BSA-MnO2 nanoprobe for identifying sentinel lymph node metastasis state of breast cancer and application of BSA-MnO2 nanoprobe

Abstract

The invention belongs to the technical field of medicine, and relates to a BSA-MnO2 nanoprobe used for identifying breast cancer sentinel lymph node metastasis state, and applications thereof. The BSA-MnO2 nano probe is obtained by carrying out a reaction on BSA (Bovine Serum Albumin) and KMnO4; the BSA-MnO2 nanoprobe is injected into a human body as a magnetic resonance lymphatic vessel contrast agent, and in combination with a nuclear magnetic resonance scanning technology, a clear nuclear magnetic resonance structure image of a sentinel lymph node of a living body and quantitative parameters related to a lymphatic drainage function are successfully obtained, such as a nuclear magnetic enhancement signal value MES, a maximum nuclear magnetic enhancement signal value, a 3h relative lymph node clearance rate and the like. A sentinel lymph node metastasis assessment framework is formed through the parameters and can be used for identifying normal sentinel lymph nodes and breast cancer metastatic sentinel lymph nodes, and more importantly, the framework can be used for identifying breast cancer sentinel lymph node tiny metastasis and breast cancer sentinel lymph node large metastasis. And a new means is provided for noninvasive identification of the breast cancer sentinel lymph node metastasis state in clinical practice.

Description

Technical Field

[0001] The present invention relates to a BSA-MnO2-based assay for identifying the metastasis status of sentinel lymph nodes in breast cancer. 2 Nanoprobes and their applications. Background Art

[0002] Sentinel lymph node (SLN) is the first drainage lymph node. Its metastatic status is unrelated to the size of the primary tumor. It is a key indicator for predicting the overall survival rate and the risk of lymph node metastasis in breast cancer patients. Therefore, early diagnosis of SLN metastasis has important clinical significance for tumor staging and treatment decisions in breast cancer patients. At present, the gold standard for clinical diagnosis of SLN metastasis is still postoperative histopathological examination, but invasive surgery and postoperative complications can lead to some problems, such as chronic lymphatic insufficiency with lymphedema. In recent years, non-invasive imaging techniques, such as ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI), optical imaging, and radionuclide imaging, have been routinely used in clinical evaluation, but these methods still have some limitations. The clinical application of CT and radionuclide imaging is limited to a certain extent due to radioactive exposure and low spatial resolution. Radionuclide imaging also has an inevitable high cost. Similarly, US is also limited by low spatial resolution, while optical imaging is limited by tissue penetration depth. In addition, conventional plain-contrast MRI has higher soft-tissue contrast and spatial resolution compared with other techniques, but its value for lymph node staging is still limited.

[0003] Magnetic resonance lymphography (MRL) is a noninvasive imaging method. Its high soft tissue contrast and spatial resolution can not only provide subtle lymph node anatomical information, but also indirectly reflect changes in lymphatic drainage function by monitoring the perfusion and metabolism of contrast agents, making it a potentially powerful noninvasive diagnostic technique for early assessment of SLN metastasis status. However, the gadolinium-based contrast agents currently used in clinical practice are complexes that are more likely to diffuse rapidly into the blood system and be rapidly metabolized by the kidneys, which is not conducive to MRL examinations. Therefore, exploring a contrast agent that is more suitable for MRL examinations is of great significance to the clinical promotion of the application of MRL. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a BSA-MnO 2 Nanoprobes and applications using the BSA-MnO 2 Nanoprobes as MRL contrast agents can accurately identify micrometastasis and macrometastasis of breast cancer sentinel lymph nodes.

[0005] One object of the present invention is achieved by the following technical solutions:

[0006] A BSA-MnO assay for identifying sentinel lymph node metastasis in breast cancer 2 Nanoprobe, the BSA-MnO 2 The nanoprobe is composed of bovine serum albumin (BSA) and potassium permanganate (KMnO 4 ) is reacted to obtain; the BSA-MnO 2 Nanoprobes are used as magnetic resonance lymphangiography contrast agents to identify the status of sentinel lymph node metastasis in breast cancer, which includes micrometastasis and macrometastasis.

[0007] The BSA-MnO 2 Nanoprobes as magnetic resonance lymphangiography contrast agents can be used to identify normal sentinel lymph nodes (sentinel lymph nodes without metastasis) and breast cancer metastatic sentinel lymph nodes, and further, can be used to identify the metastatic status of breast cancer sentinel lymph nodes, that is, BSA-MnO 2 Nanoprobes can be used as magnetic resonance lymphangiography contrast agents to identify micrometastases and macrometastases of breast cancer sentinel lymph nodes through magnetic resonance imaging technology.

[0008] In this article, the Rotterdam standard was used to define the SLN metastasis status of breast cancer. The maximum tumor diameter of 0.1-1 mm was defined as micrometastasis of breast cancer SLN, and the maximum tumor diameter of >1 mm was defined as macrometastasis of breast cancer SLN.

[0009] Preferably, the BSA-MnO 2 The preparation method of the nanoprobe comprises the following steps: placing a bovine serum albumin aqueous solution in a water bath at 25 to 50°C, then dropping a potassium permanganate aqueous solution, maintaining the temperature for 1 to 10 hours, dialyzing, and drying to obtain BSA-MnO 2 Nanoprobes.

[0010] Preferably, the BSA-MnO 2 In the nanoprobe, MnO 2 The mass fraction is 1 to 20 wt %; more preferably 2 to 10 wt %.

[0011] Preferably, the BSA-MnO 2 The nanoprobe is used as a magnetic resonance lymphangiography contrast agent to identify the metastasis status of sentinel lymph nodes in breast cancer. The identification method includes the following steps:

[0012] 4T1 cells (mouse breast cancer cells) were injected into the right sole of the mouse, and after several days, SLN metastasis mice were obtained;

[0013] The SLN-transferred mice were anesthetized and then subjected to MRI scanning to obtain T1-weighted images before injection;

[0014] Then, BSA-MnO was injected into the left and right soles of the SLN-transferred mice. 2 For nanoprobes, MRI scanning was performed 5 to 200 min after injection to obtain T1-weighted images after injection;

[0015] The T1-weighted images before and after injection were processed for consistency, and then the region of interest (ROI) was delineated on the SLN coronal image with the largest cross section, and the corresponding average MRI signal value was obtained and recorded. 2 The average NMR signal value after nanoprobe injection was subtracted from the ipsilateral injection of BSA-MnO 2 The average NMR signal value before the nanoprobe is used to obtain the MR enhanced signal value (MES);

[0016] When the nuclear magnetic enhancement signal value on the right side is greater than that on the left side, the SLN metastasis is determined to be a SLN micrometastasis. When the nuclear magnetic enhancement signal value on the right side is less than that on the left side, the SLN metastasis is determined to be a SLN macrometastasis.

[0017] The second object of the present invention is achieved by the following technical solutions:

[0018] A BSA-MnO 2 Application of nanoprobes in the preparation of magnetic resonance lymphangiography contrast agents for identifying the metastasis status of sentinel lymph nodes of breast cancer, the application method comprising:

[0019] 4T1 cells were injected into the right sole of the mouse, and SLN-transferred mice were obtained several days later;

[0020] The SLN-transferred mice were anesthetized and then subjected to MRI scanning to obtain T1-weighted images before injection;

[0021] Then, BSA-MnO was injected into the left and right soles of the SLN-transferred mice. 2 For nanoprobes, MRI scanning was performed 5 to 200 min after injection to obtain T1-weighted images after injection;

[0022] The T1-weighted images before and after injection were processed for consistency, and then the region of interest was delineated on the SLN coronal image with the largest cross section, and the corresponding average MRI signal value was obtained and recorded. 2 The average NMR signal value after nanoprobe injection was subtracted from the ipsilateral injection of BSA-MnO 2The average NMR signal value before the nanoprobe is used to obtain the NMR enhancement signal value;

[0023] When the nuclear magnetic enhancement signal value on the right side is greater than that on the left side, the SLN metastasis is determined to be a SLN micrometastasis. When the nuclear magnetic enhancement signal value on the right side is less than that on the left side, the SLN metastasis is determined to be a SLN macrometastasis.

[0024] The NMR enhanced signal value on the right side is the value of BSA-MnO injected on the right side. 2 The average NMR signal value after the nanoprobe was subtracted from the value after the injection of BSA-MnO on the right side. 2 The average NMR signal value before the nanoprobe; the NMR enhanced signal value on the left is the value of the left side injected with BSA-MnO 2 The average NMR signal value after the nanoprobe was subtracted from the left side after injection of BSA-MnO 2 Average NMR signal value before the nanoprobe.

[0025] Preferably, several days is 2 to 10 days. Generally speaking, 2-4 days after 4T1 cells are injected into the right sole of the mouse, micro-metastasis of breast cancer SLN can be obtained, and 5-10 days after 4T1 cells are injected, macro-metastasis of breast cancer SLN can be obtained.

[0026] Preferably, 0.1-1000 μg BSA-MnO is applied to each sole. 2 Nanoprobe / g mouse injection. The same amount of BSA-MnO was injected into each foot. 2 Furthermore, 1-100 μg BSA-MnO 2 Nanoprobes / g mouse injection.

[0027] BSA-MnO 2 The nanoprobes were injected in the form of solution to form BSA-MnO 2 The nanoprobe was dissolved in water to form BSA-MnO 2 Nanoprobe solution. BSA-MnO 2 In the nanoprobe solution, BSA-MnO 2 The concentration is 0.1 to 1000 mg / ml, preferably 1 to 100 mg / ml.

[0028] Preferably, the MRI scanning parameters include: TR (repetition time) = 200-400 ms, TE (echo time) = 3-20 ms, slice thickness = 0.2-3 mm, and field of view = 10-100 mm.

[0029] Preferably, the uniformization process includes uniformization of window width and window level.

[0030] Preferably, after injecting BSA-MnO 2 An NMR scan is performed at any time point from 5 to 90 minutes after the nanoprobe. When the NMR enhancement signal value on the right side is greater than the NMR enhancement signal value on the left side, the SLN metastasis is determined to be a SLN micrometastasis. When the NMR enhancement signal value on the right side is less than the NMR enhancement signal value on the left side, the SLN metastasis is determined to be a SLN macrometastasis.

[0031] The third object of the present invention is achieved by the following technical solutions:

[0032] A BSA-MnO 2 Application of nanoprobes in the preparation of magnetic resonance lymphangiography contrast agents for identifying the metastasis status of sentinel lymph nodes of breast cancer, the application method comprising:

[0033] 4T1 cells (mouse breast cancer cells) were injected into the right sole of the mouse, and after several days, SLN metastasis mice were obtained;

[0034] The SLN-transferred mice were anesthetized and then subjected to MRI scanning to obtain T1-weighted images before injection;

[0035] Then, BSA-MnO was injected into the left and right soles of the SLN-transferred mice. 2 Nanoprobe, after injection of BSA-MnO 2 MRI scans were performed at different time points within 5 to 200 min after nanoprobe injection to obtain T1-weighted images at different time points after injection;

[0036] The T1-weighted images before and after injection and at different time points were processed uniformly, and then the region of interest (ROI) was delineated on the SLN coronal image with the largest cross section, and the corresponding average nuclear magnetic signal value was obtained and recorded. 2 The average NMR signal values ​​at different time points after nanoprobe injection were subtracted from the values ​​of BSA-MnO injected on the same side. 2 The average NMR signal value before the nanoprobe was obtained to obtain the NMR enhancement signal value (MES) at different time points, and the maximum NMR enhancement signal value (referred to as “peak value”) was taken;

[0037] When the maximum nuclear magnetic enhancement signal value on the right side is ≥1.1*the maximum nuclear magnetic enhancement signal value on the left side, the SLN metastasis is judged to be a SLN micrometastasis; when the maximum nuclear magnetic enhancement signal value on the right side is ≤0.6*the maximum nuclear magnetic enhancement signal value on the left side, the SLN metastasis is judged to be a SLN macrometastasis.

[0038] BSA-MnO was injected into the right side 2 The average NMR signal values ​​at different time points after nanoprobe injection minus the value of BSA-MnO injection on the right side 2The average NMR signal value before the nanoprobe was obtained, and the NMR enhancement signal values ​​at different time points on the right were obtained. The maximum NMR enhancement signal value on the right was the largest NMR enhancement signal value; the left side was injected with BSA-MnO 2 The average NMR signal values ​​at different time points after nanoprobe injection minus the left side injection of BSA-MnO 2 The average NMR signal value before the nanoprobe is used to obtain the NMR enhancement signal values ​​at different time points on the left side. The largest NMR enhancement signal value is the maximum NMR enhancement signal value on the left side.

[0039] The fourth object of the present invention is achieved by the following technical solutions:

[0040] A BSA-MnO 2 Application of nanoprobes in the preparation of magnetic resonance lymphangiography contrast agents for identifying the metastasis status of sentinel lymph nodes of breast cancer, the application method comprising:

[0041] 4T1 cells (mouse breast cancer cells) were injected into the right sole of the mouse, and after several days, SLN metastasis mice were obtained;

[0042] The SLN-transferred mice were anesthetized and then subjected to MRI scanning to obtain T1-weighted images before injection;

[0043] Then, BSA-MnO was injected into the left and right soles of the SLN-transferred mice. 2 Nanoprobe, after injection of BSA-MnO 2 MRI scans were performed at different time points within 5 to 200 min after nanoprobe injection to obtain T1-weighted images at different time points after injection;

[0044] The T1-weighted images before and after injection and at different time points were processed uniformly, and then the region of interest (ROI) was delineated on the SLN coronal image with the largest cross section, and the corresponding average nuclear magnetic signal value was obtained and recorded. 2 The average NMR signal values ​​at different time points after nanoprobe injection were subtracted from the values ​​of BSA-MnO injected on the same side. 2 The average NMR signal value before the nanoprobe was obtained, and the NMR enhanced signal value (MES) at different time points was obtained. The maximum NMR enhanced signal value (referred to as "peak") was taken; and the relative lymph node clearance rate (%RLC) at 3 hours was calculated. The relevant calculation formula is as follows:

[0045] %RLC at 3 h = [(peak value - MES at 3 h) / peak value] × 100%;

[0046] When 70%≤%RLC of the right side at 3h<100%, the SLN metastasis is determined to be SLN micrometastasis; when 0<%RLC of the right side at 3h≤50%, the SLN metastasis is determined to be SLN macrometastasis.

[0047] Preferably, when the maximum nuclear magnetic enhancement signal value on the right side is ≥1.1*the maximum nuclear magnetic enhancement signal value on the left side, and 70%≤%RLC of 3h on the right side is <100%, the SLN metastasis is determined to be SLN micrometastasis; when the maximum nuclear magnetic enhancement signal value on the right side is ≤0.6*the maximum nuclear magnetic enhancement signal value on the left side, and 0<%RLC of 3h on the right side is ≤50%, the SLN metastasis is determined to be SLN macrometastasis.

[0048] Preferably, the accuracy of identifying the sentinel lymph node metastasis status of breast cancer is determined by ROC curve and / or confusion matrix.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1. Manganese is an essential trace element for the human body and has an excellent T1 relaxation rate. BSA is the most abundant protein in the blood, and its concentration in the blood is significantly higher than that in the tissue fluid. This natural concentration difference can drive BSA into the lymphatic vessels instead of the capillaries. In addition, bovine serum albumin has abundant functional groups, such as amino groups and carboxyl groups. These functional groups can interact with metal ions under certain conditions, participate in chemical reactions, and act as reducing agents. By utilizing these characteristics of bovine serum albumin, the present invention synthesizes BSA-MnO with good biocompatibility and excellent T1 relaxation rate through a simple preparation method. 2 Nanoprobes.

[0051] 2. BSA-MnO prepared by the present invention 2 The nanoprobe was injected into the body as a magnetic resonance lymphangiography contrast agent, and combined with nuclear magnetic resonance scanning technology, clear nuclear magnetic resonance structural images of the living sentinel lymph nodes and quantitative parameters related to the lymphatic drainage function, such as the nuclear magnetic enhancement signal value MES, the maximum nuclear magnetic enhancement signal value, and the relative lymph node clearance rate in 3 hours were successfully obtained. These parameters formed a sentinel lymph node metastasis assessment framework, which can be used to distinguish normal sentinel lymph nodes from breast cancer metastasis sentinel lymph nodes. More importantly, it can be used to distinguish the metastasis status of breast cancer sentinel lymph nodes: micrometastasis of breast cancer sentinel lymph nodes and large metastasis of breast cancer sentinel lymph nodes.

[0052] 3. The present invention uses machine learning to further detect BSA-MnO 2 The accuracy of nanoprobes as magnetic resonance lymphangiography contrast agents in identifying SLN metastatic status was demonstrated based on BSA-MnO 2Nanoprobe-enhanced MRL assessment has high sensitivity and accuracy in distinguishing normal sentinel lymph nodes, micrometastatic sentinel lymph nodes, and macrometastatic sentinel lymph nodes.

[0053] 4. In clinical practice, non-invasive differentiation of normal sentinel lymph nodes, micro-metastatic sentinel lymph nodes and macro-metastatic sentinel lymph nodes is of great significance and can avoid many complications. 2 The related research on enhanced MRL of nanoprobes provides a new means for non-invasive identification of sentinel lymph node metastasis status of breast cancer in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 T1-weighted MR images and H&E staining images of the mouse SLN metastasis model; Figure 1 (A) T1-weighted MR images of MicroT-SLN mice before, 10 min, 30 min, 1.5 h, and 3 h after injection of BM NPs or Gd-DTPA into the right foot. Figure 1 (B) T1-weighted MR images of Macro T-SLN mice before, 10 min, 30 min, 1.5 h, and 3 h after injection of BM NPs or Gd-DTPA into the right foot. Figure 1 (C) H&E staining images of normal sentinel lymph node (N-LN), micrometastatic SLN (Micro T-SLN) and macrometastatic SLN (Macro T-SLN).

[0055] Figure 2 The time-dependent MR enhancement signal curve, the maximum nuclear magnetic enhancement signal value and the %RLC at 3h of the mouse SLN metastasis model are shown in FIG. Figure 2 (A) is the TMRSCs curve of the right and left sides of the mouse SLN metastasis model before and after 10min, 30min, 1.5h, and 3h of BM NP injection. Figure 2 (B) is the curve of TMRSCs on the left and right sides of the right foot of the mouse SLN metastasis model before Gd-DTPA injection and 10min, 30min, 1.5h, and 3h after injection. Figure 2 (C) is the maximum NMR enhancement signal value on both sides after BM NPs were injected into the right sole of the mouse SLN metastasis model. Figure 2 (D) % RLC of the left and right sides 3 h after intraplantar injection of BM NPs in the right foot of the mouse SLN metastasis model. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0056] Figure 3 Detection of BSA-MnO for machine learning2 Flowchart, confusion matrix and ROC analysis curve of the accuracy of nanoprobes as MRL contrast agents in identifying SLN metastasis status; Figure 3 (A) Machine learning detection of BSA-MnO 2 Flow chart of the accuracy of nanoprobes as MRL contrast agents in identifying SLN metastasis status. Figure 3 (B) is the confusion matrix of three categories: N-LN, Micro T-SLN, and Macro T-SLN. Figure 3 C is the ROC analysis curve of N-LN, Micro T-SLN, and Macro T-SLN. DETAILED DESCRIPTION

[0057] The technical scheme of the present invention is further described below by specific examples and accompanying drawings. It should be understood that the specific examples described herein are only used to help understand the present invention and are not intended for specific limitations of the present invention. The accompanying drawings used herein are only for better illustrating the disclosure of the present invention and do not have a limiting effect on the scope of protection. If not otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used raw materials in the art, and the methods used in the embodiments are all conventional methods in the art.

[0058] Example 1: Preparation of bovine serum albumin-manganese dioxide nanoprobes

[0059] Weigh 250 mg of bovine serum albumin (BSA, Beijing Dingguo Biotechnology Co., Ltd.) and dissolve it in 7 mL of ultrapure water. Then weigh potassium permanganate (KMnO 4 , Sinopharm Chemical Reagent Co., Ltd.) 31.6 mg was dissolved in 3 mL of ultrapure water. The BSA aqueous solution was placed in a 37°C water bath and 3 mL of KMnO 4 The aqueous solution was slowly added dropwise to the BSA aqueous solution. During the addition process, the originally purple KMnO 4 The aqueous solution turned into a brown solution. Then, the above mixed solution was reacted for 2 hours. After the reaction, the obtained product was put into a dialysis bag (molecular weight cutoff value: 8-14kDa) and dialyzed in ultrapure water for 24 hours. Finally, the dialyzed material was freeze-dried and stored at 4°C for later use to obtain solid BSA-MnO 2 Nanoprobes.

[0060] BSA-MnO 2 The nanoprobe was dissolved in water to form 18 mg / mL BSA-MnO 2 Nanoprobe aqueous solution, wherein Mn=13.4mmol / L.

[0061] Example 2: Identification of sentinel lymph node metastasis status in breast cancer

[0062] BALB / c mice (18-20 g, 6 weeks) were purchased from Hangzhou Muhao Biotechnology Co., Ltd.

[0063] Experimental Group:

[0064] 50 μL of 4T1 cells (1х10 5 5 BALB / c mice injected with 4T1 cells for 7 days and 5 BALB / c mice injected with 4T1 cells for 3 days were selected to construct the SLN metastasis model (including 10 SLN metastasis mice). In the preliminary test, the tissue H&E staining examination preliminarily determined that the BALB / c mice injected with 4T1 cells for 7 days had large SLN metastasis of breast cancer, and the BALB / c mice injected with 4T1 cells for 3 days had small SLN metastasis of breast cancer.

[0065] The SLN transfer mice were anesthetized by an animal gas anesthesia machine, and after anesthesia, they were placed in a 3T magnetic resonance scanner (Siemens, MAGNETOM Skyra 3.0T) for T1-weighted coronal scanning to obtain T1-weighted images before injection. Subsequently, 50 μL of BSA-MnO in Example 1 was subcutaneously injected into the left and right soles of the SLN transfer model. 2 Nanoprobe (BMNPs) solution, respectively in BSA-MnO 2 Magnetic resonance imaging was performed 10 min, 30 min, 1.5 h, and 3 h after nanoprobe injection, and the scanning parameters were as follows: TR = 345 ms, TE = 13 ms, slice thickness = 1 mm, field of view = 40 mm, and T1-weighted images were obtained 10 min, 30 min, 1.5 h, and 3 h after injection.

[0066] After the imaging scan, the sentinel lymph node specimens on both sides of the mouse were collected and examined by tissue H&E staining. The H&E staining images of normal sentinel lymph nodes (N-LN), micro-metastatic SLN (Micro T-SLN) and macro-metastatic SLN (Macro T-SLN) are shown in Figure 2. Figure 1 (C) It was determined that BALB / c mice injected with 4T1 cells 7 days later had large SLN metastasis of breast cancer, and BALB / c mice injected with 4T1 cells 3 days later had small SLN metastasis of breast cancer.

[0067] The window width and window position of the T1-weighted images before injection and the T1-weighted images at 10 min, 30 min, 1.5 h, and 3 h after injection were unified (window width = 418, window position = 178). Then, the region of interest (ROI) was delineated on the coronal images of the sentinel lymph nodes with the largest cross-section at different time points, and the corresponding average nuclear magnetic signal values ​​were obtained and recorded.

[0068] BSA-MnO was injected into the right side 2 The average NMR signal value of the nanoprobe after 10min, 30min, 1.5h, and 3h minus the value of BSA-MnO injected on the right side 2 The average NMR signal value before the nanoprobe was obtained to obtain the NMR enhanced signal value MES at 10min, 30min, 1.5h, and 3h on the right side; BSA-MnO was injected on the left side 2 The average NMR signal value of the nanoprobe after 10min, 30min, 1.5h, and 3h minus the value of BSA-MnO injected on the left side 2 The average nuclear magnetic signal value before the nanoprobe was used to obtain the nuclear magnetic enhancement signal values ​​at 10min, 30min, 1.5h, and 3h on the left side. The magnetic resonance signal intensity curve (TMRESCs) that changes with time was drawn using MES. According to TMRESCs, the maximum nuclear magnetic enhancement signal value (peak) on the right and the maximum nuclear magnetic enhancement signal value on the left can be obtained. The relative lymph node clearance rate (%RLC) at 3h was calculated, and the relevant calculation formula is as follows:

[0069] %RLC at 3h on the right side=[(peak value on the right side-MES on the right side at 3h) / peak value on the right side]×100%;

[0070] %RLC at 3 h on the left side=[(peak value on the left side-MES on the left side at 3 h) / peak value on the left side]×100%.

[0071] Control group:

[0072] Six BALB / c mice were injected with 50 μL of 4T1 cells (1х10 5 cells), three BALB / c mice injected with 4T1 cells 7 days after injection and three BALB / c mice injected with 4T1 cells 3 days after injection were selected to construct the SLN metastasis model (including 6 SLN metastasis mice).

[0073] Following the same operation process as the experimental group, Gd-DTPA solution (50 μL, Gd = 13.4 mmol / L) was subcutaneously injected into the left and right soles of the SLN metastasis model. The MES of the nuclear magnetic enhancement signal value of Gd-DTPA injection for 10 min, 30 min, 1.5 h, and 3 h on the right side, and the MES of the nuclear magnetic enhancement signal value of Gd-DTPA injection for 10 min, 30 min, 1.5 h, and 3 h on the left side were obtained; the maximum nuclear magnetic enhancement signal value on the right side, the maximum nuclear magnetic enhancement signal value on the left side; the %RLC of 3 h on the right side, and the %RLC of 3 h on the left side.

[0074] Result analysis:

[0075] Figure 1 (A) T1-weighted MR images of the right foot of Micro T-SLN mice before (pre) and 10min, 30min, 1.5h, and 3h after injection of BM NPs or Gd-DTPA. The maximum coronal images of the left and right sentinel lymph nodes at each time point were merged with the dividing line (white dotted line) for easy observation and comparison. Figure 1 (B) is a T1-weighted MR image of the right foot of Macro T-SLN mice before (pre) and 10min, 30min, 1.5h, and 3h after injection of BM NPs or Gd-DTPA. As can be seen from the figure, the morphology of the right sentinel lymph node (i.e., breast cancer metastatic sentinel lymph node, T-SLN) is enlarged compared with the left non-metastatic sentinel lymph node (N-LN), and this morphological change is consistent with the MR images of the control group ( Figure 1 (A), 1(B)). In addition, MR images after BMNPs injection showed that the MR signals of the SLNs on both sides were significantly enhanced after BM NPs administration, and the borders of the lymph nodes became clearer. This signal enhancement could last for at least 3 h. However, no obvious MR signal changes were observed in mice with either micro or large SLN metastasis before and after Gd-DTPA administration ( Figure 1 A. Figure 1 B).

[0076] Time-dependent MR enhancement signal curves (TMRESCs) were plotted from T1-weighted MR images. Figure 2(A) It can be seen that in Micro T-SLN metastasis mice, the MES values ​​of the right T-SLN at different time points were stronger than those of the left N-LN, while in Macro T-SLN metastasis mice, the MES values ​​of the right T-SLN at different time points were lower than those of the left N-LN. This may be because, in the early stages of sentinel lymph node metastasis, the sentinel lymph nodes undergo tissue remodeling such as lymphangiogenesis and increased lymphatic drainage, which leads to an increase in the number of BM NPs entering micro-metastatic T-SLNs. However, as tumor cells continue to proliferate, the lymph node structure is further destroyed, which in turn reduces the number of BM NPs entering macro-metastatic T-SLNs. Because Gd-DTPA molecules are small, they quickly diffuse into the blood system and are metabolized by the kidneys. The TMRESCs of Gd-MRL show that there is no significant difference in the MR signal before and after Gd-DTPA administration, whether in micro or macro SLN metastasis mice ( Figure 2 B).

[0077] In addition, we extracted two quantitative parameters from TMRSCs, including the maximum NMR enhancement signal value (peak value) and %RLC at 3h, to more comprehensively evaluate the metastatic status of SLNs. Figure 2 C shows that the peak value of the right Micro T-SLN was significantly increased by 24.1% compared with the left N-LN (p<0.05). However, the peak value of the right Macro T-SLN decreased by 71.7% compared with the left N-LN (p<0.0001). Figure 2 D shows that %RLC at 3 h increased from 58.6% in left N-LN to 94.7% in right Micro T-SLN (p < 0.05), while it decreased to 24.4% in right Macro T-SLN (p < 0.01). The two quantitative parameters obtained can provide complementary information for the detailed evaluation of the metastatic status of SLN.

[0078] Example 3: Machine learning evaluation of the accuracy of identifying SLN metastasis status

[0079] Figure 3 (A) Machine learning detection of BSA-MnO 2Flow chart of the accuracy of nanoprobes as MRL contrast agents in identifying SLN metastasis status. First, the MR images of the sentinel lymph nodes on the left and right sides at different times before and after injection were imported into the 3DSlicer software. Taking the micro-metastatic SLN as an example, the segmentation in the 3D Slicer software was used to manually segment the ROI area of ​​the right micro-metastatic SLN at each time point, and then the image features of the ROI area were extracted by Radiomics and exported into an excel table. The above process was performed on the right large metastasis and normal SLN, and finally the three-category feature table of normal, micro- and large metastasis SLN was compiled. Then, the three-category feature table was imported into the python software to calculate the receiver operating characteristic curve (ROC) through a three-class support vector machine, and the accuracy of the diagnostic method was judged according to the area under the curve (AUC). AUC is a fitting graph drawn mainly based on the false positive rate (specificity) on the x-axis and the true positive rate (sensitivity) on the y-axis. The AUC values ​​are divided into five levels, including unsatisfactory (AUC < 0.50), poor (AUC 0.60-0.70), fair (AUC 0.70-0.80), good (AUC 0.80-0.90), and excellent (AUC 0.90-1.00). The AUC obtained by the three-classification support vector machine was 0.999, which indicates that the enhanced MRL based on BM NPs has a very high accuracy in distinguishing the metastatic status of SLN ( Figure 3 C).

[0080] On the other hand, the accuracy of the diagnostic method was further verified by the confusion matrix visualization, and the recognition accuracy of the confusion matrix was as high as 98.3% ( Figure 3 (the average of 100, 95, and 100 in B).

[0081] Combined with machine learning, BM NPs-based enhanced MRL showed high sensitivity and accuracy in distinguishing N-LNs, micrometastatic T-SLNs, and macrometastatic T-SLNs.

[0082] The various aspects, embodiments, and features of the present invention should be considered to be illustrative in all aspects and not limiting of the present invention, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0083] In the preparation method of the present invention, the order of each step is not limited to the order listed. For those skilled in the art, without creative work, the order of each step is also within the protection scope of the present invention. In addition, two or more steps or actions can be performed simultaneously.

[0084] Finally, it should be noted that the specific embodiments described herein are merely examples of the present invention, and are not intended to limit the implementation methods of the present invention. A person skilled in the art of the present invention may make various modifications or supplements to the specific embodiments described, or replace them in a similar manner. It is not necessary and impossible to provide all examples of all implementation methods here. However, these obvious changes or modifications derived from the essential spirit of the present invention still fall within the scope of protection of the present invention, and interpreting them as any additional limitation is contrary to the spirit of the present invention.

Claims

1. A BSA-MnO2 nanoprobe for identifying the metastasis status of sentinel lymph nodes in breast cancer, characterized in that: The BSA-MnO2 nanoprobe is obtained by reacting bovine serum albumin with potassium permanganate; the BSA-MnO2 nanoprobe is used as a magnetic resonance lymphangiography contrast agent to identify the metastasis status of breast cancer sentinel lymph nodes, and the metastasis status of breast cancer sentinel lymph nodes includes micrometastasis of breast cancer sentinel lymph nodes and macrometastasis of breast cancer sentinel lymph nodes.

2. The BSA-MnO2 nanoprobe according to claim 1, characterized in that: The preparation method of the BSA-MnO2 nanoprobe comprises the following steps: placing a bovine serum albumin aqueous solution in a water bath at 25 to 50° C., then dropping a potassium permanganate aqueous solution, maintaining the temperature for 1 to 10 hours, dialyzing, and drying to obtain a BSA-MnO2 nanoprobe; And / or, in the BSA-MnO2 nanoprobe, the mass fraction of MnO2 is 1 to 20 wt%.

3. Use of the BSA-MnO2 nanoprobe as claimed in claim 1 in the preparation of a magnetic resonance lymphangiography contrast agent for identifying the metastatic status of sentinel lymph nodes in breast cancer.

4. The use according to claim 3, characterized in that: The application method comprises: 4T1 cells were injected into the right sole of the mouse, and SLN-transferred mice were obtained several days later; The SLN-transferred mice were anesthetized and then subjected to MRI scanning to obtain T1-weighted images before injection; Then, BSA-MnO2 nanoprobes were injected into the left and right soles of the SLN-transferred mice, and MRI scans were performed 5 to 200 minutes after injection to obtain T1-weighted images after injection; The T1-weighted images before and after the injection were processed for consistency, and then the region of interest was delineated on the sentinel lymph node coronal image with the largest cross section to obtain and record the corresponding average nuclear magnetic signal value; the average nuclear magnetic signal value after the injection of the BSA-MnO2 nanoprobe was subtracted from the average nuclear magnetic signal value before the injection of the BSA-MnO2 nanoprobe on the same side to obtain the nuclear magnetic enhancement signal value; When the nuclear magnetic enhancement signal value on the right side is greater than that on the left side, the SLN metastasis is determined to be a SLN micrometastasis. When the nuclear magnetic enhancement signal value on the right side is less than that on the left side, the SLN metastasis is determined to be a SLN macrometastasis.

5. The use according to claim 3, characterized in that: The application method comprises: 4T1 cells were injected into the right sole of the mouse, and SLN-transferred mice were obtained several days later; The SLN-transferred mice were anesthetized and then subjected to MRI scanning to obtain T1-weighted images before injection; Then, BSA-MnO2 nanoprobes were injected into the left and right soles of the SLN-transferred mice, and MRI scans were performed at different time points within 5 to 200 minutes after the injection of BSA-MnO2 nanoprobes to obtain T1-weighted images at different time points after the injection; The T1-weighted images before and after injection and at different time points were processed for consistency, and then the region of interest (ROI) was delineated on the sentinel lymph node coronal image of the largest cross section to obtain and record the corresponding average nuclear magnetic signal value; the average nuclear magnetic signal value at different time points after the injection of the BSA-MnO2 nanoprobe was subtracted from the average nuclear magnetic signal value before the injection of the BSA-MnO2 nanoprobe on the same side to obtain the nuclear magnetic enhancement signal value at different time points, and the maximum nuclear magnetic enhancement signal value was taken; When the maximum nuclear magnetic enhancement signal value on the right side is ≥1.1*the maximum nuclear magnetic enhancement signal value on the left side, the SLN metastasis is judged to be a SLN micrometastasis; when the maximum nuclear magnetic enhancement signal value on the right side is ≤0.6*the maximum nuclear magnetic enhancement signal value on the left side, the SLN metastasis is judged to be a SLN macrometastasis.

6. The use according to claim 3, characterized in that: The application method comprises: 4T1 cells were injected into the right sole of the mouse, and SLN-transferred mice were obtained several days later; The SLN-transferred mice were anesthetized and then subjected to MRI scanning to obtain T1-weighted images before injection; Then, BSA-MnO2 nanoprobes were injected into the left and right soles of the SLN-transferred mice, and MRI scans were performed at different time points within 5 to 200 minutes after the injection of BSA-MnO2 nanoprobes to obtain T1-weighted images at different time points after the injection; The T1-weighted images before and after injection and at different time points were processed in a consistent manner, and then the region of interest (ROI) was delineated on the sentinel lymph node coronal image of the largest cross section to obtain and record the corresponding average nuclear magnetic signal value; the average nuclear magnetic signal value at different time points after injection of the BSA-MnO2 nanoprobe was subtracted from the average nuclear magnetic signal value before injection of the BSA-MnO2 nanoprobe on the same side to obtain the nuclear magnetic enhancement signal value at different time points, and the maximum nuclear magnetic enhancement signal value was taken; and the relative lymph node clearance rate at 3 hours was calculated, and the calculation formula was as follows: Relative lymph node clearance rate at 3 hours = [(maximum nuclear magnetic enhancement signal value - nuclear magnetic enhancement signal value at 3 hours) / maximum nuclear magnetic enhancement signal value] × 100%; When 70%≤the relative lymph node clearance rate of the right side within 3 hours is <100%, the SLN metastasis is determined to be SLN micrometastasis; when 0<the relative lymph node clearance rate of the right side within 3 hours is ≤50%, the SLN metastasis is determined to be SLN macrometastasis.

7. The use according to claim 6, characterized in that: When the maximum nuclear magnetic enhancement signal value on the right side is ≥1.1*the maximum nuclear magnetic enhancement signal value on the left side, and 70%≤the relative lymph node clearance rate of the right side within 3 hours is <100%, the SLN metastasis is judged as SLN micrometastasis; when the maximum nuclear magnetic enhancement signal value on the right side is ≤0.6*the maximum nuclear magnetic enhancement signal value on the left side, and 0<the relative lymph node clearance rate of the right side within 3 hours is ≤50%, the SLN metastasis is judged as SLN macrometastasis.

8. The use according to any one of claims 4 to 6, characterized in that: 0.1-1000 μg BSA-MnO2 nanoprobes / g mouse were injected into each sole of the foot, and the same amount of BSA-MnO2 nanoprobes were injected into each sole of the foot.

9. The use according to any one of claims 4 to 6, characterized in that: MRI scanning parameters include: TR = 200-400 ms, TE = 3-20 ms, slice thickness = 0.2-3 mm, field of view = 10-100 mm.

10. The use according to any one of claims 4 to 6, characterized in that: The accuracy of identifying sentinel lymph node metastasis status of breast cancer was determined by ROC curve and / or confusion matrix.

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