Near-infrared two-region fluorescence / magnetic resonance bimodal probe based on albumin as well as preparation method and application of near-infrared two-region fluorescence / magnetic resonance bimodal probe

Abstract

The invention belongs to the field of medical contrast agents, and relates to an albumin-based near-infrared two-region fluorescence / magnetic resonance bimodal probe as well as a preparation method and application thereof. The bimodal probe disclosed by the invention is a protein compound formed by self-assembling a near-infrared two-region fluorescent dye (IR820), a nuclear magnetic contrast agent (GdL) and albumin. The near-infrared two-region fluorescent dye (IR820) and the nuclear magnetic contrast agent (GdL) respectively show the characteristics of near-infrared two-region fluorescence enhancement and relaxation rate increase in a serum albumin solution. The near-infrared two-region fluorescent dye and the nuclear magnetic contrast agent have the advantages of simple preparation process, high relaxation rate, good stability, good biocompatibility and the like. The near-infrared two-region fluorescence / magnetic resonance bimodal probe disclosed by the invention can be used for diagnosis, real-time monitoring and surgical navigation of tumor lymph node metastasis.

Description

Technical Field

[0001] This invention relates to the field of medical contrast agents, and more particularly to an albumin-based near-infrared two-region fluorescence / magnetic resonance dual-modal probe, its preparation method, and its application. Background Technology

[0002] Cancer is one of the most serious threats to human health. Surgical resection is the preferred treatment for cancer. However, due to the invasive nature of tumors, their boundaries are often unclear. Excessive resection can severely impact postoperative recovery, while insufficient resection can lead to metastasis and recurrence. Furthermore, the sentinel lymph node (SLN) is the first site of lymphatic metastasis from primary malignant tumors; therefore, systematic lymph node dissection is often necessary during surgery. Although complete resection of the primary lesion and thorough dissection of potentially metastatic lymph nodes are crucial for radical surgery, locating those lymph nodes hidden deep beneath normal tissue remains a challenge for clinicians and scientists, significantly impacting cancer diagnosis, treatment, and postoperative recovery.

[0003] In recent years, fluorescence imaging-guided surgical resection has emerged as a novel tumor treatment method, significantly improving the detection rate of tumors and small lesions. However, most existing fluorescent probes emit wavelengths in the visible and near-infrared I region (700-950 nm), and their tissue penetration depth and spatial resolution are limited due to the absorption and scattering of photons in these wavelengths by biological tissues. In contrast, near-infrared II fluorescence (1000-1700 nm) has lower absorption and scattering in living tissues and almost no interference from biological tissue autofluorescence, thus exhibiting better tissue penetration depth and higher spatial resolution, and is considered the future direction of next-generation fluorescence imaging technology.

[0004] Traditional single-mode imaging methods, such as X-ray, ultrasound (US), optical imaging (OI), and magnetic resonance imaging (MRI), while each with its advantages, have limitations in early tumor diagnosis and intraoperative real-time imaging. For example, optical imaging technology has many advantages, such as high sensitivity and good selectivity, and can visualize microscopic biological samples such as cells and tissue pathology sections, but it suffers from insufficient optical penetration depth. Magnetic resonance imaging can perform omnidirectional, multi-angle, whole-body tomographic imaging of large living samples, providing more accurate imaging of living organs and tissues with various anatomical changes. However, MRI also has drawbacks such as insufficient spatial resolution, low sensitivity, and long imaging time. Therefore, there is a good complementarity between the advantages and disadvantages of optical imaging and MRI. Thus, developing novel magneto-optical dual-mode molecular probes can leverage the complementarity between the two imaging methods, fully utilizing the advantages of MRI's lack of tissue penetration depth limitations and optical imaging's high sensitivity, while simultaneously obtaining anatomical and molecular functional information. This allows for diagnostic tasks that are difficult to accomplish with a single imaging method and provides more comprehensive imaging information.

[0005] However, existing multimodal probes still require optimization in terms of biocompatibility, stability, and imaging performance. Therefore, this invention aims to develop a serum protein-based dual-modal protein complex probe for magnetic resonance imaging (MRI) and near-infrared II (NIR-II) imaging. This probe combines the biocompatibility and stability of serum proteins with the imaging properties of gadolinium complexes and near-infrared fluorescent dyes, aiming to achieve precise preoperative in vivo tumor localization and intraoperative navigation guided by near-infrared imaging technology, providing a new technical means for the early diagnosis and treatment of tumors. Summary of the Invention

[0006] The purpose of this invention is to prepare a near-infrared two-zone fluorescence / magnetic resonance dual-modal probe based on albumin, which can realize preoperative magnetic-optical dual-modal imaging of tumors and sentinel lymph nodes, as well as intraoperative navigation guided by near-infrared imaging technology.

[0007] The albumin-based near-infrared II fluorescence / magnetic resonance (NIR-II) dual-modal probe of this invention is a protein complex formed by loading a serum protein with a near-infrared II fluorescent dye (IR820) and a magnetic resonance contrast agent (GdL). The albumin-based NIR-II fluorescence / magnetic resonance dual-modal probe of this invention is characterized by: compared to IR820 alone, this dual-modal probe exhibits a significant near-infrared II fluorescence signal in the 920-1200 nm range; the emission peak is located at 952 nm, and its relaxation rate is significantly increased compared to GdL alone; therefore, serum protein, as a carrier, significantly enhances the imaging effect of IR820 and GdL. Using NIR-II fluorescence and MR imaging techniques, dual-modal imaging of tumors and metastatic lymph nodes in a mouse model of lymph node metastasis was successfully achieved. The research results of this invention provide valuable tools for preoperative tumor assessment and intraoperative surgical navigation.

[0008] In this invention, the infrared II fluorescent dye IR820 has the structure shown in the following formula: .

[0009] In this invention, the nuclear magnetic resonance contrast agent (GdL) has the structure shown in the following formula: .

[0010] In a preferred embodiment of the present invention, the molar ratio of serum protein, IR820 and GdL is 6-8:1-2:0.5-1.

[0011] In a preferred embodiment of the present invention, the serum protein, IR820, and GdL are bound together by non-covalent bonds. In this invention, the near-infrared II fluorescent dye (IR820) and the nuclear magnetic resonance contrast agent (GdL) are self-assembled with the serum protein through intermolecular interactions. IR820 and GdL are both located in the cracks of the three structural proteins of the serum protein, and the interactions mainly include hydrogen bonds, van der Waals forces, and π-π interactions. Self-assembly via non-covalent bonds effectively reduces the complexity of the preparation process.

[0012] In a preferred embodiment of the present invention, the serum protein includes any one or a mixture of two or more of bovine serum albumin, human serum albumin, mouse serum albumin, ovalbumin, and rabbit serum albumin; preferably, the serum protein is bovine serum albumin and / or human serum albumin. Another aspect of the present invention relates to the above-mentioned method for preparing a near-infrared two-region fluorescence / magnetic resonance dual-mode probe based on albumin, which is prepared by simply mixing serum protein, IR820 and GdL in a certain proportion. In a preferred embodiment of the present invention, the method includes the following steps: (1) Prepare stock solutions of serum protein, IR820 and GdL of a certain concentration using physiological saline; for example, BSA (9%), IR820 (2.5 mM) and GdL (5 mM).

[0013] (2) Mix 500 µL BSA (9%), 400 µL IR820 (2.5 mM), and 100 µL GdL (5 mM) in a 1.5 mL centrifuge tube and vortex mixer for 1 minute to obtain 1 mL of the near-infrared two-zone fluorescence / magnetic resonance dual-mode probe of the present invention.

[0014] This invention systematically studies the photophysical properties of a dual-modal probe, including ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy. Its absorption characteristics are analyzed using ultraviolet-visible absorption spectroscopy, and its luminescence behavior is revealed by fluorescence emission spectroscopy. These studies provide comprehensive characterization data for the photophysical performance of dual-modal probes.

[0015] This invention evaluates the light penetration depth capability of the prepared bimodal probe. Capillaries containing IR820 and BSA-IR820 were immersed in a 1% lipid solution, and near-infrared II (NIR-II) imaging was performed at different depths (1-4 mm). NIR-II imaging confirmed that the fluorescence signal of BSA-IR820 was very strong. Furthermore, it was observed that the signal intensity of the NIR-II image decreased significantly with increasing penetration depth. The experimental results demonstrate that this bimodal nanoprobe possesses high resolution and deep tissue penetration capability, which is extremely advantageous for fluorescence surgical navigation.

[0016] This invention utilizes the prepared dual-modal probe for in vivo fluorescence imaging in mice. Given the probe's excellent NIR-II imaging capability in in vivo experiments, the NIR-II fluorescence surgical navigation capability of BSA-IR820 was further verified. By establishing a subcutaneous tumor model of the back of 4T1 breast cancer cells in mice, BSA-IR820 was injected orally into the tumor. Bright near-infrared II fluorescence signals were clearly observed, and thanks to its excellent fluorescence signal, precise localization of the tumor tissue was successfully achieved, leading to surgical resection. These results confirm that the BSA-IR820 probe has good imaging performance in in vivo tumor models, demonstrating its potential application value not only in in situ tumor visualization but also in fluorescence-guided surgical navigation.

[0017] This invention utilizes the prepared dual-modal probe for in vivo fluorescence imaging and fluorescence surgical navigation testing in mice. Injection of BSA-IR820 into the paw pads clearly illuminated metastatic sentinel lymph nodes, and the excellent fluorescence signal facilitated successful lymph node resection. Bright-field imaging and fluorescence imaging of the excised lymph nodes clearly showed probe enrichment in the metastatic lymph nodes (appearing green). This effectively confirmed BSA-IR820's ability to track metastatic lymph nodes. Histological staining confirmed that all excised lymph nodes were pathologically positive, demonstrating successful resection of metastatic lymph nodes rather than normal ones. These results further demonstrate that BSA-IR820 can not only accurately identify metastatic lymph nodes but also provide reliable guidance for fluorescence-guided surgery.

[0018] This invention utilizes a prepared dual-modal probe for in vivo magnetic resonance imaging (MRI) in mice. A mouse lymph node metastasis model was constructed, and mice were subjected to MRI T1WI sequences. The results confirmed that in mice injected with BSA@GdL, the MRI signal in the popliteal lymph node metastasis region gradually increased over time, while in mice injected with GdL, almost no signal change was observed in the popliteal lymph nodes. This indicates that BSA@GdL has a better imaging effect on lymph node metastases than GdL alone.

[0019] The advantages and beneficial effects of this invention include at least one or more, or all of the following: a. The method for preparing the albumin-based near-infrared fluorescence / magnetic resonance dual-modal probe of the present invention is simple. It can be obtained simply by mixing serum protein, IR820 and GdL in a certain proportion, without the need for organic solvents or complex operations such as heating, and the preparation process is short.

[0020] b. The prepared dual-modal probe organically integrates the carrier, NMR contrast agent, and near-infrared II fluorescent dye into the protein complex, realizing magneto-optical dual-modal imaging. Its performance is significantly improved compared to that of serum protein, IR820, or GdL alone.

[0021] c. Complementary imaging characteristics: Near-infrared II fluorescence imaging provides high resolution and deep tissue penetration, while MRI offers excellent soft tissue contrast and spatial resolution. This combination can provide more comprehensive information about biological tissues.

[0022] d. Improve diagnostic accuracy: By combining two imaging techniques, the location of the tumor can be more accurately located, especially when the boundary between the tumor and the surrounding normal tissue is not clear, which helps to improve the precision of surgical resection.

[0023] e. Real-time monitoring: Near-infrared fluorescence imaging (NIIR II) allows for real-time observation of dynamic changes in the tumor, while MRI provides detailed information on tumor structure. This real-time monitoring is crucial for surgical navigation and efficacy assessment.

[0024] In summary, the dual-modal probe BSA-IR820-GdL of this invention, through its multimodal synergistic imaging mechanism, not only overcomes the long-standing challenge of balancing sensitivity and resolution in traditional imaging techniques, but more importantly, provides a novel molecular imaging tool for the early diagnosis and accurate staging of clinical lymph node metastasis. This research provides important technical reference and theoretical foundation for the development of next-generation precision oncology diagnostic and therapeutic probes. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the self-assembly of the albumin-based near-infrared two-region fluorescence / magnetic resonance dual-modal probe of the present invention; Figure 2 The hydrodynamic diameter and potential test diagram of the albumin-based near-infrared two-region fluorescence / magnetic resonance dual-mode probe of this invention are shown. Figure 3 The images show the UV absorption spectrum and the near-infrared II fluorescence emission spectrum of the albumin-based near-infrared II fluorescence / magnetic resonance dual-mode probe of this invention. Figure 4 This is an in vitro near-infrared fluorescence image of the albumin-based near-infrared fluorescence / magnetic resonance dual-modal probe of the present invention. Figure 5 This is a magnetic resonance imaging comparison of a mouse lymph node metastatic tumor model using the albumin-based near-infrared two-zone fluorescence / magnetic resonance dual-modal probe of this invention. Figure 6 This is a NIR-II fluorescence surgical navigation diagram of a mouse dorsal tumor model and a lymph node metastasis model based on an albumin-based near-infrared two-zone fluorescence / magnetic resonance dual-modal probe, according to the present invention. Figure 7 This is a comparison of lymph node resection in a mouse lymph node metastasis model based on the albumin-based near-infrared fluorescence / magnetic resonance dual-modal probe of the present invention. Detailed Implementation

[0026] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art should understand that the following examples are only for illustrating this application and should not be regarded as limiting the scope of protection of this application. Unless otherwise specified in the examples, all conditions are performed according to conventional conditions or manufacturer's recommended conditions; reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0027] Example 1: Preparation of a near-infrared II fluorescence / magnetic resonance dual-modal probe based on albumin: (1) Dissolve BSA in physiological saline to prepare a 9% stock solution 1. (2) Dissolve near-infrared II fluorescent dye (IR820) in physiological saline to prepare a 2.5 mM stock solution 2. (3) Dissolve magnetic resonance imaging contrast agent GdL in physiological saline to prepare a 5 mM stock solution 3. (4) Use a pipette to take 500 µL of stock solution 1, 400 µL of stock solution 2, and 100 µL of stock solution 3 into 1.5 mL centrifuge tubes, mix them with a vortex mixer for 1 minute, and finally obtain the near-infrared II fluorescence / magnetic resonance dual-modal probe of the present invention (the molar ratio of serum protein, IR820, and GdL is 6.8:1:0.5). The results are as follows. Figure 1 As shown. Molecular docking confirmed that the near-infrared II fluorescent dye (IR820), nuclear magnetic resonance contrast agent (GdL), and BSA albumin are self-assembled through intermolecular interactions, and the preparation process is significantly simpler than that of covalent bonding.

[0028] Example 2 The hydration size and potential of the bimodal nanoprobe prepared in Example 1 were tested. The bimodal nanoprobe prepared in Example 1 was diluted to a suitable concentration, transferred to a cuvette, and the changes in hydraulic diameter and potential were measured using a nanoparticle size analyzer. Figure 2 As shown, the bimodal probe has a similar size to the BSA protein alone, with a hydrodynamic diameter of 3.4 nm. This indicates that IR820 and GdL did not assemble with the BSA protein to form larger nanoparticles, but were instead embedded in the cracks of the BSA protein through intermolecular forces, which is completely consistent with the theoretical calculation results.

[0029] Example 3 The bimodal nanoprobe prepared in Example 1 was subjected to UV-Vis-NIR absorption spectroscopy and NIR-II fluorescence emission spectroscopy. The bimodal nanoprobe prepared in Example 1 was diluted 10-fold with deionized water, transferred to a cuvette, and measured using a UV-Vis-NIR spectrophotometer. The test results are as follows: Figure 3 As shown in Figure A, after IR820 self-assembled with BSA protein, the UV absorption spectrum changed, with a significant decrease in the absorption peak at 692 nm and a significant increase in the absorption peaks at 783 and 833 nm. The addition of GdL caused a further decrease in the 783 nm absorption peak and a further increase in the 833 nm peak. These results indicate that, compared to IR820 alone, its self-assembly with BSA protein resulted in a blue shift in the UV-Vis absorption spectrum. Furthermore, the bimodal nanoprobe prepared in Example 1 was diluted 10-fold with deionized water, transferred to a cuvette, and tested using a near-infrared fluorescence spectrometer. The test results are as follows... Figure 3As shown in B, IR820 alone showed almost no fluorescence in the 900-1300 nm range, while IR820 in 4.5% BSA protein solution exhibited a significant fluorescence emission peak, with enhanced fluorescence intensity compared to IR820 alone.

[0030] Example 4 Near-infrared II fluorescence imaging was performed on the dual-modal nanoprobe prepared in Example 1. Stock solutions of different concentrations of IR820, GdL, and the protein complex (BSA-IR820-GdL) were prepared and filled into capillaries before imaging. Figure 4 As shown in Figure A, free IR820 did not produce a detectable NIR-II imaging signal at any concentration, while the BSA-IR820-GdL protein complex showed a significant NIR-II fluorescence signal, which increased with increasing concentration. Stock solution 3 prepared in Example 1 was diluted with physiological saline and 4.5% BSA solution to prepare test solutions of different concentrations. Subsequently, the effects of different short-pass (SP) and long-pass (LP) filters on NIR-II imaging were further investigated. Compared with the long-pass filter, the short-pass filter SP1150 (<1150 nm) produced the strongest signal intensity. The signal intensities were in the order of LP1000 > LP1100 > LP1300, with LP1300 showing almost no detectable fluorescence. Accompanying this decrease, the signal-to-noise ratio (S / N) gradually decreased, and the full width at half maximum (FWHM) correspondingly broadened, which is completely consistent with the NIR-II emission spectrum. Figure 4 B). To evaluate the tissue penetration capability of the dual-modal nanoprobe, capillaries containing IR820 and BSA-IR820-GdL were immersed in a 1% lipid solution, and near-infrared II imaging was performed at different depths (1-4 mm). Figure 4 As shown in Figure C, the fluorescence signal is very prominent under NIR-II imaging. It can be observed that the signal intensity of the NIR-II image decreases significantly with increasing penetration depth, accompanied by blurring of the capillary edges. This phenomenon can be attributed to light attenuation and scattering. This dual-modal nanoprobe near-infrared II fluorescence imaging exhibits high resolution and deep tissue penetration capability, which is extremely advantageous for fluorescence surgical navigation.

[0031] Example 5 The dual-modal nanoprobe prepared in Example 1 was used for magnetic resonance imaging of lymph node metastasis in breast cancer.

[0032] 1) Constructing a model of breast cancer lymph node metastases First, 4T1 mouse breast cancer tumor cells in the logarithmic growth phase were selected. These cells were then digested with trypsin, and the digested cells were centrifuged at 1000 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the cells were washed three times with PBS. Next, serum-free 1640 medium was added to the cells to adjust the concentration of 4T1 breast cancer tumor cells to 1 × 10⁻⁶. 6 Cells / mL. The cell suspension was then gently pipetted to prepare a single-cell suspension, which was then placed on ice to maintain its viability. Next, the left hind limb paw pads of Balb / c mice were thoroughly disinfected with 75% ethanol. The prepared 4T1 breast cancer tumor cell single-cell suspension was aspirated into an insulin syringe, and approximately 25 μL of the suspension was injected into the subcutaneous tissue of the mouse hind limb paw pads to establish a 4T1 breast cancer lymph node metastasis model.

[0033] 2) Magnetic Resonance Imaging Experiment Two Balb / c mice with an established 4T1 model of breast cancer lymph node metastasis were selected. 25 μL of GdL and BSA-IR820-GdL were injected into the hind limb paw pad tumors, respectively. MRI T1WI sequences were performed on both mice at different time points after injection (0 min, 15 min, 30 min, and 1 hour). Figure 5 As shown, in mice injected with BSA-IR820-GdL, the magnetic resonance imaging signal in the popliteal lymph node metastasis area gradually increased over time, while in mice injected with GdL, almost no signal change was observed in the popliteal lymph nodes, indicating that BSA-IR820-GdL has a better imaging effect on lymph node metastases than GdL alone.

[0034] Example 6 Near-infrared fluorescence imaging was performed on a dorsal subcutaneous tumor model using the dual-modal nanoprobe prepared in Example 1.

[0035] 1) Constructing a mouse dorsal subcutaneous tumor model First, 4T1 mouse breast cancer tumor cells in the logarithmic growth phase were selected. These cells were then digested with trypsin, and the digested cells were centrifuged at 1000 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the cells were washed three times with PBS. Next, serum-free 1640 medium was added to the cells to adjust the concentration of 4T1 breast cancer tumor cells to 1 × 10⁻⁶. 6 Cells / mL. Then, the cell suspension was gently pipetted to prepare a single-cell suspension and placed on ice to maintain its activity. Next, the prepared 4T1 breast cancer tumor cell single-cell suspension was aspirated into an insulin syringe, and approximately 50 μL of the suspension was injected subcutaneously into the right back of mice to establish a mouse subcutaneous tumor model.

[0036] 2) Near-infrared II fluorescence imaging experiment Two Balb / c mice with an established 4T1 mouse model of subcutaneous breast cancer tumors on the back were selected. Each mouse underwent an orthotopic injection of 25 μL of BSA-IR820-GdL into the back tumor. Subsequently, near-infrared II (NIR-II) imaging was performed on the mice. The imaging results showed that... Figure 6 As shown in Figure A, a bright near-infrared II fluorescence signal can be observed at the tumor site, and thanks to its excellent fluorescence signal, the tumor tissue was accurately located and surgical resection was successfully completed.

[0037] Two Balb / c mice with a successfully established 4T1 mouse model of breast cancer lymph node metastasis were selected. 25 μL of IR820 and BSA-IR820-GdL were injected into the hind limb paw pad tumors of each mouse, respectively. Subsequently, near-infrared II (NIR-II) imaging was performed on the mice. The imaging results showed that, as Figure 6 As shown in Figure B, on the left hind limb inoculated with tumor cells, in addition to the particularly strong fluorescence signal in the paw pad area, the fluorescence signals in the popliteal fossa and ischial lymph node metastatic tumor areas were also very obvious. In contrast, the fluorescence signal on the right hind limb, which was not inoculated with tumor cells, was relatively weak. In summary, this dual-modal probe can clearly observe the illumination of metastatic sentinel lymph nodes, and thanks to its excellent fluorescence signal, metastatic lymph nodes can be successfully removed.

[0038] Example 7 Bright-field photography and near-infrared II fluorescence imaging were performed on the excised lymph nodes, such as... Figure 7 As shown in the results, normal lymph nodes show almost no fluorescence signal, while lymph nodes with tumor metastasis show strong fluorescence signals, with the probe enriched in metastatic lymph nodes (appearing green). This effectively confirms that BSA-IR820-GdL has the ability to track metastatic lymph nodes. Histological staining confirmed that all excised lymph nodes were pathologically positive, effectively proving that we successfully removed metastatic lymph nodes rather than normal lymph nodes. These results fully demonstrate that BSA-IR820 can not only accurately identify metastatic lymph nodes but also provide reliable guidance for fluorescence-guided surgery.

[0039] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. An albumin-based near-infrared second window fluorescence / magnetic resonance dual-modal probe, characterized in that, The bimodal probe is a protein complex of serum protein, near-infrared two-region fluorescent dye IR820 and nuclear magnetic contrast agent GdL.

2. The bimodal probe according to claim 1, wherein the molar ratio of the serum protein, IR820 and GdL is 6-8:1-2:0.5-1.

3. The bimodal probe according to claim 1, wherein the serum protein, IR820 and GdL are combined by non-covalent bond to form the protein complex.

4. The bimodal probe according to claim 1, wherein the serum protein comprises any one of bovine serum albumin, human serum albumin, mouse serum protein, ovalbumin, rabbit serum protein or a mixture of two or more thereof.

5. The bimodal probe according to claim 4, wherein the serum protein is bovine serum albumin and / or human serum albumin.

6. A method of preparing the bimodal probe according to any one of claims 1 to 5, characterized in that The bimodal probe is prepared by simply mixing the serum protein, IR820 and GdL in a certain ratio to obtain the final probe.

7. Use of the bimodal probe according to any one of claims 1-5 in the preparation of a biological imaging agent.

8. The use according to claim 7, wherein the biological imaging agent is used for locating tumor tissue.

9. Use of the bimodal probe according to any one of claims 1-5 in the preparation of a diagnostic reagent for lymph node metastasis.

10. The use according to claim 9, wherein the diagnostic reagent targets enrichment in metastatic lymph nodes.

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