Gadolinium-based magnetic resonance / near-infrared fluorescence bimodal probe as well as preparation and application thereof

Abstract

The invention discloses a gadolinium-based magnetic resonance / near-infrared fluorescence bimodal probe as well as preparation and application thereof, and relates to the field of nano materials and medical engineering. The bimodal probe comprises nanoparticles formed by coordination and self-assembly of gadolinium ions and indocyanine green through sulfonic acid groups, and the molar ratio of the gadolinium ions to the indocyanine green is (2-10): 1. The bimodal probe shows the relaxation performance which is obviously superior to that of other clinical common contrast agents in a 9.4 T ultrahigh field magnetic resonance system, and the longitudinal relaxation rate of the bimodal probe can reach 22.3 mM <-1 > s <-1 >. The bimodal probe can be directly produced on a large scale on the basis of clinically approved drugs, and the bimodal probe has excellent imaging performance and blood-brain barrier penetrating power and provides a very potential strategy for image-guided biomedical application.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterials and medical engineering, specifically to a gadolinium-based magnetic resonance / near-infrared fluorescence dual-modal probe and its preparation and application. Background Technology

[0002] The blood-brain barrier (BBB) ​​prevents 98% of small molecules and all large molecules from entering the brain, significantly increasing the complexity of diagnosing brain diseases. Currently, routine imaging techniques for examining brain structure and function include magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and fluorescence imaging. Among these techniques, the choice of contrast agent plays a crucial role in improving diagnostic efficacy, and the ability to cross the blood-brain barrier is one of the key characteristics of an ideal brain contrast agent. Although strategies such as liposome encapsulation, antibody modification, and neurotropic viral delivery have been shown to cross the blood-brain barrier, these methods mostly involve complex synthetic processes and high production costs, thus limiting their widespread application in clinical practice.

[0003] Near-infrared spectroscopy (NIR-II) imaging (wavelength range 1000-1700 nm) and magnetic resonance imaging (MRI) offer significant complementary advantages in the diagnosis of brain diseases. MRI can accurately depict the location, size, and morphological features of tumors, while NIR-II imaging has attracted considerable attention due to its high resolution and deeper tissue penetration (>9 mm) compared to traditional fluorescence imaging, and it can compensate for the limitation of MRI in real-time imaging. Therefore, the development of NIR-II / MRI dual-mode imaging probes capable of crossing the blood-brain barrier holds promise for further improving the accuracy of brain disease diagnosis.

[0004] Currently, commonly used gadolinium-based MRI contrast agents can only detect tumor lesions with severely disrupted blood-brain barriers. Furthermore, due to their poor imaging efficacy and limited specificity, higher doses are required for clinical application, potentially leading to gadolinium deposition in the brain or renal systemic fibrosis in some patients. For near-infrared imaging, indocyanine green (ICG), a cyanin-based near-infrared fluorescent dye, has been approved by the U.S. Food and Drug Administration (FDA) for human clinical research and application. ICG rapidly binds to plasma proteins after intravenous injection but also cannot cross the blood-brain barrier. Therefore, developing near-infrared II fluorescence / T1-magnetic resonance-enhanced dual-modal probes with blood-brain barrier-crossing properties, based on current clinical medical drugs, is crucial for further improving the diagnostic and therapeutic efficacy of these drugs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as the lack of bimodal probes that can cross the blood-brain barrier, complex synthesis conditions, and the need to improve the performance of clinical contrast agents, this application proposes a gadolinium-based magnetic resonance / near-infrared fluorescence bimodal probe and its preparation and application.

[0006] According to a first aspect of the present invention, a gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe is proposed, comprising nanoparticles formed by the self-assembly of gadolinium ions and indocyanine green through coordination of sulfonic acid groups, wherein the molar ratio of gadolinium ions to indocyanine green is (2-10):1.

[0007] Furthermore, the molar ratio of the gadolinium ion to the indocyanine green is (4-10):1.

[0008] Furthermore, the molar ratio of the gadolinium ion to the indocyanine green is (4-5):1.

[0009] Furthermore, the molar ratio of the gadolinium ion to the indocyanine green is 5:1.

[0010] Furthermore, water molecules exist in the bimodal probe through physical adsorption and / or coordination with gadolinium ions. The bimodal probe has a water content of 15±5wt% and an average hydrated particle size of 14-15nm. The particle size of the bimodal probe, as determined by transmission electron microscopy, is 4-5nm, and it is suitable for pH 6.5-7.4.

[0011] Furthermore, the dual-mode probe exhibits near-infrared II fluorescence emission characteristics after being excited by 800–820 nm light.

[0012] Furthermore, the longitudinal relaxation rate r1 was obtained by adjusting the component feeding ratio of the dual-mode probe. Under a 9.4T magnetic field, the longitudinal relaxation rate r1 of the dual-mode probe was >15 mM. -1 s -1 .

[0013] Furthermore, the longitudinal relaxation rate r1 is preferably >20mM. -1 s -1 Further preferred is ≥22mM -1 s -1 .

[0014] According to a second aspect of the present invention, a method for preparing a gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe is provided, comprising:

[0015] S1. Slowly add the gadolinium salt solution dropwise to the indocyanine green mother liquor, controlling the molar ratio of gadolinium ions to indocyanine green to be (2-10):1, and stir the reaction magnetically under light-protected conditions.

[0016] S2. The reaction product is initially separated by centrifugation, and the precipitate is then dispersed by ultrasonication. A second centrifugation is performed to obtain the dual-mode probe.

[0017] Furthermore, the molar ratio of the gadolinium ion to the indocyanine green is (4-10):1.

[0018] Furthermore, the molar ratio of the gadolinium ion to the indocyanine green is (4-5):1.

[0019] Furthermore, the molar ratio of the gadolinium ion to the indocyanine green is 5:1.

[0020] Furthermore, the magnetic stirring time in step S1 is 2-4 hours; the conditions for the initial centrifugation and the second centrifugation in step S2 are both 10000-12000 rpm for 5-30 minutes.

[0021] According to a third aspect of the present invention, a method for evaluating the ability of a probe to cross the blood-brain barrier is provided, comprising the steps of: applying an effective amount of the probe to a research subject, and acquiring brain images and / or brain tissue distribution data by magnetic resonance imaging, near-infrared fluorescence imaging and / or fluorescence microscopy.

[0022] According to a fourth aspect of the present invention, a biological tissue imaging method is provided, comprising the following steps: applying an effective amount of the probe to a research subject, and then acquiring image data of a target tissue region by magnetic resonance imaging and / or near-infrared II fluorescence imaging; the target tissue region includes lesion sites and / or normal tissue sites.

[0023] According to a fifth aspect of the invention, there is provided the use of the probe in the preparation of reagents for disease diagnosis, wherein the disease is a tumor or a neurological disease.

[0024] Compared with the prior art, the beneficial effects of this invention are as follows:

[0025] This invention utilizes a one-step metal coordination-assisted self-assembly technique to construct a dual-modal probe capable of both MRI and NIR-II fluorescence imaging. This probe exhibits significantly superior relaxation performance compared to other commonly used clinical contrast agents in a 9.4T ultra-high field MRI system, with a longitudinal relaxation rate reaching 22.3 mM. -1 s -1More importantly, ICG-Gd achieves efficient and stable blood-brain barrier penetration. In orthotopic glioma models, ICG-Gd can precisely delineate brain tumor boundaries and enable accurate near-infrared II (NIR-II) / magnetic resonance imaging (MRI) diagnosis of microgliomas (<1.5 mm). Overall, ICG-Gd can be directly mass-produced based on clinically approved drugs, and its superior NIR-II / MRI imaging performance and blood-brain barrier penetration provide a highly promising strategy for image-guided biomedical applications. Attached Figure Description

[0026] Figure 1 A flowchart illustrating the preparation of ICG-Gd NPs according to a specific embodiment of the present invention is shown;

[0027] Figure 2 The diagram shows the Tyndall effect of ICG-Gd NPs prepared according to a specific embodiment of the present invention;

[0028] Figure 3 The figure shows the results of synthesizing ICG-GdNPs under different precursor molar ratios in a specific embodiment of the present invention;

[0029] Figure 4 A transmission electron microscope image of ICG-Gd NPs according to a specific embodiment of the present invention is shown;

[0030] Figure 5 A graph showing the hydration particle size of ICG-Gd NPs according to a specific embodiment of the present invention is provided.

[0031] Figure 6 The ultraviolet-visible-near-infrared absorption spectra of ICG-Gd NPs according to a specific embodiment of the present invention are shown;

[0032] Figure 7 The NIR-I and NIR-II fluorescence spectra of ICG-Gd NPs according to a specific embodiment of the present invention are shown;

[0033] Figure 8 The figure shows the longitudinal relaxation rate r1 value and T1-weighted MR image under a field strength of 9.4T in a specific embodiment of the present invention;

[0034] Figure 9 The diagram shows the longitudinal relaxation rate r1 values ​​and T1-weighted MR images of ICG-Gd NPs at different pH values ​​under a 9.4T field strength according to a specific embodiment of the present invention.

[0035] Figure 10Thermogravimetric analysis and differential thermal analysis according to a specific embodiment of the present invention are shown;

[0036] Figure 11 A stability result diagram of ICG-Gd NPs according to a specific embodiment of the present invention is shown;

[0037] Figure 12 The graph shows the long-term stability results of ICG-Gd NPs in PBS, DMEM and 10% FBS according to a specific embodiment of the present invention;

[0038] Figure 13 The following image shows the results of fluorescence scanning microscopy and flow cytometry of ICG-Gd NPs according to a specific embodiment of the present invention;

[0039] Figure 14 The diagram illustrates the toxicity of different concentrations of ICG-Gd NPs to different cells according to a specific embodiment of the present invention;

[0040] Figure 15 T1-weighted MR images of the brains of healthy mice at 0 h before and 0.25 h, 0.5 h, 1 h, 2 h and 4 h after tail vein injection of ICG-Gd NPs and Gd-DTPA according to a specific embodiment of the present invention are shown.

[0041] Figure 16 The diagram illustrates the signal-to-noise ratio (SNR) of the corresponding MR grayscale value of the pituitary gland and the corresponding MR grayscale value of the parenchyma at 0 h before and 0.25 h, 0.5 h, 1 h, 2 h, and 4 h after tail vein injection of ICG-Gd NPs and Gd-DTPA in mice according to a specific embodiment of the present invention.

[0042] Figure 17 The NIR-I fluorescence imaging and fluorescence signal intensity map of isolated brain tissue 0.5 h after tail vein injection of ICG-Gd NPs, ICG and saline are shown in a specific embodiment of the present invention.

[0043] Figure 18 The following is an illustration of fluorescence imaging results of a section of normal mouse brain tissue according to a specific embodiment of the present invention;

[0044] Figure 19 The NIR-II fluorescence imaging and fluorescence signal intensity map of U87MG glioma mice injected with ICG-Gd NPs via tail vein, 0h before ICG injection, and 0.5h, 1h, 2h, 4h and 8h after injection are shown in a specific embodiment of the present invention.

[0045] Figure 20The following are axial MR images of U87MG glioma mice obtained at different time points after intravenous injection, according to a specific embodiment of the present invention.

[0046] Figure 21 This diagram illustrates a quantitative analysis of MRI signals in gliomas at different time points according to a specific embodiment of the present invention.

[0047] Figure 22 An image of an SDS-PAGE electrophoresis gel exposure according to a specific embodiment of the present invention is shown;

[0048] Figure 23 The thermal analysis of ICG-Gd nanoparticles and ICG-adsorbed proteins according to a specific embodiment of the present invention is shown. Detailed Implementation

[0049] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0050] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0051] The bimodal probe provided by this invention is uniformly abbreviated as ICG-Gd NPs in the following specific embodiments. The bimodal probe includes nanoparticles formed by the self-assembly of gadolinium ions and indocyanine green through coordination of sulfonic acid groups. The molar ratio of gadolinium ions to indocyanine green is (2-10):1, such as 2:1, 4:1, 6:1, 8:1, 10:1 or any value between them.

[0052] In some embodiments, the molar ratio of gadolinium ions to indocyanine green is preferably (4–10):1, such as 4:1, 6:1, 8:1, 10:1, or any value between them; more preferably (4–5):1, such as 4:1, 5:1, or any value between them; and even more preferably 5:1. Regarding the selection of the range of the molar ratio, this invention uses material relaxation efficiency as the main reference, and uses the longitudinal relaxation rate r1 value as the core evaluation index for multiple rounds of verification. Parameter optimization is achieved by continuously adjusting the molar ratio of the gadolinium-based precursor to the organic ligand, such as… Figure 3 As shown, when the molar ratio of gadolinium ions to indocyanine green is 5:1, the dual-modal probe exhibits significantly better T1-weighted imaging sensitivity in a 9.4T ultra-high field magnetic resonance system than other ratio schemes, with a longitudinal relaxation rate reaching 22.3 mM. -1 s-1 .

[0053] Water molecules exist in the bimodal probe through physical adsorption and / or coordination with gadolinium ions. The water content of the bimodal probe is 15±5wt%, such as 10wt%, 15wt%, 20wt%, or any value between them; the average hydrated particle size is 14-15nm, such as 14nm, 15nm, or any value between them; the water content is positively correlated with the longitudinal relaxation rate r1 of the bimodal probe.

[0054] The particle size of the dual-modal probe, as determined by transmission electron microscopy, is 4–5 nm, such as 4 nm, 5 nm, or any value between them; it is suitable for pH values ​​of 6.5–7.4, such as 6.5, 7.0, 7.4, or any value between them.

[0055] The dual-mode probe exhibits near-infrared II fluorescence emission characteristics after being excited by 800–820 nm light.

[0056] The longitudinal relaxation rate r1 was obtained by adjusting the component feeding ratio of the dual-mode probe. The longitudinal relaxation rate r1 of the dual-mode probe under a 9.4T magnetic field is greater than 15mM. -1 s -1 Preferably >20mM -1 s -1 Further preferred is ≥22mM -1 s -1 The MRI signal intensity of the dual-modal probe is positively correlated with the concentration of gadolinium ions.

[0057] The method for preparing a dual-modal probe provided by this invention includes:

[0058] S1. Slowly add the gadolinium salt solution dropwise to the indocyanine green mother liquor, controlling the molar ratio of gadolinium ions to indocyanine green to be (2-10):1, such as 2:1, 4:1, 6:1, 8:1, 10:1 or any value between them, and stir the reaction magnetically under light-protected conditions.

[0059] S2. The reaction product is initially separated by centrifugation, and the precipitate is then dispersed by ultrasonication. A second centrifugation is performed to obtain the dual-mode probe.

[0060] In some embodiments, the molar ratio of the gadolinium ion to the indocyanine green is preferably (4–10):1, such as 4:1, 6:1, 8:1, 10:1, or any value between them; more preferably (4–5):1, such as 4:1, 5:1, or any value between them; and even more preferably 5:1. The parameters are optimized by continuously adjusting the molar ratio of the gadolinium precursor to the organic ligand, such as… Figure 3As shown, the longitudinal relaxation rate r1 was obtained by adjusting the composition ratio of the dual-modal probe. When the molar ratio of gadolinium ions to indocyanine green was 5:1, the dual-modal probe exhibited significantly better T1-weighted imaging sensitivity than other formulations in a 9.4T ultra-high field magnetic resonance system, with a longitudinal relaxation rate reaching 22.3 mM. -1 s -1 .

[0061] The magnetic stirring time in step S1 is 2 to 4 hours; such as 2 hours, 3 hours, 4 hours or any value between them; the conditions for the first centrifugation and the second centrifugation in step S2 are both 10,000 to 12,000 rpm for 5 to 30 minutes, such as 10,000 rpm, 11,000 rpm, 12,000 rpm or any value between them, such as 5 minutes, 10 minutes, 20 minutes, 30 minutes or any value between them.

[0062] The present invention also provides a method for evaluating the ability of a probe to cross the blood-brain barrier, comprising the following steps: applying an effective amount of the probe to a research subject, and acquiring brain images and / or brain tissue distribution data by magnetic resonance imaging, near-infrared fluorescence imaging and / or fluorescence microscopy.

[0063] The present invention also provides a biological tissue imaging method, comprising the following steps: applying an effective amount of the probe to the research subject, and then acquiring image data of the target tissue region by magnetic resonance imaging and / or near-infrared II fluorescence imaging; the target tissue region includes lesion sites and / or normal tissue sites.

[0064] The present invention also provides the application of the probe described herein in the preparation of reagents for disease diagnosis, wherein the disease is a tumor or a neurological disease.

[0065] Example 1: Preparation method of ICG-Gd NPs

[0066] The large-scale preparation and purification steps of ICG-Gd NPs are as follows. First, accurately weigh 100 mg of indocyanine green (ICG) powder and place it in a 2 L glass beaker. Add 1 L of ultrapure water and stir magnetically until completely dissolved to obtain an ICG stock solution with a concentration of 100 mg / L. Then, dissolve 246.4 mg of gadolinium chloride hexahydrate (GdCl3·6H2O) in 20 mL of ultrapure water and slowly add it dropwise to the above ICG solution system through a separatory funnel. The entire reaction process is carried out under light-protected conditions with continuous magnetic stirring for 3 h to promote coordination self-assembly. After the reaction is completed, the product is purified by centrifugation. First, centrifuge at 11000 rpm for 5 min and filter. Discard the supernatant containing unreacted ICG and free gadolinium ions. Collect the precipitate and resuspend it in an appropriate amount of ultrapure water. After ultrasonic dispersion, centrifuge again at 11000 rpm for 30 min to further remove residual impurities. The resulting ICG-Gd nanoparticle precipitate was redispersed in a small amount of ultrapure water for subsequent experiments. Figure 1 and Figure 2 ).

[0067] This study used the relaxation efficiency of materials as the main reference and the longitudinal relaxation rate r1 as the core evaluation index for multiple rounds of verification. Parameter optimization was achieved by continuously adjusting the molar ratio of gadolinium-based precursor to organic ligand. Experimental results show that when the molar ratio is 1:5, the synthesized nanomaterial exhibits significantly better T1-weighted imaging sensitivity in a 9.4T ultra-high field magnetic resonance system than other ratio schemes, with a longitudinal relaxation rate reaching 22.3 mM. -1 s -1 ( Figure 3 ).

[0068] Example 2: Material Characterization of ICG-Gd NPs

[0069] Transmission electron microscopy (TEM) and high-resolution TEM characterization: A small amount of material was ultrasonically dispersed and dropped onto a standard copper mesh support film. For high-resolution TEM characterization, an ultrathin carbon support film is typically used. The material was allowed to air dry to ensure complete solvent evaporation. The size and morphology of the nanomaterials were then observed under an electron microscope. The synthesized nanomaterials were small in size, with a particle size of 4–5 nm, and exhibited uniform morphology, demonstrating good monodispersity. Figure 4 ).

[0070] Hydrated particle size analysis: The sample was diluted to a suitable concentration and detected using a dynamic light scattering particle size analyzer. The results, including numerical values ​​and distribution maps, were analyzed to obtain the average particle size. The average hydrated particle size was 14.6 nm. Figure 5 ).

[0071] Optical property analysis: Nanoparticles were ultrasonically dispersed in an aqueous solution, and the UV-Vis absorption and fluorescence spectra were measured using a UV spectrophotometer and a fluorescence spectrophotometer. The NIR-I fluorescence excitation spectrum had an emission wavelength of 810 nm and an excitation wavelength of 780 nm. The NIR-II fluorescence emission spectrum had an excitation wavelength of 808 nm. The results showed that the synthesized probe and the starting material (ICG) had overlapping absorption peaks at the same wavelength (780 nm). Simultaneously, the increased absorption peak width of the nanomaterial was observed in the figure, which is attributed to the coordination of ICG with Gd ions. Figure 6 Furthermore, further testing of the optical properties of the ICG-Gd nanoparticles showed that they retained the fluorescent properties of ICG. They exhibited maximum fluorescence emission at 810 nm and significant NIR-II fluorescence emission. Figure 7 )

[0072] Magnetic resonance imaging and relaxation rate studies: ICG-Gd NPs solutions, Gd-BOPTA solutions, and Gd-DTPA solutions with different concentration gradients were prepared and placed in 200 μL PCR tubes. The relaxation rate was measured using a Bruker Biospin 9.4T animal MRI scanner (Bruker, Germany). Experimental parameters were as follows: 256×256 matrix, slice thickness 0.6 mm, repeat time (TR) / echo time (TE) = 3000 / 100 ms, field of view (FOV) = 40 mm × 40 mm. The results showed that the longitudinal relaxation (r1) of ICG-Gd NPs was as high as 19.50 mM. -1 s -1 (9.4T). Furthermore, with increasing Gd concentration, the corresponding T1-weighted image brightness significantly increased, indicating that its MRI enhancement signal is concentration-dependent. The r1 value of ICG-Gd NPs was 5.3 times higher than that of the commercial contrast agent Gd-DTPA and 4.5 times higher than that of Gd-BOPTA (Table 2-3). Higher r1 values ​​were detected at lower magnetic fields. Figure 8 Furthermore, ICG-Gd NPs maintained excellent T1 contrast characteristics in an acidic microenvironment. Figure 9 ).

[0073] Thermogravimetric analysis and differential thermal analysis: Gd-ICG nanoparticles ( Figure 10 a) and ICG ( Figure 10Thermogravimetric analysis and differential thermal analysis results in (b) indicate that during the heating process, the mass loss due to water evaporation occurs around 300℃ as the temperature increases. Gd-ICG nanomaterials lose approximately 15 wt% due to dehydration at 300℃, indicating a high water content in these nanoparticles. Furthermore, the pyrolysis of ICG occurs within the temperature range of 300–500℃. The two sets of thermogravimetric analysis data are compared (…). Figure 10 In section c), when the temperature reaches above 500℃, the difference in residual mass fraction between Gd-ICG NPs and ICG is mainly related to the Gd content. Analysis shows that the gadolinium mass fraction in Gd-ICG nanoparticles can be estimated from the difference in mass fraction within this temperature range, and is approximately 10 wt%. Therefore, thermogravimetric analysis indicates that Gd-ICG NPs contain approximately 10% Gd and 15% water, and the high water content may be one of the reasons for the high relaxation rate of the material.

[0074] Stability analysis: Stability was assessed in a series of buffer solutions (SBF solution) with pH values ​​ranging from 6.5 to 7.4, simulating body fluids. pH 7.4 is close to physiological pH, and pH 6.5 is close to the pH of the tumor microenvironment. SBF simulated body fluids. The release assay used a 1000 Da dialysis bag containing 2 mL of ICG-Gd NPs sample solution. The dialysis bag containing the sample was placed in a 50 mL blue-capped bottle containing the corresponding buffer solution and shaken at 37°C. At each time point (0h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 48h), 3 mL of the solution outside the dialysis bag was collected, and an equal volume of buffer solution was added. The dialyzed Gd... 3+ The concentration of Gd was determined by ICP-MS. In all test solutions, Gd was detected by dialysis. 3+ The release ratio of the material Gd 3+ The stability of the components was assessed. Results showed no Gd leakage within two days under the experimental conditions, demonstrating the stability of ICG-Gd in an acidic microenvironment and simulated body fluid conditions. This indicates that ICG can stabilize Gd. 3+ Reduce Gd in the body 3+ Security risks caused by leakage Figure 11 ).

[0075] Stability analysis in DMEM, 10% fetal bovine serum, and PBS: The material was dispersed in DMEM, PBS, and 10% fetal bovine serum (FBS) and allowed to stand. The particle size was measured using a dynamic light scattering particle size analyzer (DLS) on days 1, 3, and 7 to analyze the stability of the material in different biological environments. The results showed that ICG-Gd maintained a stable particle size in the above solutions for 7 days, without significant dissociation or aggregation. Figure 12 ).

[0076] Fluorescence microscopy results demonstrated that ICG-Gd can be effectively taken up by cells and enter the cell, mainly concentrated in lysosomes. Figure 13 The effect of different concentrations of ICG-Gd nanoparticles on cell viability was evaluated using the MTT assay. Figure 14 The results showed that ICG-Gd nanoparticles exhibited low toxicity to U87-MG, Renca, HepG2, and LO2 cells, demonstrating excellent biocompatibility and low cytotoxicity.

[0077] Example 3: Study on crossing the blood-brain barrier in normal mice

[0078] MRI imaging: ICG-Gd NPs and Gd-DTPA (10 μmol / kg Gd) were injected into the tail vein, respectively. 3+ T1-weighted MR images of the mouse brain were obtained using a 9.4T animal MRI scanner at 0h, 0.25h, 0.5h, 1h, 2h, and 4h after drug administration. The main experimental parameters were: 256×256 matrix, slice thickness 0.5mm, echo time 8.5ms, repetition time 1000ms, field of view (FOV) 30mm×30mm, and 25 slices. After injection of ICG-Gd NPs, the T1 MRI signal in the pituitary gland and brain parenchyma of mice significantly increased. This indicates that ICG-Gd NPs successfully penetrated the blood-brain barrier and entered the brain parenchyma of healthy mice. In contrast, the signal change in the brain of mice injected with Gd-DTPA was not significant, with no significant signal enhancement or reduction observed. This indicates that Gd-DTPA failed to effectively cross the blood-brain barrier and could not enter the brain parenchyma. Figure 15 and Figure 16 This experimental result provides preliminary information on the diffusion of ICG-Gd nanomaterials in the brain.

[0079] Fluorescence Imaging: Female BALB / c mice were injected via tail vein with ICG-Gd NPs and ICG solution (ICG content was the same, 2 mg / kg), and an equal volume of saline was injected as a blank control group. Thirty minutes after injection, the mice were perfused with saline (containing heparin sodium) to remove residual dye from the blood vessels. Brain tissue was then collected for in vitro imaging analysis. NIR-I fluorescence imaging of the isolated brain tissue showed that the ICG-Gd NPs group exhibited a high fluorescence signal in the brain, with a signal intensity significantly higher than the ICG group and the saline group. This further indicates that ICG-Gd nanoparticles can effectively penetrate the intact blood-brain barrier and enter brain tissue. Figure 17 ).

[0080] Brain tissue sections: Female BALB / c mice were pre-injected via the tail vein with 100 μL FITC-Dextran (MW 70000, 5 mg / mL) to mark blood vessels, and allowed to stand for a period of time after injection to allow for full distribution. Subsequently, ICG-Gd nanoparticles (0.3 mM Gd) were injected. 3+ 200 μL of ammonium chloride was injected, and mice were sacrificed 0.5 h later. Brain tissue was completely removed, frozen, and prepared into 20 μm sections using a cryostat. The sections were then covered with paraformaldehyde and fixed for approximately half an hour. After washing twice with PBS, the sections were observed under an upright fluorescence microscope. The histological fluorescence images of the brain tissue clearly showed the accumulation of ICG-Gd NPs in the endothelial walls of brain microvessels and their diffusion via the blood-brain barrier (BBB). ICG-Gd NPs also accumulated in the surrounding brain environment. These results demonstrate that ICG-Gd can effectively cross the blood-brain barrier in normal mice. Figure 18 ).

[0081] Example 4: Imaging Study of In Situ Glioma

[0082] Methods for establishing an orthotopic glioma mouse model: Male nude mice (BALB / c, 18–20 g) were anesthetized with 5% chloral hydrate and placed on a stereotaxic device (RWD, Life Science Co., Ltd.). A burr hole was drilled in the skull (2.0 mm at the anterior fontanelle, 2.0 mm on the right side, 2.5 mm deep). The injection burr hole was adjusted, and 4 μL of PBS containing 2 × 10⁵ U87MG-Luc cells was slowly injected into the right side of the mouse brain. After injection, the skull hole was sealed with bone wax, and the scalp was sutured with absorbable surgical sutures. A heating pad was used to maintain the body temperature at 37°C until the mice awoke. Seven days after inoculation, potassium D fluorescein was injected intraperitoneally for bioluminescence imaging and MRI to monitor glioma growth, thus confirming the successful establishment of the orthotopic glioma and its use in the following in vivo imaging.

[0083] Near-infrared II imaging study of a mouse model of orthotopic glioma: U87MG-Luc mice with orthotopic glioma were anesthetized with 2% isoflurane / oxygen. Bioluminescence imaging was performed by intraperitoneal injection of potassium fluorescein D. Two groups of mice with similar signals were selected for near-infrared II imaging experiments. ICG-Gd NPs and ICG solution (with the same ICG content, 2 mg / kg) were injected via the tail vein, respectively. Near-infrared II imaging was performed at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h after administration. The main experimental parameters were as follows: excitation wavelength 808 nm, exposure time 100 ms, 1200 nm long-pass filter, and laser power density 30 mW / cm². 2Using the IVIS Lumina II small animal imaging system, we were able to monitor fluorescence signals at mouse tumor sites in real time. We selected two groups of mice with similar glioma sizes and signal strengths, and then compared imaging results after injecting ICG-Gd NPs and ICG (with the same ICG content) via the tail vein. Near-infrared II imaging technology revealed significant enrichment of ICG-Gd nanoparticles at the tumor site, and their retention time within the tumor was significantly longer than that of traditional fluorescent probes such as ICG (e.g., near-infrared II imaging). Figure 19 These experimental results further demonstrate that ICG-Gd nanoparticles exhibit superior performance compared to ICG in the diagnosis of in situ gliomas, not only enhancing the imaging signal of the tumor but also improving its ability to target the tumor.

[0084] Magnetic resonance imaging study of a mouse model of orthotopic glioma: Three groups of mice with orthotopic gliomas were anesthetized with 2% isoflurane / oxygen, and ICG-Gd NPs (Gd) were injected into the tail vein. 3+ (Concentration of 0.01 mmol / kg), Gd-DTPA (Gd 3+ (Concentration of 0.01 mmol / kg), Gd-DTPA (Gd 3+ The concentration was 0.1 mmol / kg. T1-weighted MR images of the mouse brain were obtained using a 9.4T animal MRI scanner at 0 h, 0.5 h, 1 h, 2 h, and 4 h after drug administration. The main experimental parameters were as follows: 256 × 256 matrix, slice thickness 0.6 mm, repetition time / echo time = 3000 / 100 ms, field of view (FOV) = 30 mm × 30 mm, and number of slices 25.

[0085] T1-weighted magnetic resonance imaging (MRI) was used to evaluate gliomas in situ by intravenous administration of Magnevist (gadolinium concentrations of 0.1 mmol / kg and 0.01 mmol / kg) and ICG-Gd (gadolinium concentration of 0.01 mmol / kg), both commonly used clinical MRI contrast agents. Results showed that within 4 hours of ICG-Gd (gadolinium concentration of 0.01 mmol / kg) injection, a glioma area approximately 1.5 mm in diameter exhibited significant signal enhancement (see [link to relevant documentation]). Figure 20 and Figure 21 This study successfully detected gliomas and clearly distinguished them from normal brain tissue. At the clinically standard dose (gadolinium concentration of 0.1 mmol / kg), Gd-DTPA also achieved enhanced imaging of gliomas. However, when using the same dose of Gd-DTPA as ICG-Gd (gadolinium concentration of 0.01 mmol / kg), the signal changes in the glioma region were weaker. Magnetic resonance imaging results indicate that ICG-Gd demonstrates superior diagnostic efficacy at only one-tenth of the clinical dose, significantly reducing the safety risks associated with gadolinium-based contrast agents.

[0086] Example 5: Study on protein-mediated blood-brain barrier crossing

[0087] To verify whether ICG-Gd nanoparticles can adsorb relevant proteins during blood circulation to achieve blood-brain barrier crossing, in this embodiment, ICG-Gd NPs were co-incubated with mouse plasma to simulate their in vivo environment. After multiple high-speed centrifugations to remove unbound proteins, the nano-protein crown complex was obtained. The resulting product was then analyzed using SDS-PAGE electrophoresis, stained with Coomassie brilliant blue, and prepared for subsequent protein quantification analysis. Figure 22 As can be seen, compared with the ICG control group, the SDS-PAGE bands of ICG-Gd NPs were slightly darker, indicating that ICG-Gd has a more significant ability to adsorb proteins during blood circulation. Further LC-MS / MS was used to quantitatively analyze the types and quantities of adsorbed proteins. Figure 23 As shown, there are significant differences in the types of proteins adsorbed by ICG-Gd NPs and ICG. Gd-ICG NPs adsorb far more proteins related to blood-brain barrier crossing than ICG, including albumin, serum transferrin, and low-density lipoprotein receptor-associated protein 1 (LDL-A1). In summary, ICG-Gd nanoparticles can adsorb a variety of blood-brain barrier crossing-related proteins during blood circulation, forming a nano-protein corona complex. Furthermore, by binding to relevant protein receptors on the BBB, they exhibit a significant blood-brain barrier crossing ability.

[0088] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0089] In the description of this application, it should be understood that the terms "upper," "lower," "inner," "outer," etc., indicating orientation or positional relationships based on the orientation or positional relationships shown in the accompanying drawings, are used only for the convenience of describing this application and for simplification, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. The word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The simple fact that certain measures are recited in mutually different dependent claims does not indicate that combinations of these measures cannot be used for improvement. Any reference signs in the claims should not be construed as limiting the scope.

Claims

1. A gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe, characterized in that, The invention includes nanoparticles formed by the self-assembly of gadolinium ions and indocyanine green through coordination with sulfonic acid groups, wherein the molar ratio of gadolinium ions to indocyanine green is (2-10):

1.

2. The gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to claim 1, characterized in that, The molar ratio of gadolinium ions to indocyanine green is (4-10):

1.

3. The gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to claim 2, characterized in that, The molar ratio of gadolinium ions to indocyanine green is (4-5):

1.

4. The gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to claim 3, characterized in that, The molar ratio of gadolinium ions to indocyanine green is 5:

1.

5. The gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to claim 1, characterized in that, Water molecules exist in the bimodal probe through physical adsorption and / or coordination with gadolinium ions. The bimodal probe has a water content of 15±5wt% and an average hydrated particle size of 14-15nm. The particle size of the bimodal probe is 4-5nm as determined by transmission electron microscopy, and it is suitable for pH 6.5-7.

4.

6. The gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to any one of claims 1-5, characterized in that, The dual-mode probe exhibits near-infrared II fluorescence emission characteristics after being excited by 800–820 nm light.

7. The gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to any one of claims 1-5, characterized in that, The longitudinal relaxation rate r1 was obtained by adjusting the component feeding ratio of the dual-mode probe. Under a 9.4T magnetic field, the longitudinal relaxation rate r1 of the dual-mode probe was >15 mM. -1 s -1 .

8. The gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to claim 7, characterized in that, The longitudinal relaxation rate r1 is preferably >20 mM. -1 s -1 Further preferred is ≥22mM -1 s -1 .

9. A method for preparing a gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe, characterized in that, include: S1. Slowly add the gadolinium salt solution dropwise to the indocyanine green mother liquor, controlling the molar ratio of gadolinium ions to indocyanine green to be (2-10):1, and stir the reaction magnetically under light-protected conditions. S2. The reaction product is initially separated by centrifugation, and the precipitate is then dispersed by ultrasonication. A second centrifugation is performed to obtain the dual-mode probe.

10. The method for preparing the gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to claim 9, characterized in that, The molar ratio of gadolinium ions to indocyanine green is (4-10):

1.

11. The method for preparing the gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to claim 10, characterized in that, The molar ratio of gadolinium ions to indocyanine green is (4-5):

1.

12. The method for preparing the gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to claim 11, characterized in that, The molar ratio of gadolinium ions to indocyanine green is 5:

1.

13. The method for preparing the gadolinium-based magnetic resonance / near-infrared fluorescence dual-mode probe according to claim 9, characterized in that, The magnetic stirring time in step S1 is 2-4 hours; the conditions for the first centrifugation and the second centrifugation in step S2 are both 10000-12000 rpm for 5-30 minutes.

14. A method for evaluating the ability of a probe to cross the blood-brain barrier, characterized in that, Includes the following steps: Apply an effective amount of the probe according to any one of claims 1-5 to the research subject, and acquire brain images and / or brain tissue distribution data by magnetic resonance imaging, near-infrared fluorescence imaging and / or fluorescence microscopy.

15. A biological tissue imaging method, characterized in that, Includes the following steps: An effective amount of the probe according to any one of claims 1-5 is applied to the research subject, and then image data of the target tissue region is acquired by magnetic resonance imaging and / or near-infrared II fluorescence imaging; the target tissue region includes lesion sites and / or normal tissue sites.

16. The use of a probe as described in any one of claims 1-5 in the preparation of a reagent for disease diagnosis, wherein the disease is a tumor or a neurological disease.

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