A magnetic resonance NIR-II fluorescence imaging dual-mode nanoprobes and a preparation method and application thereof

Abstract

This invention belongs to the field of biomedical nanomaterials technology, and provides a magnetic resonance / NIR-II fluorescence imaging dual-modal nanoprobe, its preparation method, and its application. The magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe of this invention comprises amino- or thiol-modified mesoporous silica nanoparticles, an NIR-II fluorescent dye linked to the mesoporous silica nanoparticles, bovine serum albumin linked to the NIR-II fluorescent dye, and a gadolinium-based magnetic resonance contrast agent linked to the bovine serum albumin. This invention uses bovine serum albumin as a "molecular bridge," utilizing various intermolecular forces to stably and firmly bind the NIR-II fluorescent dye and gadolinium-based magnetic resonance contrast agent to the mesoporous silica nanoparticles, forming a highly stable and biocompatible MRI / NIR-II fluorescence dual-modal imaging nanoprobe. This improves the contrast agent loading rate and stability, and avoids the problem of contrast agent leakage during in vivo circulation.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, and in particular to a magnetic resonance NIR-II fluorescence imaging dual-modal nanoprobe, its preparation method, and its application. Background Technology

[0002] Precision diagnosis and treatment of tumors, especially the early detection, accurate localization, and effective assessment of lymph node metastases, are crucial for improving patient survival rates. Medical imaging technology plays an irreplaceable role in this clinical process. Among them, magnetic resonance imaging (MRI) and near-infrared-II (NIR-II) fluorescence imaging are two highly promising techniques.

[0003] MRI imaging boasts significant advantages, including being radiation-free and offering extremely high soft tissue resolution. It provides detailed anatomical information, making it ideal for preoperative surgical planning and clearly demonstrating the relationship between the tumor and surrounding tissues and blood vessels. However, MRI imaging is relatively slow, hindering true real-time dynamic monitoring. In contrast, NIR-II fluorescence imaging offers high sensitivity, real-time performance, is radiation-free, and has good tissue penetration depth, making it highly promising for intraoperative surgical navigation. It helps surgeons distinguish tumor boundaries from normal tissue in real time, ensuring complete tumor resection and maximizing the preservation of vital functional tissues. Furthermore, early assessment of postoperative treatment outcomes is crucial, requiring an imaging method capable of dynamically and sensitively monitoring biological changes in the lesion area (such as apoptosis and altered metabolic activity). This is precisely where the high sensitivity of NIR-II imaging excels. Therefore, combining the high resolution of MRI imaging with the high sensitivity and real-time performance of NIR-II fluorescence imaging to construct a dual-modal imaging nanoprobe is an ideal strategy for achieving an integrated precision medicine solution encompassing precise preoperative localization, real-time intraoperative navigation, and timely postoperative assessment.

[0004] While the MRI / NIR-II fluorescence dual-modal imaging concept offers significant advantages, its technical implementation faces substantial challenges. The core difficulty lies in how to efficiently and stably integrate two contrast agents (MRI contrast agent and NIR-II fluorescent dye) onto a single nanocarrier. Existing strategies often employ simple physical embedding or stepwise chemical coupling, which can easily lead to problems such as low contrast agent loading, poor stability, and leakage during in vivo circulation. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe, its preparation method, and its application. This invention uses bovine serum albumin (BSA) as a "molecular bridge" and utilizes various intermolecular forces (covalent bonds, hydrogen bonds, and hydrophobic interactions) to stably and firmly bind NIR-II fluorescent dyes and gadolinium-based magnetic resonance contrast agents onto mesoporous silica nanoparticles, forming a highly stable and biocompatible MRI / NIR-II fluorescence dual-modal imaging nanoprobe. This improves the contrast agent loading rate and stability, avoids the problem of "leakage" of the contrast agent during in vivo circulation, and meets the key needs of precise diagnosis and treatment of tumors (especially metastatic lymph nodes) throughout the preoperative, intraoperative, and postoperative stages.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe, comprising amino or thiol-modified mesoporous silica nanoparticles, an NIR-II fluorescent dye connected to the mesoporous silica nanoparticles, bovine serum albumin connected to the NIR-II fluorescent dye, and a gadolinium-based magnetic resonance contrast agent connected to the bovine serum albumin.

[0007] Preferably, the NIR-II fluorescent dye includes IR820, which has the following structure: .

[0008] Preferably, the gadolinium-based magnetic resonance contrast agent is GdL, and the GdL has the following structure: .

[0009] Preferably, the diameter of the mesoporous silicon nanoparticles is 30~200nm.

[0010] This invention also provides a method for preparing the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe described in the above technical solution, comprising the following steps: Provides amino- or thiol-modified mesoporous silica nanoparticles; The amino or thiol-modified mesoporous silica nanoparticles were covalently coupled with NIR-II fluorescent dye to obtain dye-modified mesoporous silica nanoparticles. The dye-modified mesoporous silica nanoparticles were mixed with bovine serum albumin and water for coating to obtain bovine serum albumin-coated composite nanoparticles. The bovine serum albumin-coated composite nanoparticles were mixed with a gadolinium-based magnetic resonance contrast agent and anchored to obtain the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe.

[0011] Preferably, the mass ratio of the amino or thiol-modified mesoporous silica nanoparticles to the NIR-II fluorescent dye is 3~10:1; The covalent coupling reaction is carried out at a temperature of 70-90°C for 2-10 hours.

[0012] Preferably, the mass ratio of the dye-modified mesoporous silica nanoparticles to bovine serum albumin is 0.5~10:1, the coating temperature is 20~40℃, and the coating time is 6~12h.

[0013] Preferably, the mass ratio of the bovine serum albumin-coated composite nanoparticles to the gadolinium-based magnetic resonance contrast agent is 10~100:1; The anchoring temperature is 20~40℃, and the time is 6~12h.

[0014] This invention also provides the application of the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe described in the above technical solution in the preparation of imaging formulations.

[0015] Preferably, the imaging agent is used for the diagnosis of metastatic lymph nodes in tumors.

[0016] This invention provides a magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe.

[0017] Compared with the prior art, the present invention has the following significant advantages: 1. Innovative integration strategy: This invention is the first to propose and realize modular assembly using BSA protein with excellent biocompatibility as a "molecular bridge" and ingeniously utilizing various intermolecular forces (covalent bonds, hydrogen bonds, hydrophobic interactions); it can effectively prevent leakage of contrast agents during in vivo circulation and ensure the stability and accuracy of imaging signals.

[0018] 2. Superior Dual-Modal Imaging Performance: Highly Efficient MRI Contrast: Thanks to the high-concentration loading of local gadolinium-based magnetic resonance contrast agent mediated by BSA, the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe of this invention exhibits significantly enhanced T1-weighted contrast effects, with a relaxation efficiency (r1) far exceeding that of clinically commonly used small-molecule gadolinium contrast agents; Bright NIR-II Fluorescence: The BSA coating effectively avoids the aggregation quenching effect of NIR-II fluorescent dyes, allowing them to maintain strong fluorescence emission in the NIR-II window, achieving high signal-to-noise ratio fluorescence imaging.

[0019] 3. Ideal in vivo diagnostic and therapeutic properties: The BSA protein crown on the magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe endows the probe with good water solubility and biocompatibility, prolongs blood circulation time, and promotes efficient targeting and retention in metastatic lymph nodes.

[0020] 4. Synergistic Clinical Application Value: The probe of this invention successfully combines the high spatial resolution of MRI with the high spatiotemporal resolution of NIR-II imaging. In application, preoperative MRI provides clear anatomical structures and accurately locates metastatic lesions; intraoperative NIR-II imaging can guide surgeons to precisely remove tumors in real time and dynamically; and postoperative NIR-II imaging can dynamically monitor treatment effects. The synergy of these two technologies provides a powerful tool for the precise diagnosis and treatment of tumor lymph node metastasis throughout the entire process.

[0021] Furthermore, the gadolinium-based magnetic resonance contrast agent is GdL. In this invention, the high relaxation efficiency of GdL itself enables the magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe to exhibit significantly enhanced T1-weighted imaging effects, with a relaxation efficiency (r1) far exceeding that of clinically commonly used small-molecule gadolinium contrast agents.

[0022] Furthermore, the mesoporous silica nanoparticles have a diameter of 30-100 nm and a pore size of 2-3 nm. The mesoporous silica nanoparticles in the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe of the present invention have a diameter of 30-100 nm, a size suitable for drainage through the lymphatic system and accumulation in lymph nodes.

[0023] In vitro and in vivo experimental results demonstrate that the magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe provided by this invention not only possesses highly efficient magnetic resonance T1-weighted imaging performance (high relaxation efficiency) but also emits a strong NIR-II fluorescence signal. In a tumor metastatic lymph node model, this magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe can be efficiently enriched, achieving clear anatomical localization of lesions via magnetic resonance imaging and real-time dynamic monitoring of the drainage process via NIR-II imaging. The two work synergistically and complementaryly, significantly improving the accuracy and reliability of diagnosis. This invention provides a novel, efficient, and safe dual-modality imaging nanoreagent for the precise diagnosis of tumor lymph node metastasis, possessing significant clinical translational value.

[0024] The present invention also provides a method for preparing the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe described in the above technical solution. The present invention uses the biocompatible BSA protein as a "molecular bridge" and cleverly utilizes a variety of intermolecular forces (covalent bonds, hydrogen bonds, hydrophobic interactions) for modular assembly; it avoids cumbersome chemical modifications and has a simple process with good reproducibility. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the principle of fabricating magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes according to the present invention; Figure 2 Transmission electron microscope (TEM) images of mesoporous silicon materials with different particle sizes prepared for this invention; Figure 3 The image shows a transmission electron microscope (TEM) image of the MSN-NH2-IR820-BSA-GdL obtained in Example 1. Figure 4 The image shows a transmission electron microscope (TEM) image of the MSN-SH-IR820-BSA-GdL obtained in Example 2. Figure 5 This is a hydration particle size distribution diagram of different materials in Example 1; Figure 6 This is a hydration particle size distribution diagram of different materials in Example 2; Figure 7 Zeta potential diagrams for different materials in Examples 1 and 2; Figure 8 The ultraviolet-visible-near-infrared absorption spectra of MSN-NH2-IR820-BSA-GdL and MSN-SH-IR820-BSA-GdL; Figure 9 The NIR-II fluorescence emission spectra of MSN-NH2-IR820-BSA-GdL and MSN-SH-IR820-BSA-GdL are shown. Figure 10 MRI images of GdL, MSN-NH2-IR820-BSA-GdL, and MSN-SH-IR820-BSA-GdL at different concentrations; Figure 11 In vitro magnetic resonance T1 relaxation efficiency assessment graphs for MSN-NH2-IR820-BSA-GdL and MSN-SH-IR820-BSA-GdL; Figure 12 NIR-II fluorescence images of MSN-NH2-IR820-BSA-GdL and MSN-SH-IR820-BSA-GdL at different concentrations; Figure 13 The effect of different filters on NIR-II imaging of MSN-NH2-IR820-BSA-GdL and MSN-SH-IR820-BSA-GdL is shown in the figure. Figure 14 Comparison of signal-to-noise ratios of different magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes under different filter conditions; Figure 15 A schematic diagram of the tissue penetration depth model; Figure 16 NIR-II fluorescence imaging of capillaries at different depths; Figure 17 for Figure 16 Signal-to-noise ratio and length statistics of medium capillaries; Figure 18 Analysis chart of capillary scribing intensity at different depths (0-5mm); Figure 19 Cell survival rate at different concentrations of dual-modal imaging nanoprobes; Figure 20 Images of mixed dual-modal imaging nanoprobes and mouse erythrocytes at different concentrations; Figure 21 The ultraviolet absorption spectra of the mixture of dual-modal imaging nanoprobes at different concentrations and mouse erythrocytes; Figure 22 The hemolysis rate of mouse erythrocytes by dual-modal imaging nanoprobes at different concentrations; Figure 23 A schematic diagram illustrating the establishment of a subcutaneous tumor model of colorectal cancer on the back of CT26 mice; Figure 24 NIR-II fluorescence imaging of mouse dorsal tumors at different time points; Figure 25 Fluorescence statistics of mouse dorsal tumors at different time points; Figure 26 A schematic diagram illustrating fluorescent surgical navigation for the removal of a tumor on the back of a mouse. Figure 27 NIR-II fluorescence imaging at different time points (0-24h) in a mouse lymph node metastasis model; Figure 28 Statistical analysis of fluorescence signals in metastatic lymph nodes at different time points; Figure 29 This is a schematic diagram illustrating the use of a dual-modal imaging nanoprobe for surgical navigation in a tumor-bearing mouse model of lymph node metastases. Figure 30 This is a T1-weighted magnetic resonance imaging (MRI) image of a tumor-bearing mouse with a dual-modal imaging nanoprobe. Figure 31 The results of NIR-II fluorescence imaging and MRI imaging of mice with lymph node metastases prepared from MSN-SH-IR820-BSA-Gd mesoporous silica particles of different particle sizes in Example 1 are shown. Detailed Implementation

[0026] This invention provides a magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe, comprising amino or thiol-modified mesoporous silica nanoparticles, an NIR-II fluorescent dye connected to the mesoporous silica nanoparticles, bovine serum albumin connected to the NIR-II fluorescent dye, and a gadolinium-based magnetic resonance contrast agent connected to the bovine serum albumin.

[0027] The magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe provided by this invention comprises mesoporous silica nanoparticles modified with amino or thiol groups. In this invention, the diameter of the mesoporous silica nanoparticles is preferably 30-200 nm, more preferably 50-100 nm; the pore size is preferably 2-3 nm. Controlling the diameter of the mesoporous silica nanoparticles to 30-200 nm in this invention can achieve efficient targeted enrichment of lymph nodes.

[0028] The magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe provided by this invention comprises an NIR-II fluorescent dye connected to the mesoporous silicon nanoparticles. In this invention, the NIR-II fluorescent dye preferably comprises IR820, and IR820 preferably has the following structure: .

[0029] In this invention, the NIR-II fluorescent dye and the amino or thiol-modified mesoporous silica nanoparticles are preferably connected by covalent bonds.

[0030] The magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe provided by this invention comprises bovine serum albumin (BSA) linked to the NIR-II fluorescent dye. In this invention, the BSA and the NIR-II fluorescent dye are preferably linked via hydrogen bonds and hydrophobic interactions.

[0031] The magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe provided by this invention comprises a gadolinium-based magnetic resonance contrast agent linked to bovine serum albumin. In this invention, the gadolinium-based magnetic resonance contrast agent is preferably GdL, and the GdL preferably has the following structure: .

[0032] In this invention, the BSA and the gadolinium-based magnetic resonance contrast agent are preferably linked by hydrogen bonds. Compared to clinically commonly used small-molecule gadolinium reagents, this invention uses GdL as a gadolinium-based magnetic resonance contrast agent, which exhibits higher relaxation efficiency and hydration stability, laying a solid foundation for constructing high-performance MRI imaging modules.

[0033] The magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe provided by this invention uses amino- or thiol-modified mesoporous silica nanoparticles (MSNs) as the core carrier and bovine serum albumin (BSA) as a multifunctional molecular bridge to sequentially and stably integrate NIR-II fluorescent dyes and gadolinium-based magnetic resonance contrast agents onto the core carrier, forming a magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe with a "core-shell" structure. This invention uses bovine serum albumin (BSA) as a "molecular bridge" and utilizes various intermolecular forces (covalent bonds, hydrogen bonds, and hydrophobic interactions) to stably and firmly bind NIR-II fluorescent dyes and gadolinium-based magnetic resonance contrast agents onto the mesoporous silica nanoparticles, forming a highly stable, highly biocompatible, and highly efficient MRI / NIR-II fluorescence dual-modality imaging nanoprobe. This achieves complementarity between high-resolution anatomical imaging and high-sensitivity real-time dynamic imaging, meeting the key needs of precise diagnosis and treatment of tumors (especially metastatic lymph nodes) throughout the preoperative, intraoperative, and postoperative stages.

[0034] This invention also provides a method for preparing the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe described in the above technical solution, comprising the following steps: Provides amino- or thiol-modified mesoporous silica nanoparticles; The amino or thiol-modified mesoporous silica nanoparticles were covalently coupled with NIR-II fluorescent dye to obtain dye-modified mesoporous silica nanoparticles. The dye-modified mesoporous silica nanoparticles were mixed with bovine serum albumin and water for coating to obtain bovine serum albumin-coated composite nanoparticles. The bovine serum albumin-coated composite nanoparticles were mixed with a gadolinium-based magnetic resonance contrast agent and anchored to obtain the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe.

[0035] This invention provides mesoporous silica nanoparticles modified with amino or thiol groups.

[0036] In this invention, the preparation method of the amino-modified mesoporous silica nanoparticles (denoted as MSN-NH2) preferably includes the following steps: Hexadecyltrimethylammonium bromide (CTAB) was dispersed in water to obtain a surfactant solution; The surfactant solution, tetraethyl orthosilicate, and triethanolamine (TEA) were mixed and hydrolyzed to obtain mesoporous silica nanoparticles. The mesoporous silica nanoparticles, 3-aminopropyltriethoxysilane (APTES), and toluene were mixed and modified to obtain the amino-modified mesoporous silica nanoparticles.

[0037] This invention disperses hexadecyltrimethylammonium bromide (CTAB) in water to obtain a surfactant solution. In this invention, the preferred ratio of CTAB to water is 950 mg:50-100 mL, more preferably 950 mg:60 mL. The preferred dispersion temperature is 70-90°C, more preferably 80°C. The dispersion time is not specifically limited, as long as it allows CTAB to dissolve completely. Preferably, the surfactant solution also includes sodium salicylate (NaSal); the preferred mass ratio of sodium salicylate to CTAB is 0-300:950, specifically preferably 50:950, 100:950, 200:950, 250:950, or 300:950. The addition of sodium salicylate is to adjust the particle size of the mesoporous silica nanoparticles.

[0038] After obtaining the surfactant solution, the present invention mixes the surfactant solution, tetraethyl orthosilicate, and TEA, and hydrolyzes them to obtain mesoporous silica nanoparticles. In the present invention, the molar ratio of hexadecyltrimethylammonium bromide to tetraethyl orthosilicate is preferably 0.065:1; the molar ratio of tetraethyl orthosilicate to TEA is preferably 40:1. In the present invention, the hydrolysis temperature is preferably 70-90℃, more preferably 80℃; the time is preferably 6-12 hours, specifically 12 hours; the hydrolysis is preferably carried out under stirring conditions, and the stirring speed is preferably 500-1000 rpm, more preferably 700 rpm. After hydrolysis, the present invention preferably further includes: centrifuging and washing the obtained hydrolysate, collecting the suspension; mixing the suspension, ethanol, and concentrated hydrochloric acid, reacting, and then washing and drying the resulting reaction solution sequentially to obtain the mesoporous silica nanoparticles. In the present invention, the centrifugation speed is preferably 11000 rpm, the time is preferably 10 minutes, and the reagent used for centrifugation and washing is preferably ethanol. In this invention, the reaction temperature is preferably 60°C, the reaction time is preferably 3 hours, and the reaction is preferably carried out under oil bath conditions. In this invention, the washing preferably includes sequential water washing and ethanol washing; the drying is preferably freeze drying.

[0039] After obtaining mesoporous silica nanoparticles, the present invention mixes the mesoporous silica nanoparticles, 3-aminopropyltriethoxysilane (APTES), and toluene for modification to obtain the amino-modified mesoporous silica nanoparticles. In the present invention, the preferred ratio of the mesoporous silica nanoparticles to 3-aminopropyltriethoxysilane is 300 mg: 0.2-0.4 mL, more preferably 300 mg: 0.3 mL; the preferred ratio of the mesoporous silica nanoparticles to toluene is 300 mg: 20-40 mL, more preferably 300 mg: 30 mL. In the present invention, mixing the mesoporous silica nanoparticles, 3-aminopropyltriethoxysilane (APTES), and toluene preferably includes the following steps: dispersing the mesoporous silica nanoparticles in toluene and adding 3-aminopropyltriethoxysilane. In the present invention, the dispersion is preferably carried out under ultrasonic conditions. In this invention, the modification temperature is preferably 80-90℃, more preferably 85℃, and the modification time is preferably 12-24h, more preferably 18h. The modification is preferably carried out under stirring conditions, and the stirring speed is preferably 400-600rpm, more preferably 500rpm. After the modification is completed, the invention preferably further includes: centrifuging the obtained modified liquid to collect the precipitate; washing and drying the precipitate sequentially to obtain the amino-modified mesoporous silica nanoparticles. In this invention, the centrifugation speed is preferably 11000rpm, and the centrifugation time is preferably 10min; the washing preferably includes sequential water washing and ethanol washing. In this invention, the drying is preferably vacuum drying.

[0040] In this invention, the method for preparing the thiol-modified mesoporous silica nanoparticles is preferably the same as the method for preparing the amino-modified mesoporous silica nanoparticles described in the above technical solution, except that 3-aminopropyltriethoxysilane (APTES) is replaced with 3-mercaptopropyltrimethoxysilane (MPTMS).

[0041] After providing amino- or thiol-modified mesoporous silica nanoparticles, this invention involves covalently coupling the amino- or thiol-modified mesoporous silica nanoparticles with an NIR-II fluorescent dye to obtain dye-modified mesoporous silica nanoparticles. In this invention, the raw materials for the covalent coupling reaction preferably further include triethylamine and a polar solvent. In this invention, the mass ratio of the amino- or thiol-modified mesoporous silica nanoparticles to the NIR-II fluorescent dye is preferably 3~10:1, more preferably 4~6:1, and specifically preferably 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In this invention, the molar ratio of the amino- or thiol-modified mesoporous silica nanoparticles to triethylamine is preferably 300 mg: 10~50 μL, more preferably 300 mg: 32 μL. In this invention, the polar solvent preferably includes DMF; the ratio of the amino- or thiol-modified mesoporous silica nanoparticles to the polar solvent is preferably 300 mg: 10-30 mL, more preferably 300 mg: 20 mL. In this invention, the temperature of the covalent coupling reaction is preferably 70-90°C, more preferably 80°C; the time is preferably 2-10 h, more preferably 3 h; the covalent coupling reaction is preferably carried out under stirring conditions, the stirring speed is preferably 500-700 rpm, more preferably 600 rpm; the covalent coupling reaction is preferably carried out under sealed, light-protected conditions. In this invention, the covalent coupling reaction of the amino- or thiol-modified mesoporous silica nanoparticles with the NIR-II fluorescent dye is specifically preferably carried out by mixing the amino- or thiol-modified mesoporous silica nanoparticles, the NIR-II fluorescent dye, triethylamine, and the polar solvent to carry out the covalent coupling reaction. After the covalent coupling reaction is completed, the present invention preferably further includes: centrifuging the obtained covalent coupling reaction solution to collect the solid; washing and freeze-drying the solid sequentially to obtain the dye-modified mesoporous silica nanoparticles. In the present invention, the centrifugation speed is preferably 11000 rpm and the time is preferably 10 min; the washing preferably includes washing with pure water and washing with ethanol sequentially; the present invention does not specifically limit the number of times the pure water washing and ethanol washing are performed, as long as the supernatant is colorless.

[0042] After obtaining dye-modified mesoporous silica nanoparticles, the present invention mixes the dye-modified mesoporous silica nanoparticles with bovine serum albumin and water for coating, thereby obtaining bovine serum albumin-coated composite nanoparticles. In the present invention, the mass ratio of the dye-modified mesoporous silica nanoparticles to bovine serum albumin is preferably 0.5~10:1, more preferably 1~5:1, and specifically preferably 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In the present invention, the volume ratio of the dye-modified mesoporous silica nanoparticles to water is preferably 100mg:5~15mL, more preferably 100mg:10mL. In the present invention, mixing the dye-modified mesoporous silica nanoparticles with bovine serum albumin and water preferably includes the following steps: ultrasonically dispersing the dye-modified mesoporous silica nanoparticles in water, followed by adding bovine serum albumin. In this invention, the coating temperature is preferably 20-40°C, more preferably room temperature, i.e., no additional heating or cooling is required; the coating time is preferably 6-12 hours, more preferably 12 hours; the coating is preferably carried out under stirring conditions, and the stirring speed is preferably 600-800 rpm, more preferably 700 rpm. After the coating is completed, the invention preferably further includes: centrifuging the obtained coating liquid to obtain a solid; washing and dispersing the solid sequentially to obtain the bovine serum albumin-coated composite nanoparticles. In this invention, the centrifugation speed is preferably 11000 rpm, and the centrifugation time is preferably 10 minutes; the washing reagent is preferably pure water; the dispersion reagent is preferably water. In this invention, the bovine serum albumin-coated composite nanoparticles preferably exist in the form of a bovine serum albumin-coated composite nanoparticle dispersion; the concentration of the bovine serum albumin-coated composite nanoparticle dispersion is preferably 10-20 mg / mL.

[0043] After obtaining bovine serum albumin-coated composite nanoparticles, this invention mixes the bovine serum albumin-coated composite nanoparticles with a gadolinium-based magnetic resonance contrast agent and anchors them to obtain the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe. In this invention, the mass ratio of the bovine serum albumin-coated composite nanoparticles to the gadolinium-based magnetic resonance contrast agent is preferably 10~100:1, specifically preferably 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1. In this invention, the anchoring temperature is preferably 20~40℃, more preferably room temperature; the time is preferably 6~12h, more preferably 12h; the anchoring is preferably carried out under stirring conditions. After the anchoring is completed, this invention preferably further includes: centrifuging the obtained anchoring reaction solution, and freeze-drying the obtained precipitate to obtain the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe. In this invention, the centrifugation speed is preferably 11,000 rpm and the centrifugation time is preferably 10 min.

[0044] Figure 1 This is a schematic diagram illustrating the principle of preparing magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes according to the present invention.

[0045] This invention also provides the application of the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe described in the above-described technical solution in the preparation of imaging formulations. In this invention, the imaging formulation is preferably used for the diagnosis of metastatic lymph nodes in tumors. In this invention, the imaging formulation is preferably used for magnetic resonance T1-weighted imaging and / or NIR-II fluorescence imaging. In this invention, the tumor is preferably a colorectal tumor.

[0046] The following detailed description, in conjunction with embodiments, illustrates the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe, its preparation method, and its applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0047] Example 1 Preparation of a composite nanomaterial for magnetic resonance / NIR-II fluorescence dual-modal imaging: (1) Preparation of mesoporous silica nanoparticles with different particle sizes. Hexadecyltrimethylammonium bromide (CTAB, 950 mg, 2.6 mmol) and sodium salicylate (0, 100 mg, 200 mg, 300 mg, or 400 mg) were added to 60 mL of water and stirred in an oil bath at 80 °C until completely dissolved. Triethanolamine (150 mg, 1 mmol) and tetraethyl orthosilicate (TEOS, 9 mL, 40 mmol) were added to the solution, and the mixture was reacted at 80 °C and 700 rpm for 12 h. The solid was collected by centrifugation (11000 rpm, 10 min), and washed with ethanol and concentrated hydrochloric acid or calcined at high temperature to obtain mesoporous silica nanoparticles with different particle sizes (diameters of 50 nm, 70 nm, 120 nm, 200 nm, 260 nm, and 300 nm, respectively). The diameter of the mesoporous silica nanoparticles was directly proportional to the content of sodium salicylate; that is, the more sodium salicylate, the larger the particle size.

[0048] Transmission electron microscopy (TEM) was performed on mesoporous silica nanoparticles of different sizes, and the results are as follows: Figure 2 As shown, Figure 2 In the image, the images from top to bottom and from left to right correspond to TEM images with diameters of 50nm, 70nm, 120nm, 200nm, 260nm, and 300nm, respectively. As the sodium salicylate content increases during the preparation process, the diameter of the mesoporous silica nanoparticles gradually increases, showing a positive correlation.

[0049] (2) Preparation of amino- or thiol-modified mesoporous silica nanoparticles. 300 mg of the above mesoporous silica nanoparticles were mixed with 30 mL of toluene, and 0.3 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was reacted at 85 °C and 500 rpm for 18 h. After centrifugation (11000 rpm, 10 min), the precipitate was collected, washed multiple times with water and ethanol, and then freeze-dried to obtain amino-modified mesoporous silica nanoparticles, denoted as MSN-NH2. Similarly, for thiol-modified mesoporous silica nanomaterials, simply replace 3-aminopropyltriethoxysilane (APTES) in the above steps with 3-mercaptopropyltrimethoxysilane (MPTMS) to obtain thiol-modified mesoporous silica nanomaterials (MSN-SH).

[0050] (3) Preparation of mesoporous silica material modified with near-infrared dye IR820. Take 300 mg of amino or thiol-modified mesoporous silica nanoparticles obtained in step (2), 50 mg of NIR-II fluorescent dye IR820, 20 mL of DMF, and 32 μL of triethylamine and react them at 80 °C and 600 rpm for 3 hours. The process is closed and protected from light. The final product is centrifuged at high speed (11000 rpm, 10 minutes) to collect the solid product. The solid product is washed multiple times with pure water and ethanol until the supernatant is colorless to remove residual reactants. The product is freeze-dried to obtain mesoporous silica nanoparticles modified with dye IR820, which are denoted as MSN-NH2-IR820.

[0051] (4) Take 100 mg of the mesoporous silica nanoparticles modified by the above dye IR820, disperse them in 10 mL of water to prepare a dispersion, add 100 mg of bovine serum albumin (BSA), react at room temperature and 700 rpm for 12 h, centrifuge the obtained reaction solution (11000 rpm, 10 min) to obtain a solid, wash the solid once with pure water, and add 10 mL of deionized water to obtain a bovine serum albumin-coated composite nanoparticle dispersion (10 mg / mL), which is denoted as MSN-NH2-IR820-BSA.

[0052] (5) Add 10 mg of gadolinium-based magnetic resonance contrast agent GdL to the 10 mL bovine serum albumin-coated composite nanoparticle dispersion obtained in step (4), and stir for 12 h. Utilize the interaction between gadolinium-based magnetic resonance contrast agent and BSA molecules to anchor the gadolinium-based magnetic resonance contrast agent on the surface of the composite nanoparticles. Centrifuge (11000 rpm, 10 min) the next day to precipitate the precipitate, and freeze-dry it to obtain the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe, denoted as MSN-NH2-IR820-BSA-GdL.

[0053] Example 2 The difference from Example 1 is that the amino-modified mesoporous silica nanoparticles are replaced with thiol-modified mesoporous silica nanoparticles (MSN-SH); wherein, the preparation method of thiol-modified mesoporous silica nanoparticles includes the following steps: replacing 3-aminopropyltriethoxysilane (APTES) in the above amino-mesoporous silica nanoparticle synthesis step with γ-mercaptopropyltrimethoxysilane (MPTMS) to obtain thiol-modified mesoporous silica nanoparticles, and the final magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobe is denoted as MSN-SH-IR820-BSA-GdL.

[0054] Comparative Example 1 The difference from Example 2 is that bovine serum albumin is replaced with human serum albumin, otherwise it is the same as Example 2.

[0055] Test Example 1 The magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Examples 1 and 2 were tested by transmission electron microscopy (TEM) at different magnifications, and the results are as follows: Figure 3 and Figure 4 As shown. Figure 3 and Figure 4 As shown, the obtained magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes MSN-NH2-IR820-BSA-GdL and MSN-SH-IR820-BSA-GdL have a regular spherical mesoporous structure and a suitable size of about 50 nm.

[0056] Test Example 2 Hydration size tests were performed on the different materials prepared in Examples 1 and 2: The different materials prepared in Examples 1 and 2 (mesoporous silica nanoparticles with a diameter of 50 nm) were diluted to appropriate concentrations, transferred to cuvettes, and their hydrodynamic diameters were measured using a nanoparticle size analyzer. The results are as follows: Figure 5 and Figure 6 As shown, Figure 5 and Figure 6 As shown, the hydrodynamic diameter of individual MSN-SH-IR820 nanoparticles is approximately 60–70 nm, while that of MSN-NH2-IR820 nanoparticles is approximately 90 nm. In contrast, the hydrodynamic diameters of the dual-modal imaging nanoprobes MSN-SH-IR820-BSA-GdL loaded with BSA protein and GdL are 81 nm and 91 nm, respectively. These results indicate that the materials prepared have similar sizes and do not assemble with BSA to form larger nanoparticles; rather, they are embedded in the cracks of the BSA protein through intermolecular forces.

[0057] Potential tests were performed on the different materials prepared in Examples 1 and 2: The different materials prepared in Examples 1 and 2 (mesoporous silica nanoparticles with a diameter of 50 nm) were diluted to appropriate concentrations, transferred to cuvettes, and the change in potential was measured using a nanoparticle size analyzer. The results are as follows: Figure 7 As shown. From Figure 7 It can be seen that the Zeta potential analysis shows that the surface charge undergoes a systematic change after each functionalization step.

[0058] Test Example 3 The magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Examples 1 and 2 were subjected to UV-Vis and NIR-NIR absorption spectroscopy and NIR-NIR II fluorescence emission spectroscopy tests. The specific procedure was as follows: the two magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes prepared in Examples 1 and 2 were dissolved in deionized water to prepare a solution with a concentration of 1 mg / mL, transferred to a cuvette, and tested using a UV-Vis and NIR-NIR spectrophotometer. The test results are as follows. Figure 8 As shown, since the subsequently coated BSA and GdL do not absorb in the 400-1000 nm range, the UV absorption spectra of the two magnetic resonance / NIR-II fluorescence dual-modality imaging nanoprobes prepared in Examples 1 and 2 are mainly determined by MSN-NH2-IR820 and MSN-SH-IR820. After IR820 is covalently coupled with amino or thiol-modified mesoporous silica nanoparticles, the UV absorption spectrum changes. The UV absorption peak of MSN-NH2-IR820 is at 678 nm, while that of MSN-SH-IR820 is at 833 nm, showing a very significant difference. When BSA protein and GdL are loaded, their absorption spectra are similar to those before loading.

[0059] Furthermore, the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Examples 1 and 2 were diluted 5 times with deionized water, transferred to a cuvette, and tested using a near-infrared fluorescence spectrometer with excitation by an 808 nm laser. The test results are as follows: Figure 9 As shown, the fluorescence emission of the thiol-modified bimodal imaging nanoprobe is significantly stronger than that of the amino-modified bimodal imaging nanoprobe.

[0060] Test Example 4 The in vitro magnetic resonance T1 relaxation efficiency (relaxation rate r1) of the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Examples 1 and 2 was evaluated: GdL solutions of different concentrations (0.2, 0.15, 0.1, 0.05, 0.025, and 0 mM) and 3 mL of magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe dispersions of different concentrations (1, 0.75, 0.5, 0.25, 0.125, and 0 mg / mL) were prepared, and MRI imaging and relaxation rate tests were performed. The results are as follows: Figure 10 and Figure 11 As shown, Figure 10 In the image, A represents MRI images of GdL solutions at different concentrations, and B represents MRI images of magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe dispersions at different concentrations. Figure 11In the middle, the left image shows the in vitro magnetic resonance T1 relaxation efficiency of MSN-NH2-IR820-BSA-GdL; the right image shows the in vitro magnetic resonance T1 relaxation efficiency of MSN-SH-IR820-BSA-GdL. Figure 10 and Figure 11 It can be seen that the relaxation rate r1 of the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe is significantly higher than that of GdL alone, which proves the high efficiency of the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe provided by this invention as a T1 contrast agent.

[0061] Test Example 5 In vitro near-infrared II fluorescence imaging was performed on the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Examples 1 and 2. Stock solutions of magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes at different concentrations (3.125, 6.25, 12.5, 25, 50 μM) were prepared and filled into capillaries for NIR-II fluorescence imaging. The results are as follows: Figure 12 As shown, A represents the fluorescence and pseudo-color images of NIR-II fluorescence imaging for different concentrations of MSN-SH-IR820-BSA-GdL; B represents the fluorescence and pseudo-color images of NIR-II fluorescence imaging for different concentrations of MSN-NH2-IR820-BSA-Gd; C represents the capillary streak intensity analysis image in A; and D represents the capillary streak intensity analysis image in B. From... Figure 12 It can be seen that the fluorescence signal of the thiol-modified bimodal imaging nanoprobe is significantly stronger than that of the amino-modified bimodal imaging nanoprobe, and the higher the concentration, the stronger the fluorescence signal.

[0062] Subsequently, the effects of different filters on NIR-II imaging were further investigated. Two magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Examples 1 and 2 were dissolved in water to form a 10 μM solution and placed in a 1.5 mL EP tube for NIR-II fluorescence imaging. The results are as follows: Figure 13 As shown, from Figure 13 It can be seen that LP900 has the strongest signal, while LP1200 has almost no detectable fluorescence signal. Therefore, subsequent NIR-II fluorescence imaging uses a thiol-modified dual-modal imaging nanoprobe MSN-SH-IR820-BSA-GdL, and LP900 is selected because it has the strongest fluorescence signal.

[0063] Selected using LAS X software Figure 13 Intensity was statistically analyzed at the white framed and black background locations marked on the EP tube, three times. The obtained signal values ​​were then imported into Origin 2021 for plotting and analysis. The results are as follows. Figure 14 As shown, Figure 14 This study compares the signal-to-noise ratio of different magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobes (mesoporous silicon nanoparticles with a diameter of 50 nm) under different filter conditions. Figure 14 It can be seen that the signal-to-noise ratio of MSN-SH-IR820-BSA-GdL is higher than that of MSN-NH2-IR820-BSA-GdL under different filter conditions. Among them, LP900 has the highest signal-to-noise ratio, and LP900 can be selected for subsequent experiments.

[0064] To evaluate the tissue penetration capability of the dual-modal imaging nanoprobe, in Figure 15 The tissue penetration capability was tested using the tissue penetration depth model shown. Specifically, the capillary of the thiol-modified dual-modal imaging nanoprobe (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Example 2 was immersed in a 1% lipid solution, and near-infrared II imaging was performed at different depths (1-5 mm). The results are as follows. Figure 16 As shown. Figure 16 As shown, 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 imaging nanoprobe's near-infrared II fluorescence imaging possesses high resolution and deep tissue penetration capability, which is extremely advantageous for fluorescence surgical navigation.

[0065] The intensity analysis of capillaries was performed using LAS X software, and the data was imported into Origin 2024 software for plotting. The results are as follows. Figure 17 As shown, Figure 17 for Figure 16 Statistical analysis of signal-to-noise ratio and length of capillaries, from Figure 17 It can be seen that the signal-to-noise ratio gradually decreases with increasing depth, and the signal becomes almost undetectable at 4 mm. In contrast, the full width at half maximum (FWHM) of the NIR-II fluorescence intensity profile shows the opposite trend.

[0066] The scribing intensity of capillaries at different depths (0-5 mm) was analyzed using LAS X software, and the results are as follows: Figure 18 As shown, from Figure 18 It can be seen that as the penetration depth increases, the signal intensity of the NIR-II image decreases significantly, accompanied by blurring of the capillary edges.

[0067] Test Example 6 In vitro cytotoxicity assays (MTS method) were performed on the two dual-modal imaging nanoprobes (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Examples 1 and 2: Stock solutions of dual-modal imaging nanoprobes at different concentrations (400, 300, 200, 100, and 50 μg / mL) were prepared. Cells were incubated at a concentration of 1 × 10⁻⁶ cells / mL. 4 The microplates were seeded at a density of [number] wells in 96-well plates and incubated for 24 hours. Then, stock solutions of dual-modal imaging nanoprobes at different concentrations were added. After 48 hours of incubation, each well was washed three times with PBS, and then 100 μL of fresh culture medium and 10 μL of MTS solution were added to each well. Incubation was continued for another 3 hours under the same conditions. The absorbance at 500 nm was measured using a microplate reader. Results are as follows: Figure 19 As shown, Figure 19 Cell viability at different concentrations of dual-modal imaging nanoprobes. Figure 19 It can be seen that even at higher concentrations, this dual-modal imaging nanoprobe exhibits low toxicity to the test cells, demonstrating its good biocompatibility.

[0068] Different concentrations of dual-modal imaging nanoprobes (mesoporous silica nanoparticles with a diameter of 50 nm, 62.5, 125, 250, 500, and 1000 mg / mL) and mouse erythrocytes were added together, mixed, and allowed to stand for 2 hours. The mixture was then centrifuged, photographed, and the supernatant was collected for UV testing. The hemolysis rate was calculated based on Equation 1 using UV absorption and plotted.

[0069] Formula 1.

[0070] In Formula 1, A = ultraviolet absorption intensity at 576 nm.

[0071] Figure 20 Images of mixed dual-modal imaging nanoprobes and mouse erythrocytes at different concentrations; Figure 21 The ultraviolet absorption spectra of the mixture of dual-modal imaging nanoprobes at different concentrations and mouse erythrocytes; Figure 22 The hemolysis rate of mouse erythrocytes by dual-modal imaging nanoprobes at different concentrations. Figures 20-22 It can be seen that the nanoprobe concentration was below 5% at all tested concentrations, demonstrating good blood compatibility.

[0072] Test Example 7 The thiol-modified bimodal nanoprobe MSN-SH-IR820-BSA-GdL (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Example 2 was used for NIR-II fluorescence imaging and fluorescence surgical navigation of mouse dorsal tumors.

[0073] 1) Constructing a mouse dorsal subcutaneous tumor model First, CT26 mouse colorectal 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 CT26 colorectal 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 CT26 colorectal cancer tumor cell single-cell suspension was aspirated into an insulin injector, and approximately 50 μL of the suspension was injected subcutaneously into the right back of mice to establish a mouse subcutaneous tumor model. Figure 23 A schematic diagram for establishing a CT26 colorectal cancer dorsal subcutaneous tumor model.

[0074] 2) Near-infrared II fluorescence imaging experiment Balb / c mice with an established CT26 colorectal cancer subcutaneous tumor model on the back were selected. 25 μL of a thiol-modified dual-modality imaging nanoprobe dispersion (1 mg / mL) was injected orally into the tumor on the back of the mice. Subsequently, near-infrared II (NIR-II) imaging was performed on the mice at different time points (0-2 h). The results are as follows: Figure 24 As shown, Figure 24 NIR-II fluorescence imaging of mouse dorsal tumors at different time points, from Figure 24 It can be seen that the fluorescence signal lasts for at least 2 hours after injection.

[0075] The fluorescence intensity of mouse dorsal tumors at different time points (0-2h) was statistically analyzed using ImageJ software. The results are as follows: Figure 25 , Figure 25 Statistical analysis of fluorescence at different time points in mouse dorsal tumors, from Figure 25 It can be seen that the fluorescence signal lasts for at least 2 hours after injection.

[0076] Balb / c mice with an established CT26 colorectal cancer subcutaneous tumor model on the back were selected. 25 μL of a thiol-modified dual-modality imaging nanoprobe dispersion (1 mg / mL) was injected orally into the tumor on the back of the mice. Near-infrared II (NIR-II) imaging was performed on the mice, and the tumor on the back of the mice was completely removed based on the fluorescence signal. The results are as follows: Figure 26 ,from Figure 26 As can be seen, a bright near-infrared II fluorescence signal was observed at the tumor site, and thanks to its excellent fluorescence signal, the tumor tissue was accurately located and surgical resection was successfully completed.

[0077] Test Example 8 The thiol-modified dual-modal imaging nanoprobe MSN-SH-IR820-BSA-GdL (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Example 2 was used for NIR-II fluorescence imaging and fluorescence surgical navigation of mouse lymph node metastases.

[0078] 1) Constructing a model of colorectal cancer lymph node metastases First, CT26 colorectal cancer 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 CT26 colorectal cancer cells to 1 × 10⁻⁶. 7 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 CT26 colorectal 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 CT26 colorectal cancer lymph node metastasis model.

[0079] 2) Near-infrared II fluorescence imaging experiment Two Balb / c mice with a successfully established 4T1 colorectal cancer lymph node metastasis model were selected. 25 μL of a thiol-modified dual-modality imaging nanoprobe MSN-SH-IR820-BSA-GdL dispersion (concentration 1 mg / mL) was injected into the paw pad tumor of each mouse's hind limb. Subsequently, near-infrared II (NIR-II) imaging was performed on the mice at different time points (0-24 h), and the results are shown below. Figure 27 As shown, Figure 27 To obtain NIR-II fluorescence images of a mouse lymph node metastasis model at different time points (0-24h), ImageJ software was used to statistically analyze the fluorescence intensity of the popliteal and ischial lymph nodes at different time points (0-2h). The results are as follows: Figure 28 As shown. Mice inoculated with a dispersion of the thiol-modified dual-modal imaging nanoprobe MSN-SH-IR820-BSA-GdL were sacrificed according to... Figure 29 The diagram shown illustrates the fluorescent surgical navigation system used for tumor resection during metastatic lymph node removal. Figures 27-29As can be seen, on the left hind limb side 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 side, which was not inoculated with tumor cells, was relatively weak. In summary, this dual-modal imaging nanoprobe can clearly observe the illumination of metastatic sentinel lymph nodes, and thanks to its excellent fluorescence signal, metastatic lymph nodes can be successfully removed.

[0080] Test Example 9 The dual-modal imaging nanoprobes (mesoporous silica nanoparticles with a diameter of 50 nm) prepared in Examples 1 and 2 were used for magnetic resonance imaging of colorectal cancer lymph node metastases: Two Balb / c mice with established CT26 mouse models of colorectal cancer lymph node metastases were selected. 25 μL of the dual-modal imaging nanoprobe dispersion (concentration of 3 mg / mL) was injected into the hind limb paw pad tumors. MRI T1WI sequences were performed on both mice at different time points after injection (0 minutes and 1 hour). The results are as follows: Figure 30 As shown, Figure 30 The top row of images shows the results of MSN-NH2-IR820-BSA-GdL injection. From left to right, the images are: T1-weighted magnetic resonance imaging (MRI) images before injection (0 minutes), T1-weighted MRI images 1 hour after injection, and a statistical chart of the intensity of T1-weighted MRI images before and 1 hour after injection. The bottom row of images shows the results of MSN-SH-IR820-BSA-GdL injection. From left to right, the images are: T1-weighted MRI images before injection, T1-weighted MRI images 1 hour after injection, and a statistical chart of the intensity of T1-weighted MRI images before and 1 hour after injection. Figure 30 As shown, in mice injected with the dual-modal imaging nanoprobe, the magnetic resonance imaging signal in the popliteal lymph node metastasis area gradually increased over time; it was also shown that after the injection of the dual-modal imaging nanoprobe, the metastatic lymph node area showed significant T1 signal enhancement, which contrasted sharply with the contralateral normal lymph nodes, demonstrating its high-resolution anatomical localization capability.

[0081] Test Case 10 MSN-SH-IR820-BSA-GdL, prepared from mesoporous silica nanoparticles of different particle sizes in Example 1, were subjected to NIR-II fluorescence imaging and MRI imaging in mice with lymph node metastases. The results are as follows: Figure 31 As shown, Figure 31In the image, A shows the NIR-II fluorescence imaging of MSN-SH-IR820-BSA-GdL, prepared from mesoporous silica nanoparticles with particle sizes of 70 nm, 120 nm, and 260 nm, in a mouse model of lymph node metastases. The image shows that the 70 nm and 120 nm materials exhibit similar results to the previously observed 50 nm material, both showing fluorescence signals in the popliteal and ischial lymph nodes. However, the 260 nm material failed to detect any signal in metastatic lymph nodes. Furthermore, the MRI of these three materials in mice with lymph node metastases was investigated, and the results are as follows: Figure 31 As shown in Figure B, the results are similar to those of NIR-II imaging. Only materials with particle sizes of 70 nm and 120 nm can detect popliteal lymph node signals, while no MRI signal can be observed in the 260 nm MSN-SH-IR820-BSA-Gd. This clearly demonstrates that when the particle size of mesoporous silica nanoparticles is less than 200 nm, it is easier to image and detect metastatic lymph nodes, while it is more difficult to detect fluorescence or MRI signals when the particle size exceeds 200 nm. This indicates that the particle size of mesoporous silica nanoparticles has a certain influence on the detection of lymph node metastasis.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe, characterized in that, The mixture includes amino- or thiol-modified mesoporous silica nanoparticles, an NIR-II fluorescent dye linked to the mesoporous silica nanoparticles, bovine serum albumin linked to the NIR-II fluorescent dye, and a gadolinium-based magnetic resonance contrast agent linked to the bovine serum albumin.

2. The magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe according to claim 1, characterized in that, The NIR-II fluorescent dye includes IR820, which has the following structure: 。 3. The magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe according to claim 1, characterized in that, The gadolinium-based magnetic resonance contrast agent is GdL, and GdL has the following structure: 。 4. The magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe according to claim 1, characterized in that, The diameter of the mesoporous silicon nanoparticles is 30~200nm.

5. The method for preparing the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe according to any one of claims 1 to 4, characterized in that, Includes the following steps: Provides amino- or thiol-modified mesoporous silica nanoparticles; The amino or thiol-modified mesoporous silica nanoparticles were covalently coupled with NIR-II fluorescent dye to obtain dye-modified mesoporous silica nanoparticles. The dye-modified mesoporous silica nanoparticles were mixed with bovine serum albumin and water for coating to obtain bovine serum albumin-coated composite nanoparticles. The bovine serum albumin-coated composite nanoparticles were mixed with a gadolinium-based magnetic resonance contrast agent and anchored to obtain the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the amino or thiol-modified mesoporous silica nanoparticles to the NIR-II fluorescent dye is 3~10:1; The covalent coupling reaction is carried out at a temperature of 70-90°C for 2-10 hours.

7. The preparation method according to claim 5, characterized in that, The mass ratio of the dye-modified mesoporous silica nanoparticles to bovine serum albumin is 0.5~10:1, and the coating temperature is 20~40℃ for 6~12h.

8. The preparation method according to claim 5, characterized in that, The mass ratio of the bovine serum albumin-coated composite nanoparticles to the gadolinium-based magnetic resonance contrast agent is 10~100:1; The anchoring temperature is 20~40℃, and the time is 6~12h.

9. The application of the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe according to any one of claims 1 to 4 or the magnetic resonance / NIR-II fluorescence dual-modal imaging nanoprobe prepared by the preparation method according to any one of claims 5 to 8 in the preparation of imaging formulations.

10. The application according to claim 9, characterized in that, The imaging agent is used for the diagnosis of metastatic lymph nodes in tumors.

Images