C) after injection of ZIPV-3.

[0033] Figure 12 In vivo fluorescence imaging of nude mice after ICG injection.

[0034] Figure 13 Image showing the weight monitoring of mice over 28 days after imaging.

[0035] Figure 14 HE staining of heart, liver, spleen, lung, and kidney at different time points after injection of ZIPV-1 into nude mice. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0037] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0038] I. Experimental methods and materials.

[0039] 1. Reagents.

[0040] Zinc nitrate hexahydrate (Zn(NO3)2•6H2O) and 2-methylimidazole (2-MIM) were purchased from Maclean Biotechnology Co., Ltd. (Shanghai, China). Indocyanine green (ICG) was purchased from MCE Biotechnology Co., Ltd. (New Jersey, USA). DSPE-PEG-COOH was purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China). Rabbit anti-VEGFCPolyclonal Antibody was purchased from Apex Biotech Co., Ltd. (Shanghai, China). GA-0518 (human gastric cancer cells), fetal bovine serum (FBS), RPMI-1640 medium, trypsin, and cell culture antibiotics were all purchased from Pronosei Biotechnology Co., Ltd. (Wuhan, China). All reagents did not require further purification, and the water used in the experiments was 18 MΩ cm deionized water.

[0041] 2. Instruments.

[0042] .

[0043] 3. Methods.

[0044] 3.1 Immunohistochemical staining.

[0045] Baking: Place the slices in an oven and bake at 67°C for 30 minutes to melt the paraffin. Let them cool naturally before slicing.

[0046] Dewaxing and rehydration: The sections were placed in xylene I, xylene II, and xylene III for 10 min each, then in anhydrous ethanol I, anhydrous ethanol II, 90% ethanol, 80% ethanol, and 70% ethanol for 5 min each, and washed with water 3 times for 5 min each time.

[0047] Elimination of endogenous peroxidase: Prepare a 3% hydrogen peroxide solution (30% hydrogen peroxide: methanol = 1:9), soak the slices for 20 min, and wash with water 3 times, 5 min each time.

[0048] Antigen retrieval: Prepare citrate buffer, immerse the slides in the citrate buffer, place them in an autoclave, and perform antigen retrieval using the autoclaving method. After completion, remove the slides and allow them to cool naturally to room temperature. Wash the slides three times with PBS for 5 minutes each time.

[0049] Blocking: Prepare a blocking solution with a ratio of 10% donkey serum and 1% fetal bovine serum, using PBS as the solvent. Circle the tissue with an immunohistochemical pen, add the blocking solution to the tissue, place the slide in a humidified chamber filled with water, cover, and incubate at room temperature.

[0050] Primary antibody incubation: Dilute the target primary antibody with blocking buffer, aspirate the blocking buffer from the tissue, and drop the diluted primary antibody onto the tissue, ensuring complete coverage. Place the slide in a humidified chamber filled with water, cover, and incubate overnight at 4°C.

[0051] Rewarming: Remove the slides and allow them to return to room temperature naturally. Then wash the slides three times with PBS for 5 minutes each time to remove the primary antibody. Wipe the slides dry of any excess liquid.

[0052] Secondary antibody incubation: Use a secondary antibody corresponding to the species of the primary antibody, diluted 1:200 (the diluent is a blocking solution). Add the diluted secondary antibody solution to the tissue, place it in a humidified chamber filled with water, cover, and incubate at room temperature for 1 hour. Wash the sections three times with PBS for 5 minutes each time.

[0053] AB solution incubation: Prepare AB solution 20 minutes in advance by diluting solution A and solution B to a ratio of 1:1:100 (A:B:blocking solution). Add the diluted AB solution dropwise to the tissue, place the sections in a humidified chamber, and incubate at room temperature for 30 minutes. Wash the sections three times with PBS for 5 minutes each time.

[0054] DAB staining: Dilute DAB staining solution and diluent at a ratio of 30:1000 (prepare fresh before use). Wipe off excess liquid from the slides, add DAB staining solution, and observe the tissue staining under a microscope. When positive tissues turn dark brown and negative tissues turn colorless or light brown, wash away the staining solution with PBS to stop the reaction.

[0055] Hematoxylin staining: Place the slide in hematoxylin staining solution for about 2 minutes, rinse off excess staining solution with running water, observe the staining under a microscope, then immerse the slide in hydrochloric acid alcohol differentiation solution for 1-2 seconds, rinse with running water, and then place it in ammonia water for about 2 minutes. Under a microscope, the cell nuclei should appear blue.

[0056] Gradient dehydration: The slices were placed in 70% ethanol, 80% ethanol, and 90% ethanol for 5 min each, and then in anhydrous ethanol I and anhydrous ethanol II for 10 min each.

[0057] Transparency: Place the slices in xylene I and xylene II sequentially for 10 min each.

[0058] Mounting: Apply an appropriate amount of neutral resin to the tissue, cover with a coverslip, and gently press the coverslip to ensure it adheres firmly, avoiding the formation of air bubbles.

[0059] 3.2 Preparation of ICG@ZIF (ZI), ICG@ZIF@PEG (ZIP) and ICG@ZIF@PEG-VEGFC (ZIPV).

[0060] 3.2.1 Preparation of ICG@ZIF (ZI).

[0061] ICG@ZIF was prepared using a one-pot method. 650 mg of 2-MIM was dissolved in 14 mL of methanol, and then 6 mL of a mixed solution of ICG and Zn(NO3)2•6H2O (ICG content 3 mg, Zn(NO3)2•6H2O mass 100 mg, 200 mg, 300 mg, respectively) was added. The mixture was stirred at 750 rpm for 40 min in the dark, then centrifuged at 7000 rpm for 10 min, and the supernatant was discarded. The solution was vacuum dried at 120 °C to obtain powdered ICG@ZIF. Adjusting the Zn(NO3)2•6H2O feed amount yielded a series of ZI-1, ZI-2, and ZI-3 nanoparticles. The preparation method for ZIF-8 nanoparticles was similar, but without the addition of ICG to the mixed solution. The final concentrations of 2-MIM in the synthesis system were 32.5 mg / mL, ICG was 0.15 mg / mL, and Zn(NO3)2•6H2O were 5 mg / mL, 10 mg / mL, and 15 mg / mL, respectively.

[0062] 3.2.2 Preparation of ICG@ZIF@PEG (ZIP).

[0063] 20 mg of ZI was dissolved in 8 mL of methanol and vortexed until completely dissolved. 5 mg of DSPE-PEG-COOH (Mw = 2000 Da) powder was dissolved in 2 mL of methanol and slowly added dropwise to the ZI solution prepared in section 3.1.1 over a period of 10 min. The mixture was stirred at 660 rpm for 12 h in the dark, followed by centrifugation at 3000 rpm for 10 min to obtain a series of ZIP-1, ZIP-2, and ZIP-3 nanoparticles. The final concentration of ZI in the synthesis system was 2 mg / mL, and the final concentration of DSPE-PEG-COOH was 0.5 mg / mL.

[0064] 3.2.3 Preparation of ICG@ZIF@PEG-VEGFC (ZIPV).

[0065] Take the ZIP precipitate after centrifugation, dissolve it in 6 mL of water, then add 6 µL of VEGFC antibody stock solution with a concentration of 1 mg / mL, and place it in an ultrasonic cleaner and sonicate for 30 min to obtain ZIPV-1, ZIPV-2, and ZIPV-3 solutions.

[0066] 3.3 Cell culture.

[0067] This experiment used the human gastric cancer cell line (GA-0518). Cells were cultured in RPMI 1640 complete medium (containing 10% FBS and 1% penicillin antibiotics) and incubated in a cell culture incubator at 37°C and 5% CO2.

[0068] 3.4 Intracellular fluorescence imaging of ZIPV-1.

[0069] GA-0518 cells were seeded in confocal culture dishes and cultured at 37°C and 5% CO2 for 12 h. After incubation for 1 h with 40 µg / mL ZIPV-1 medium, images were acquired using an FV-1200 confocal laser scanning microscope with a 100× objective lens. .

[0070] 3.5 Hemolysis test.

[0071] Fresh mouse blood (containing the anticoagulant EDTA) was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. The precipitate was then washed 20 times with PBS (pH 7.4, 10 mM) to obtain red blood cells (RBCs). Before use, the RBCs were diluted 10-fold with PBS. 200 µL of the RBC dispersion was mixed with 800 µL of ZIPV at different concentrations (10, 20, 40, 60, and 100 µg / mL) and gently shaken at 37 °C for 4 h. Then, the mixture was centrifuged at 2000 rpm for 3 min, and the absorbance of the supernatant was measured at 540 nm. Water and PBS were used as positive and negative controls, respectively. The hemolysis rate of different concentrations of ZIPV was calculated. ; It is the absorbance value of the sample. and These are the absorbance values ​​for the positive and negative controls, respectively.

[0072] 3.6 Tumor modeling.

[0073] The 5-6 week old BALB / c nude mice (female, weighing 18±2g) used in this experiment served as an animal model for tumor treatment evaluation. The mice were fed SPF-grade laboratory animal feed and drank sterile ultrapure water. All treatment and care procedures for the laboratory animals were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" issued by China Medical University and the "Regulations on the Management of Laboratory Animal Affairs in China".

[0074] Female Balb / c nude mice, aged 5-6 weeks, were acclimatized in an SPF environment for one week before being placed in 2.5×10⁻⁶ urination tanks. 6 GA-0518 cells, pre-transfected with the Luciferase reporter gene, were suspended in 25 µL of RPMI 1640 complete medium and injected into the left paw pad of BALB / c nude mice to establish a paw pad tumor-popliteal lymph node metastasis model. Starting from the first week of injection, mice were intraperitoneally injected weekly with D-luciferin potassium salt (15 mg / mL, at a dosage of 150 mg / kg luciferin / body weight). After a 5-minute wait, bioluminescence imaging was performed using a small animal in vivo imaging system to monitor paw pad tumor formation and popliteal lymph node metastasis.

[0075] 3.7 In vivo bioimaging.

[0076] At week 9 after tumor cell injection, in vivo imaging was performed using mice with metastatic popliteal lymph nodes. ZIPV-1, ZIPV-2, and ZIPV-3 (5 mg / kg) were injected subcutaneously into the bilateral thighs of mice near the paw pads at multiple sites (equal dose bilaterally), and images were taken at 0h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, and 48h. (Filter 840nm).

[0077] 3.8 In vivo safety testing.

[0078] 3.8.1 HE staining.

[0079] After the in vivo imaging experiment, mice were anesthetized with isoflurane. The apex of the mouse heart was perfused with 20 mL of PBS (pH 7.4, 10 mM) to remove blood and contaminants. The mice were then perfused with 4% paraformaldehyde, and the major organs (heart, liver, spleen, lungs, and kidneys) were removed. These organs were then fixed with 4% paraformaldehyde for 24 h, followed by gradient dehydration, clearing, paraffin permeation, embedding, sectioning, and H&E staining. The stained sections were then observed under an upright fluorescent biological microscope after mounting.

[0080] 3.8.2 Biochemical detection of mouse serum.

[0081] Blood was collected from the orbital region of mice and placed in 1.5 mL EP tubes. The blood was allowed to stand at room temperature for 1 hour until coagulation. After coagulation, the blood was centrifuged at 4°C and 3000 rpm / min for 15 minutes. The supernatant was transferred to labeled sample tubes for storage; this was the serum. Serum levels of GLU, UREA, TP, ALB, ALT, and ALKP were measured using an automated biochemical analyzer according to the kit instructions.

[0082] II. Experimental Results.

[0083] 1. Screening of targeted lymph node markers.

[0084] To screen for potential target proteins for identifying metastatic lymph nodes, this patent reviewed a large amount of literature and selected VEGFC, VEGFD, VEGFR3, LYVE-1, NRP-2, and PROX-1, which showed significantly higher differential expression in metastatic and non-metastatic lymph nodes or were closely related to lymph node metastasis. Additionally, CRIP-1 and ALOX12, indicators closely related to lymph node metastasis identified in previous research by our team, were selected for further validation. To further screen for differentially expressed substances, we collected tissues from patients who underwent radical gastrectomy for gastric cancer. Immunohistochemical staining was performed on metastatic and non-metastatic lymph nodes. Five fields of view were randomly selected from each tissue, and the percentage of positive area was calculated for statistical analysis. In metastatic lymph node tissues, the expression level of VEGFC was significantly higher than that in non-metastatic lymph nodes (P < 0.05), while other indicators did not show significantly higher expression. Figure 1 ).

[0085] 2. Preparation and characterization of nano-tracers.

[0086] 2.1 Preparation of ZI, ZIP and ZIPV.

[0087] ICG@ZIF@PEG-VEGFC (abbreviated as ZIPV) according to Figure 2 The method shown is used for synthesis.

[0088] 2.2 Scanning electron microscope (SEM).

[0089] The morphology of ZI, ZIP, and ZIPV series nanocarriers was analyzed using scanning electron microscopy (SEM). Figure 3 SEM results showed that the average diameters of ZI-1, ZI-2, and ZI-3 nanocrystals were 32 nm, 70 nm, and 102 nm, respectively; the average diameters of ZIP-1, ZIP-2, and ZPI-3 nanocrystals were approximately 42 nm, 77 nm, and 120 nm; and the average diameters of ZIPV-1, ZIPV-2, and ZIPV-3 nanocrystals were approximately 63 nm, 84 nm, and 125 nm.

[0090] 2.3 Transmission electron microscopy (TEM).

[0091] The morphology of ZI, ZIP, and ZIPV series nanocarriers was analyzed using transmission electron microscopy (TEM). Figure 4 The results showed that the ZIP and ZIPV nanocarriers retained the morphological characteristics of ZIF-8 and had a PEG shell of about 5 nm.

[0092] 2.4 Zeta potential.

[0093] In addition, we also detected the zeta potential of ZI, ZIP, and ZIPV series particles. Figure 5 As shown, the zeta potential of ZI-1 was 20.8 mV. After coating with a PEG shell, the zeta potential of ZIP-1 changed to 17.8 mV. After antibody insertion, the zeta potential of ZIPV-1 continued to decrease (3.29 mV). This demonstrates that the PEG was successfully encapsulated in the ZIP and the VEGFC antibody was successfully inserted into the ZIPV.

[0094] 2.5 UV-Vis absorption and fluorescence spectra of ZI.

[0095] The successful loading of ICG into ZI nanoparticles can be further verified by UV-Vis absorption spectroscopy. Figure 6 As shown in Figure A, ICG exhibits a characteristic absorption peak at 815 nm, and this peak also appears in ZI. Furthermore, under excitation at 790 nm, ZI shows a maximum fluorescence emission peak at 825 nm, further confirming the successful loading of ICG into the tracer. Figure 6 B). Simultaneously, the absorbance of different concentrations of ICG was measured using a microplate reader, and a standard curve was plotted. Figure 6 C), and calculate the encapsulation efficiency of the ICG. For example... Figure 6 As shown in Figure D, the encapsulation efficiencies of ZI-1, ZI-2, and ZI-3 were 68.57%, 63.83%, and 68.76%, respectively. ICG was successfully encapsulated into the ZI samples, and the amount of ICG encapsulated in the three groups was approximately the same.

[0096] 3. Intracellular imaging studies.

[0097] Using GA-0518 cells as a model, the imaging capabilities of this tracer were further evaluated. Figure 7 As shown, after treating GA-0518 cells with ZIPV-1 for 1 hour, the red fluorescence of ZIPV could be clearly observed using STED super-resolution confocal microscopy. The results indicate that ZIPV can perform near-infrared I imaging within tumor cells.

[0098] 4. In vivo imaging.

[0099] 4.1 Biosafety.

[0100] To verify the biocompatibility of ZIPV particles, an in vitro hemolysis assay was performed. Mouse red blood cells were washed with PBS and mixed with different concentrations of ZIPV particles. The mixture was gently shaken at 37°C for 4 hours. After centrifugation, the supernatant was collected and the absorbance was measured at 542 nm. Water and PBS were used as positive and negative controls, respectively, and the hemolysis rate of each group was calculated. The ZIPV particle supernatant in each group appeared pale red to the naked eye, while the positive control group appeared bright red. The hemolysis rate in all groups was less than 5%, indicating that the ZIPV particles have high biocompatibility. Figure 8 A, B, C).

[0101] 4.2 Construction and monitoring of a mouse model of footpad tumor metastasis in the popliteal lymph nodes.

[0102] To assess the imaging performance of the nanotracer in vivo, a nude mouse model of footpad tumor metastasis in the popliteal lymph nodes of human gastric cancer cell line GA-0518-Balb / c was constructed. Tumor cells were injected into the left footpad of mice, while PBS was injected into the right footpad as a control group.

[0103] Starting from the first week of injection, mice were intraperitoneally injected weekly with D-fluorescein potassium salt (15 mg / mL, at a dosage of 150 mg / kg fluorescein / body weight). After a 5-10 minute interval, luminescence imaging was performed using a multimodal animal in vivo imaging system to monitor footpad tumor formation and popliteal lymph node metastasis. Metastasis to the popliteal lymph nodes was observed in nude mice at week eight, and the metastasis persisted into week nine. Figure 9 ).

[0104] Nine weeks after tumor injection, mice were dissected, and tumor tissue and bilateral popliteal lymph nodes were collected, dehydrated, paraffin-embedded, and sectioned. Luciferase fluorescent staining was performed on the tissue sections. The results showed that Luciferase-positive staining was found in the tumor tissue and the popliteal lymph nodes on the side of tumor cell injection in nude mice, indicating tumor cell infiltration. No Luciferase-positive staining was found in the popliteal lymph nodes of the nude mouse control group. Figure 10 The study demonstrated that at week 9, the nude mice injected with the tumor developed popliteal lymph node metastasis, while the control group remained negative, indicating successful model construction.

[0105] 4.3 In vivo fluorescence imaging of ZIPV nanotracers.

[0106] At week 9 after tumor cell injection, popliteal lymph node metastasis was observed in mice. ZIPV-1, ZIPV-2, and ZIPV-3 at a concentration of 1 mg / mL were injected subcutaneously into the bilateral thighs of mice at a dose of 5 mg / kg body weight per side (identical dose to both sides). Fluorescence imaging was performed at 740 nm (840 nm filter) at 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h. Three nude mice were injected with each group of particles.

[0107] The results showed that in the ZIPV-1-nude mouse group, no significant difference in fluorescence intensity was observed in the bilateral lymph node regions after injection at 0h, 0.5h, 1h, and 2h, indicating that ZIPV-1 was gradually absorbed into the lymphatic system. Four hours after injection, the fluorescence intensity of the popliteal lymph nodes on the tumor-bearing side was significantly higher than that on the control side, and a significant difference in fluorescence intensity remained between the two sides at 8h. Forty-eight hours after injection, fluorescence disappeared on the control side, indicating that ZIPV-1 on that side was completely metabolized. Fluorescence remained on the tumor-bearing side, and a small amount of ZIPV-1 remained in the lymph nodes. Figure 11 A); In the ZIPV-2 nude mouse group, no significant difference in fluorescence intensity was observed in the bilateral lymph node regions 0h, 0.5h, 1h, and 2h after injection, indicating that ZIPV was gradually absorbed into the lymphatic system. 4h and 8h after injection, the fluorescence intensity of the lymph nodes on the tumor-bearing side was higher than that on the control side, but the fluorescence was less dispersed than in the ZIPV-1 nude mouse group, indicating ZIPV-2 leakage. 48h after injection, fluorescence was still present in both lymph nodes, with the fluorescence intensity on the tumor-bearing side higher than that on the control side (…). Figure 11 B); In the ZIPV-3-nude mouse group, the fluorescence range expanded 2 hours after injection, indicating that ZIPV-3 was not completely absorbed into the lymphatic system. At 4 and 8 hours after injection, the fluorescence range significantly expanded, indicating particle escape and incomplete absorption by the lymphatic system; no significant difference in fluorescence intensity and range was observed in bilateral lymph node imaging. At 48 hours after injection, fluorescence was still present in the bilateral popliteal lymph node regions, but the difference in fluorescence intensity was not significant, indicating that ZIPV-3 was not completely metabolized. Figure 11 C).

[0108] Meanwhile, to compare the imaging effects of ZIPV and ICG, ICG was injected subcutaneously into mice using the same method for fluorescence imaging. Figure 12 As shown, in the nude mouse model, the fluorescence range was significantly expanded 4 hours after injection, and ICG was emitted. 24 hours after injection, ICG showed no fluorescence in the popliteal lymph node area, indicating that it was completely metabolized.

[0109] 4.4 ZIPV security testing.

[0110] To further evaluate the in vivo toxicity of ZIPV, the weight of the mice was monitored after imaging, such as... Figure 13As shown, there was no significant change in the body weight of the mice after injection, indicating that ZIPV has high biosafety and low toxicity. Then, the heart, liver, spleen, lungs, and kidneys of the mice were collected at different time points and stained with hematoxylin and eosin (H&E) to observe the histomorphological changes of the major organs. Figure 14 It can be seen that there was no obvious tissue damage in the mice's organs. This demonstrates that ZIPV has good biocompatibility and can serve as a potential nano-tracer. Simultaneously, blood samples were collected from mice 48 hours after particle injection for biochemical analysis. The results are shown in Tables 1-4. Serum levels of GLU, UREA, TP, ALB, ALT, and ALKP in the mice were all within the normal range, and no liver or kidney function damage was observed after ZIPV injection.

[0111] Table 1. Blood biochemical analysis of mice 48 hours after ZIPV injection. .

[0112] Table 2. Blood biochemical analysis of mice 7 days after ZIPV injection. .

[0113] Table 3. Blood biochemical analysis of mice 14 days after ZIPV injection. .

[0114] Table 4. Blood biochemical analysis of mice 28 days after ZIPV injection. .

[0115] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A highly specific fluorescent tracer for metastatic lymph nodes, characterized in that, It contains the following components: a. ZIF-8 nanoparticles; b. Indocyanine green (ICG) encapsulated within ZIF-8 nanoparticles; c. Surface-modified polyethylene glycol (PEG); d. VEGFC antibody inserted into PEG.

2. The fluorescent tracer according to claim 1, characterized in that, The tracer has a particle size of 30-150 nm and a near-infrared fluorescence emission wavelength of 800-840 nm.

3. A highly specific fluorescent tracer for metastatic lymph nodes as described in claim 1, characterized in that, The method includes the following steps: Step 1, Preparation of ZI: Dissolve 2-MIM in methanol, then add a mixed solution of ICG and Zn(NO3)2•6H2O, protect from light, stir at 750 r / min for 40 min, centrifuge at 7000 r / min for 10 min, discard the supernatant; vacuum dry at 120℃ to obtain ZI powder; Step 2, ZIP preparation: (1) Measure 20 mg of ZI obtained in step one and dissolve it in 8 mL of methanol. Shake the solution on a vortex shaker until it is completely dissolved to obtain a ZI solution. (2) Dissolve DSPE-PEG-COOH powder in methanol, and slowly and uniformly add it to the ZI solution obtained in (1). Protect from light, stir at 660 r / min for 12 h, and then centrifuge at 3000 r / min for 10 min to obtain a series of ZIP nanoparticles. Step 3, Preparation of ZIPV: Take the ZIP precipitate obtained in Step 2 after centrifugation, dissolve it in water, then add 6 μL of VEGFC antibody stock solution with a concentration of 1 mg / mL, and place it in an ultrasonic cleaner for ultrasonication for 30 min to obtain ZIPV nanoparticles.

4. The preparation method according to claim 3, characterized in that, In step one, the final concentration of 2-MIM in the synthesis system is 32.5 mg / mL, the final concentration of ICG is 0.15 mg / mL, and the final concentration of Zn(NO3)2•6H2O is 5~15 mg / mL.

5. The preparation method according to claim 3, characterized in that, In step two, the final concentration of ZI in the synthesis system is 2 mg / mL, and the final concentration of DSPE-PEG-COOH is 0.5 mg / mL.

6. The preparation method according to claim 3, characterized in that, The final particle size of ZIPV in the synthesis system in step three is 60~130nm.

7. The use of the highly specific fluorescent tracer for metastatic lymph nodes as described in claim 1 in the preparation of diagnostic reagents for metastatic lymph nodes, characterized in that, The applications include: a. Delivery of tracers to the target area via subcutaneous injection; b. Use near-infrared fluorescence imaging to differentiate between metastatic and non-metastatic lymph nodes.

8. Application of VEGFC antibody in the preparation of highly specific fluorescent tracers for metastatic lymph nodes.

9. The application according to claim 8, characterized in that, The tracer includes a ZIF-ICG-VEGFC antibody.

10. The application according to claim 8, characterized in that, The VEGFC antibody specifically recognizes metastatic lymph nodes, enabling metastatic lymph node imaging.

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