Bimetallic oxide nanoprobe based on BSA biomimetic mineralization as well as preparation method and application thereof

Abstract

The invention discloses a bimetal oxide nanoprobe based on BSA biomimetic mineralization and a preparation method and application thereof, and relates to the technical field of nanoprobes. The preparation method of the nanoprobe comprises the following steps: (1) respectively dissolving gadolinium chloride hexahydrate and manganese chloride in triple distilled water to obtain a gadolinium chloride solution and a manganese chloride solution; and (2) adding a gadolinium chloride solution and a manganese chloride solution into the bovine serum albumin solution under a stirring condition at room temperature to form a mixed solution, adjusting the pH value to 12, reacting, and purifying and freeze-drying to obtain the bimetal oxide nanoprobe based on BSA biomimetic mineralization. According to the invention, a long-acting time window is realized by utilizing long blood metabolism, and is passively enriched into a middle tumor microenvironment through an EPR effect, and a T1WI magnetic resonance imaging signal which is disintegrated and intensified at a tumor position is responded in a weakly acidic tumor microenvironment, so that accurate diagnosis of tumors is realized.

Description

Technical Field

[0001] This invention relates to the field of nanoprobe technology, specifically to a bimetallic oxide nanoprobe based on BSA biomimetic mineralization, its preparation method, and its application. Background Technology

[0002] Statistics show that approximately 70% of deaths are caused by cardiovascular and cerebrovascular diseases and malignant tumors. Early, accurate, and dynamic diagnosis of these serious diseases can provide timely and abundant disease information, thereby prolonging patient survival. Currently, accurately delineating the anatomical structures and spatial relationships around lesions is crucial for the diagnosis of cardiovascular and cerebrovascular diseases and malignant tumors. Magnetic resonance imaging (MRI), due to its excellent soft tissue resolution and post-processing capabilities, has become the preferred imaging modality for the diagnosis of cardiovascular and cerebrovascular diseases and tumors. High-quality MRI diagnosis of tumors and cardiovascular and cerebrovascular diseases is highly dependent on the performance of contrast agents. An ideal MRI contrast agent not only needs high contrast performance to improve the signal-to-noise ratio of the image, but also needs a long blood circulation time to ensure a sufficient imaging time window and enrichment at the tumor target. These characteristics are essential for supporting the accurate diagnosis and timely treatment of cardiovascular and cerebrovascular diseases and early-stage tumors. However, clinical gadolinium chelate small molecule contrast agents have some inherent limitations, including relatively low relaxation rates, short plasma half-lives, and potential nephrotoxicity. Therefore, developing highly efficient MRI contrast agents will compensate for the shortcomings of clinical contrast agents and improve the ability of MRI to diagnose tumors and cardiovascular and cerebrovascular diseases.

[0003] Over the past decade, the development of MRI nanoprobes has shown significant advantages, such as higher relaxation rates, ease of surface modification, and longer blood circulation times. For example, Lu's research group developed a long-circulating polyethylene glycol-modified ultrasmall iron oxide nanoparticle for monitoring thrombolytic therapy, demonstrating excellent vascular contrast imaging performance. To enhance tumor contrast, the effective enrichment of contrast agents at the tumor site is a key consideration, and several strategies have been designed. For instance, by coupling with targeting ligand molecules, MRI contrast agents can be endowed with the ability to actively recognize tumors. Alternatively, the EPR effect can be improved by rationally designing the surface properties and hydrodynamic dimensions of MRI nanocontrast agents, thereby enhancing their passive tumor enrichment efficiency. However, the synthesis of current MRI nanocontrast agents is generally complex and time-consuming. In recent years, biomineralization methods have been developed for the synthesis of nanoparticles, showing unique advantages, including mild conditions, simple processes, and high controllability. Furthermore, bovine serum albumin, due to its excellent biocompatibility and abundant surface functional groups, has become one of the commonly used templates for preparing biomineralized nanoparticles. Recently, a series of biomineralized nanoprobes based on BSA have been developed for diagnostic imaging of diseases such as tumors, such as BSA@Gd2O3, BSA@MnO2, and BSA@Fe2O3. However, nanoprobes derived from single-metal ion biomineralization still face challenges in the accurate diagnosis of cardiovascular and cerebrovascular diseases and tumors due to insufficient contrast performance and limited tumor-specific response. Bimetallic biomineralized nanoprobes that integrate the advantages of two metal ions may achieve enhanced MRI contrast performance and tumor-specific response contrast behavior, thereby overcoming these limitations. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a bimetallic oxide nanoprobe based on BSA biomimetic mineralization, its preparation method, and its application. This nanoprobe utilizes its long blood metabolism to achieve a long-term time window and passively accumulates in the tumor microenvironment through the EPR effect. It responds to the disintegration in the weakly acidic tumor microenvironment and enhances the T1WI magnetic resonance imaging signal at the tumor location, thereby achieving precise tumor diagnosis.

[0005] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for preparing bimetallic oxide nanoprobes based on BSA biomimetic mineralization is provided, comprising the following steps:

[0006] (1) Dissolve gadolinium chloride hexahydrate and manganese chloride in triple-distilled water to obtain gadolinium chloride solution and manganese chloride solution respectively;

[0007] (2) Under stirring conditions at room temperature, gadolinium chloride solution and manganese chloride solution were added to bovine serum albumin solution to form a mixed solution. After adjusting the pH to 12, the solution was reacted, purified and freeze-dried to obtain a bimetallic oxide nanoprobe based on BSA biomimetic mineralization (BSA@Gd / Mn).

[0008] Furthermore, in step (1), the concentrations of gadolinium chloride solution and manganese chloride solution are 0.1-0.3 mM, respectively.

[0009] Preferably, the concentrations of the gadolinium chloride solution and the manganese chloride solution are both 0.1 mM.

[0010] Furthermore, in step (2), the stirring speed is 500-700 r / min.

[0011] Preferably, the stirring speed is 600 r / min.

[0012] Furthermore, in step (2), the molar ratio of gadolinium to manganese in the mixed solution is 1-3:1-2.

[0013] Furthermore, the concentration of bovine serum albumin solution is 10-75 mg / mL.

[0014] Furthermore, in step (2), the molar ratio of gadolinium to manganese is 2:1; the concentration of bovine serum albumin solution is 50 mg / mL.

[0015] Furthermore, in step (2), a 1 M sodium hydroxide solution is added to adjust the pH to 12.

[0016] Furthermore, in step (2), a dialysis bag with a molecular weight cutoff of 3400-3600 kDa is used to add triple-distilled water for purification for 24 h, and the dialysis solution is replaced 3-4 times.

[0017] Preferably, a dialysis bag with a molecular weight cutoff of 3500 kDa is used to purify the solution with triple-distilled water for 24 hours, and the dialysis solution is replaced 3 times.

[0018] Furthermore, in step (2), during freeze drying, after freezing to a solid state, the water is sublimated and dehydrated under vacuum.

[0019] The present invention also provides the bimetallic oxide nanoprobe based on BSA biomimetic mineralization prepared by the above-mentioned method for preparing bimetallic oxide nanoprobes based on BSA biomimetic mineralization.

[0020] This invention also provides the application of the above-mentioned bimetallic oxide nanoprobes based on BSA biomimetic mineralization in the preparation of vascular display and / or tumor imaging probes.

[0021] Furthermore, the application of bimetallic oxide nanoprobes based on BSA biomimetic mineralization in the preparation of weakly acidic tumor imaging probes.

[0022] Furthermore, the application of bimetallic oxide nanoprobes based on BSA biomimetic mineralization in the preparation of MRI contrast agents.

[0023] The present invention has the following beneficial effects:

[0024] 1. This invention utilizes the biomineralization of BSA with bimetallic ions to synthesize bimetallic oxide nanoprobes for long-lasting MRA imaging and precise tumor diagnosis. The integration of Gd and Mn metals significantly enhances the r1 relaxation rate of BSA@Gd / Mn, facilitating contrast imaging. The BSA@Gd / Mn nanoprobes possess an extended plasma half-life, providing an ample imaging time window, allowing for sustained, high-quality vascular visualization with a single low-dose injection. BSA@Gd / Mn can be effectively internalized by tumor cells through a passive EPR effect. After entering tumor cells, due to the presence of Mn ions, BSA@Gd / Mn exhibits a strong responsive contrast enhancement capability to the weakly acidic tumor microenvironment, which will achieve tumor-specific signal amplification, thereby enabling precise MRI diagnosis of tumors. Therefore, BSA@Gd / Mn, as a highly efficient MRI probe, holds promise for significantly advancing the precise diagnosis of vascular-related diseases and early-stage tumors.

[0025] 2. The bimetallic oxide nanoprobe based on BSA biomimetic mineralization of this invention, compared with single-component metal oxide nanoprobes, exhibits significantly enhanced spin-spin processes due to the superposition of gadolinium and manganese metals, thus achieving an ultra-high longitudinal relaxation rate (r1). Furthermore, utilizing the high homology (76%) between BSA and human plasma albumin, it achieves long-term blood circulation, providing a time window for high-quality vascular imaging. In addition, the r1 value of BSA@Gd / Mn exhibits a pH-dependent increase, which enables tumor-specific signal amplification, thereby achieving precise magnetic resonance imaging diagnosis of tumors.

[0026] 3. This invention uses a biomineralization method to synthesize a bimetallic composite nanoprobe BSA@Gd / Mn. In terms of applications, this invention can achieve continuous high-quality vascular visualization and high-contrast tumor imaging through a single low-dose injection. The resulting nanoprobe has good biocompatibility and excellent magnetic resonance imaging performance; the preparation process is simple, the cost is relatively low, and it is easy to produce; the raw materials are environmentally friendly and free from chemical pollution. Attached Figure Description

[0027] Figure 1 Fourier transform infrared spectra of the products obtained in Example 1 and Comparative Examples 1-3;

[0028] Figure 2 The UV-Vis absorption spectra of the products obtained in Example 1 and Comparative Examples 1-3 are shown below.

[0029] Figure 3 The longitudinal relaxation time of the products obtained in Examples 1-5;

[0030] Figure 4 The longitudinal relaxation time of the products obtained in Examples 1 and 6-8;

[0031] Figure 5 The longitudinal relaxation time of BSA@Gd / Mn, BSA@Mn, BSA@Gd and Gd-DTPA solutions at different concentrations was measured under 3T MRI.

[0032] Figure 6 The manganese ion content of BSA@Gd / Mn in buffer solutions under different pH conditions;

[0033] Figure 7 Gadolinium ion content of BSA@Gd / Mn in buffer solutions under different pH conditions;

[0034] Figure 8 The longitudinal relaxation times of BSA@Gd / Mn, BSA@Gd, and Gd-DTPA solutions at different pH buffers are given for different concentrations.

[0035] Figure 9 Results showing the effect of BSA@Gd / Mn on cell viability;

[0036] Figure 10 Results showing the effect of BSA@Gd / Mn on the migration and repair capacity of HUVEC cells;

[0037] Figure 11 The effect of BSA@Gd / Mn on the in vitro tube formation and differentiation ability of HUVECs;

[0038] Figure 12 CLSM image of BSA@Gd / Mn in CAL27 cells;

[0039] Figure 13 The results of flow cytometry analysis of BSA@Gd / Mn in CAL27 cells;

[0040] Figure 14 The nuclear magnetic resonance imaging effect of BSA@Gd / Mn cells;

[0041] Figure 15 The in vivo magnetic resonance angiography effect of BSA@Gd / Mn;

[0042] Figure 16 The in vivo tumor magnetic resonance imaging effect of BSA@Gd / Mn;

[0043] Figure 17 The results are for the biosafety testing of BSA@Gd / Mn.

[0044] Figure 18 The results are for the blood half-life assay of BSA@Gd / Mn. Detailed Implementation

[0045] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0046] Example 1:

[0047] A bimetallic oxide nanoprobe based on BSA biomimetic mineralization is prepared by the following steps:

[0048] (1) Dissolve gadolinium chloride hexahydrate and manganese chloride in triple-distilled water to obtain a 0.1 mM gadolinium chloride solution and a 0.1 mM manganese chloride solution;

[0049] (2) At room temperature, under stirring at 600 r / min, gadolinium chloride solution and manganese chloride solution were added to bovine serum albumin solution to form a mixed solution (the molar ratio of gadolinium to manganese in the mixed solution was 2:1, and the concentration of bovine serum albumin solution was 50 mg / mL). The pH was adjusted to 12 by adding 1 M sodium hydroxide solution and reacting for 4 h. The solution was purified for 24 h by using a dialysis bag with a molecular weight cutoff of 3500 kDa and adding triple-distilled water. The dialysis solution was replaced 3 times to completely remove unbound metal ions. After freezing to solid, the solution was sublimated and dehydrated under vacuum to obtain a bimetallic oxide nanoprobe based on BSA biomimetic mineralization (BSA@Gd / Mn).

[0050] Example 2:

[0051] The difference between Example 2 and Example 1 is that the molar ratio of gadolinium to manganese in the mixed solution of Example 2 is 3:1.

[0052] Example 3:

[0053] The difference between Example 3 and Example 1 is that the molar ratio of gadolinium to manganese in the mixed solution of Example 3 is 3:2.

[0054] Example 4:

[0055] The difference between Example 4 and Example 1 is that the molar ratio of gadolinium to manganese in the mixed solution of Example 4 is 1:1.

[0056] Example 5:

[0057] The difference between Example 5 and Example 1 is that the molar ratio of gadolinium to manganese in the mixed solution of Example 5 is 1:2.

[0058] Example 6:

[0059] The difference between Example 6 and Example 1 is that the concentration of bovine serum albumin solution in the mixed solution of Example 6 is 10 mg / mL.

[0060] Example 7:

[0061] The difference between Example 7 and Example 1 is that the concentration of bovine serum albumin solution in the mixed solution of Example 7 is 25 mg / mL.

[0062] Example 8:

[0063] The difference between Example 8 and Example 1 is that the concentration of bovine serum albumin solution in the mixed solution of Example 8 is 75 mg / mL.

[0064] Comparative Example 1:

[0065] The difference between Comparative Example 1 and Example 1 is that no gadolinium chloride was added in Comparative Example 1, and the resulting product is denoted as BSA@Mn.

[0066] Comparative Example 2:

[0067] The difference between Comparative Example 2 and Example 1 is that no manganese chloride was added in Comparative Example 2, and the resulting product was denoted as BSA@Gd.

[0068] Comparative Example 3:

[0069] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 does not add gadolinium chloride and manganese chloride, but only bovine serum albumin (BSA).

[0070] Experimental Example 1:

[0071] The Fourier transform infrared (FT-IR) and ultraviolet-visible absorption spectra of the products obtained in Examples 1 and Comparative Examples 1-3 are as follows: Figures 1-2 As shown.

[0072] Depend on Figure 1 It can be seen that after biomineralization synthesis, the infrared spectrum of BSA@Gd / Mn is almost identical to that of natural BSA, both around 1650 cm⁻¹. -1 and 1540 cm -1 The presence of characteristic absorption peaks for amide I and amide II bands confirms the successful modification of BSA. Figure 2 Further compositional evidence was provided in the UV absorption spectrum. The characteristic shoulder peak at ~280 nm is attributed to the π-π* transitions of aromatic amino acids such as tryptophan and tyrosine in BSA. The broad absorption band in the 300-400 nm range is likely related to the plasmon resonance absorption of Gd₂O₃ and MnO₂ in the nanoprobe, which preliminarily indicates the successful formation of Gd and Mn oxides.

[0073] Experimental Example 2:

[0074] 1. Two mL of the products obtained in Examples 1-5 were placed in separate 2 mL centrifuge tubes and labeled. The longitudinal relaxation time of each sample was measured using 3T MRI. The result was calculated using the formula r1=(R1) / C, where the relaxation rate R1 is the reciprocal of T1, and C is the concentration of BSA@Gd / Mn. Figure 3 As shown in the figure. The longitudinal relaxation time of the products obtained in Examples 1 and 6-8 was measured using the same method, and the results are shown in the figure. Figure 4 As shown.

[0075] Depend on Figures 3-4 It can be seen that when the Gd:Mn molar ratio is 2:1 and the BSA concentration is 50 mg / mL, the synthesized BSA@Gd / Mn nanoprobe exhibits the highest longitudinal relaxation rate (r1), which can reach 8.90 mM. -1 •s -1 (3 T field strength).

[0076] 2. The longitudinal relaxation time (T1) of BSA@Gd / Mn, BSA@Mn, BSA@Gd, and Gd-DTPA solutions of different concentrations (0, 0.03125, 0.0625, 0.125, and 0.25 mM, respectively) was measured under 3T MRI. The r1 value for different materials was calculated using the formula. Figure 5 As shown, BSA@Gd / Mn has the highest r1 value (9.28 mM). -1 •s -1 The T1 contrast ratios are approximately 1.5 times that of BSA@Gd, 2 times that of Gd-DTPA, and 4 times that of BSA@Mn, respectively. These results indicate that the superposition of Gd and Mn in BSA@Gd / Mn can significantly enhance the spin-spin process, thereby achieving ultra-high T1 contrast performance.

[0077] 3. Weak acid responsiveness of BSA@Gd / Mn

[0078] Add an appropriate amount of BSA@Gd / Mn to a dialysis bag. Place the dialysis bag (molecular weight cutoff, MWCO=3500) in buffer solutions of different pH values ​​(4.5, 5.5, 6.5, 7.4) and magnetically stir at 37 °C. Remove the PBS solution at different time points (3 h, 6 h, 9 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h) and simultaneously add an equal volume of fresh buffer solution. Measure the Gd and Mn ion content in the solution at each time point using ICP-OES and calculate the cumulative release rate. Figure 6 and Figure 7 As shown.

[0079] Depend on Figure 6 and Figure 7It can be seen that significant Mn was observed under weakly acidic conditions. 2+ Ion release, and as pH decreases, the amount of Mn released from BSA@Gd / Mn increases. 2+ The amount of ions gradually increases. In contrast, even at pH 4.5, the amount of gadolinium ions released by BSA@Gd / Mn is extremely low and negligible, which ensures the in vivo biosafety of BSA@Gd / Mn.

[0080] Different concentrations of BSA@Gd / Mn, BSA@Mn, and BSA@Gd solutions (concentrations of 0, 0.03125, 0.0625, 0.125, and 0.25 mM, respectively) were placed in 10 mM PBS buffer (pH 4.5, 5.5, 6.5, and 7.4; n=3), and then incubated at 37 °C for 12 hours. The longitudinal relaxation time T1 was measured under 3T MRI. The r1 value for different materials was calculated using the formula. Figure 8 As shown, the r1 of the BSA@Gd / Mn nanocomposite at pH 7.4 is 9.28 mM. -1 •s -1 However, at pH 6.5, 5.5, and 4.5, the relaxation rate increased significantly, with r1 rising to 16.74 mM. -1 •s -1 It is significantly higher than the relaxation rate of single-component materials at the same pH.

[0081] 4. Biosafety of BSA@Gd / Mn

[0082] (1) CCK8 assay to detect the effect of BSA@Gd / Mn on cell viability

[0083] The cytotoxicity of BSA@Gd / Mn nanoparticles was investigated using HUVEC and VERO, THLE-2. DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin was used to culture the cells. On day 1, cells were cultured at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [insert density here] in 96-well plates and cultured at 37 °C under a 5% CO2 atmosphere. The next day, dead cells were washed twice with PBS (10 mM, pH 7.4). BSA@Gd / Mn nanoprobes were dissolved in fresh culture medium at concentrations ranging from 25, 50, 100, 300, 600, to 900 mg / L, and the washed cells were then incubated in the same medium for 24 hours. Cell viability was determined using the Cell Counting Kit-8 (CCK-8). A 1-minute shaking procedure was required to ensure uniform distribution of the azo dye. Cell viability was measured at a wavelength of 450 nm. Figure 9 As shown.

[0084] Depend on Figure 9 It can be seen that even at high concentrations (900 mg / L) -1 Under these conditions, the survival rate of all three types of normal cells remained above 75%, and no obvious dose-dependent toxicity trend was observed.

[0085] (2) Scratch assay to detect the effect of BSA@Gd / Mn on the migration and repair ability of HUVEC cells

[0086] HUVEC cells were loaded at a rate of 6 × 10 5 Cells were seeded at a density of 10 cells / well in 6-well plates. A straight wound was drawn on the confluent cell monolayer using a sterile 200 μL pipette tip. After washing with cold PBS, the wound was imaged at 10x magnification using an inverted microscope (IX-53, Olympus). Cells were then co-incubated for 12 hours with different concentrations (0, 300, 600, and 900 µg / mL) of BSA@Gd / Mn nanoprobes; the same wound area was then observed again under a microscope. Images were analyzed using ImageJ software (National Institutes of Health, Bethesda, Maryland). HUVEC cell migration was calculated using the following formula: [(0-hour wound width - 12-hour wound width) / 0-hour wound width] × 100%. The experiment was performed in three independent biological replicates. The results are as follows: Figure 10 As shown.

[0087] Depend on Figure 10 It is known that even at high concentrations of nanoprobes (900 mg / L), -1 Under exposure, the relative migration rate of HUVEC cells was not significantly different from that of the control group, indicating that the nanoprobes do not affect the migration and repair function of endothelial cells.

[0088] (3) Tube formation experiment to detect the effect of BSA@Gd / Mn on the in vitro tube formation differentiation ability of HUVECs

[0089] Human umbilical vein endothelial cells (HUVECs) were stored at 4 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. Cells were then treated with different concentrations (0, 300, 600, and 900 µg / mL) of BSA@Gd / Mn nanoprobes for 24 hours. Liquefied Matrigel (100 µL) was carefully added to 24-well plates, which were then transferred to a 37 °C incubator for curing for 30 minutes. BSA@Gd / Mn-treated cells were gently added to the 24-well plates, and the plates were incubated at 37 °C for 4 to 12 hours. Lumen formation was observed at different time intervals, and images were captured using an Olympus IX-53 microscope. The number of lumens formed in the captured images was quantitatively analyzed using ImageJ software. Figure 11As shown, the results indicate that even in the presence of high concentrations of BSA@Gd / Mn, HUVEC cells can still effectively form a complete vascular network structure. Quantitative analysis of the key vascularization parameter, the number of vascularization nodes, showed no statistically significant difference between the experimental group and the control group.

[0090] 5. Internalization efficiency of BSA@Gd / Mn in CAL27 cells

[0091] (1) Culture of tumor cells

[0092] CAL27 cells were fed at a dose of 1×10 7 CAL-27 cells were seeded at a density of 0.5 h in 10 cm culture dishes. Based on the cytotoxicity results, CAL-27 cells were treated with different concentrations of BSA@Gd / Mn probe (0, 100, 200, 400 mg / L) and at different times (0.5 h, 1 h, 2 h).

[0093] (2) Internalization efficiency was detected by confocal microscopy and flow cytometry.

[0094] After treatment, peripheral airborne matter was removed by washing with cold PBS, followed by fixation in 4% paraformaldehyde for 15 min. The cells were then washed three times with PBS and stained with DAPI in the dark for 15 min. Finally, the uptake of the probe by cells was determined by observation under a confocal laser scanning microscope (CLSM) and visualization and quantitative analysis by flow cytometry. Figure 12 and Figure 13 As shown, the uptake of the nanoprobe in CAL27 cells exhibited a significant dose- and time-dependent pattern. CLSM images clearly showed that the RhB fluorescence signal (red) was mainly concentrated in the cytoplasm, and the fluorescence intensity significantly increased with prolonged incubation time and probe concentration. Flow cytometry quantification further confirmed that the average intracellular fluorescence intensity increased with increasing dose and time, indicating that CAL27 cells can effectively and efficiently internalize the BSA@Gd / Mn nanoprobe.

[0095] 6. Cell nuclear magnetic resonance imaging effect of BSA@Gd / Mn.

[0096] (1) Culture of tumor cells

[0097] CAL27 cells were fed at a dose of 1×10 7 CAL-27 cells were seeded at a density of 10 cm in culture dishes. Based on the cytotoxicity results, CAL-27 cells were treated with different concentrations of BSA@Gd / Mn probe (10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL GD) and at different times (0 h, 0.5 h, 1 h, 2 h, 4 h).

[0098] (2) Cell nuclear magnetic resonance imaging

[0099] The supernatant was aspirated, and the cells were washed twice with 2 mL PBS. Cells were then digested with trypsin, centrifuged to collect the cells, resuspended in 1 mL of 1% agarose solution, and labeled. T1-weighted imaging was then performed on 3.0 T MRI. T1-weighted imaging was used with the following parameters: repetition time (TR) = 150 ms, echo time (TE) = 11 ms, matrix size (Width × Height) = 229 × 229, flip angle = 150°, and slice thickness = 3.0 mm. After imaging, pseudocolor processing was performed using Niumag NMR Image Processing Software. Figure 14 As shown, the T1 signal intensity of the cell clusters significantly increased in a concentration-dependent manner with increasing probe concentration, further confirming the effective uptake of the nanoprobes by cells and the resulting significant relaxation enhancement effect. Furthermore, a consistent time-dependent signal enhancement trend was observed in assessments at different incubation time points.

[0100] 7. In vivo magnetic resonance angiography results of BSA@Gd / Mn.

[0101] (1) Selection of animal models

[0102] This experiment used male SD rats as an imaging model, with an age of 5 weeks.

[0103] (2) In vivo magnetic resonance imaging of small animals

[0104] MRA images of healthy male rats were scanned using 7T MRI. SD rats were anesthetized with isoflurane at a dose of 3–4%. After 3–5 minutes, the rats were transferred to the scanning coil, and the isoflurane concentration was maintained at 1–1.5% during MRI scanning. For MRA imaging, SD rats were injected with BSA@Gd / Mn (0.05 mmol Gd / kg) via a tail vein catheter, with the same doses of Gd-DTPA, BSA@Mn, and BSA@Gd used as controls. All tested rats were placed in a prone position during MRI. Angiographic data were acquired using a volumetric radiofrequency coil with a TOF-3D sequence at 7T. Scanning parameters were as follows: repetition time (TR) = 11.264 ms, echo time (TE) = 1.714 ms, matrix size (Width × Height) = 500 × 500, flip angle = 30°. Image post-processing was performed using Radiant DicomViewer software, and vascular reconstruction was performed using MIP and MPR analysis techniques. The acquired images are adjusted to fit within the same image window. For example... Figure 15As shown, the major head and neck vessels, such as the superficial temporal vein, supraorbital vein, external jugular vein, and common carotid artery, can be clearly visualized 5 minutes after injection. The BSA@Gd / Mn nanoprobe achieves simultaneous arteriovenous imaging of the brain through a single TOF scan. Thanks to its high T1 relaxation rate and long circulation characteristics, the imaging not only clearly displays the main head and neck vessels but also clearly visualizes microvessels as small as 0.15 mm in diameter. Similarly, the imaging effect of BSA@Gd / Mn was compared with single-component control materials (Gd2O3@BSA, BSA@Mn) and clinically commonly used small molecule contrast agents (Gd-DTPA) under the same conditions. The vascular enhancement effects (including signal intensity, vascular clarity, and duration) of Gd2O3@BSA and BSA@Mn were weaker than those of the bimetallic BSA@Gd / Mn, while clinically commonly used Gd-DTPA did not show significant cerebral vascular imaging under TOF sequences. This significant performance difference highlights the unique advantages of BSA@Gd / Mn nanoprobes as long-acting vascular imaging contrast agents. This ultra-high spatial resolution imaging capability has important clinical significance for the accurate diagnosis and treatment guidance of small vessel diseases (such as microaneurysms, early arteriovenous malformations, telangiectasia, etc.).

[0105] 8. In vivo tumor magnetic resonance imaging effect of BSA@Gd / Mn.

[0106] (1) Selection of animal models

[0107] CAL-27 cells in logarithmic growth phase were digested with 0.25% trypsin-EDTA solution and centrifuged at 300×g for 5 min. The supernatant was discarded, and the cell pellet was resuspended in PBS. After cell counting, the cells were centrifuged at 300×g for 5 min. The cell pellet was resuspended in DMEM medium, and the cell suspension was added to a high concentration of Matrigel, ensuring a final Matrigel concentration of not less than 10 mg / mL and a cell density of 2-3×10⁶ cells / mL. 6 / 200 μL. 200 μL of cell-Matrigel mixture was injected subcutaneously into 4-6 week old female BALB / c-nu nude mice for in vivo magnetic resonance imaging.

[0108] (2) In vivo magnetic resonance imaging of small animals

[0109] Before administering contrast agents to tumor model mice, 7.0 T MRI with a volumetric radiofrequency coil and T1-weighted imaging was used to obtain the baseline signal intensity of tumor and normal tissues. Then, the target probes BSA@Gd / Mn were administered to the mice via tail vein injection. The scanning parameters were as follows: repetition time (TR) = 2400 ms, echo time (TE) = 15 ms, matrix size (Width×Hight) = 400×400, and flip angle = 90°. The same doses of Gd-DTPA, BSA@Mn, and BSA@Gd were used as controls. Images were re-scanned at different time points after contrast agent administration using the same parameters. Regions of interest (ROIs) were plotted in the tumor and normal tissue areas using image analysis software, and signal intensity changes were measured. The degree of change in signal intensity of tumor and normal tissues at different time points was statistically analyzed and compared with Gd-DTPA, BSA@Mn, and BSA@Gd to clarify the contrast efficacy. Figure 16 As shown, the tumor signal in all groups gradually increased over time, indicating that the contrast agent was enriched in the tumor to some extent. However, the enhancement effect of the BSA@Gd / Mn experimental group was significantly better than that of Gd-DTPA and the single group. Approximately 2 hours after injection, the tumor area in the BSA@Gd / Mn group exhibited the brightest T1 signal, indicating that the probe concentration within the tumor reached its peak at this time. To objectively quantify the contrast enhancement effect, the signal-to-noise ratio (SNR) of the tumor area was analyzed using RadiAnt DICOM Viewer software. The maximum ΔSNR of the tumors in the free Gd-DTPA, BSA@Gd, and BSA@Mn groups were 18.05±3.28, 14.65±2.01, and 14.03±5.94, respectively, significantly lower than that of the BSA@Gd / Mn group (47.43±6.84). This trend is consistent with the findings of tumor MRI image observations, jointly confirming that Gd2O3 / BSA@Mn nanoprobes, as tumor microenvironment-responsive nanomagnetic resonance imaging contrast agents (nanoMRI CAs), can significantly enhance the diagnostic contrast of tumors.

[0110] 9. Biosafety of BSA@Gd / Mn

[0111] Male Sprague-Dawley (SD) rats were randomly divided into two groups, one injected with physiological saline and the other with BSA@Gd / Mn (dose 0.05 mM(Gd) / kg). Seven days later, blood was collected from the SD rats for complete blood count and blood biochemical analysis. All rats were subsequently euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys were collected for hematoxylin-eosin (H&E) staining analysis. Figure 17As shown, H&E staining results indicated no significant pathological changes in any tissues, confirming that BSA@Gd / Mn has excellent tissue biocompatibility.

[0112] 10. Blood half-life of BSA@Gd / Mn

[0113] Male SD rats were intravenously injected with a single dose (10 mg / kg) of BSA@Gd / Mn. Blood samples were collected from the SD rats at different time intervals and digested with concentrated nitric acid. Subsequently, the Gd content in the blood was quantified using inductively coupled plasma optical emission spectrometry (ICP-OES). The circulating half-life of BSA@Gd / Mn was calculated using a fitted curve of the Gd content in the blood over time. Figure 18 As shown, by monitoring the change in gadolinium concentration in the blood over time, we calculated the blood half-life (t1 / 2) of the BSA@Gd / Mn nanoprobe to be 122.00 ± 7.00 min. This value is approximately 6 times that of Gd-DTPA (approximately 20 minutes). Twenty hours after administration, the gadolinium concentration in the blood had decreased to a negligible level, confirming that the nanoprobe has no in vivo accumulation characteristics and avoiding concerns about long-term toxicity.

[0114] In summary, the bimetallic oxide nanoprobe BSA@Gd / Mn based on BSA biomimetic mineralization of this invention can be used for the precise diagnosis of head and neck vascular diseases and tumors. The superposition of Gd and Mn bimetals significantly enhances the spin-spin relaxation process, giving BSA@Gd / Mn an ultra-high r1 value (9.28 mM at 3.0T). -1 •s -1 Due to the presence of Mn, the r1 value of BSA@Gd / Mn gradually increases with decreasing pH, exhibiting pH-sensitive contrast enhancement properties. Furthermore, BSA@Gd / Mn possesses a long blood circulation time, providing an ample time window for high-resolution vascular and tumor imaging. In in vivo MRA experiments, BSA@Gd / Mn clearly visualized the vascular network in the mouse head and neck, even effectively diagnosing microvessels as small as 0.15 mm. Simultaneously, the pH-responsive contrast behavior of this nanoprobe significantly improves the tumor signal-to-noise ratio, achieving tumor-specific signal amplification and promoting accurate tumor diagnosis. Finally, the BSA@Gd / Mn nanoprobe exhibits negligible systemic toxicity and blood compatibility, demonstrating excellent biosafety. Overall, the development of BSA@Gd / Mn nanoprobes will greatly enhance the accuracy of diagnosis for cardiovascular and cerebrovascular diseases and tumors.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a bimetal oxide nanoprobe based on BSA biomimetic mineralization, characterized in that, The method comprises the following steps: (1) Dissolve gadolinium chloride hexahydrate and manganese chloride in triple distilled water respectively to obtain a gadolinium chloride solution and a manganese chloride solution; (2) Under stirring at room temperature, add the gadolinium chloride solution and the manganese chloride solution into a bovine serum albumin solution to form a mixed solution, adjust the pH to 12 for reaction, and then purify and freeze-dry to obtain a BSA biomimetic mineralized bimetal oxide nanoprobe.

2. The method for preparing BSA biomimetic mineralized bimetal oxide nanoprobe according to claim 1, characterized in that, In step (1), the concentration of the gadolinium chloride solution and the manganese chloride solution is 0.1-0.3 mM respectively.

3. The method for preparing BSA-biomimetic mineralized bimetal oxide nanoprobe according to claim 1, wherein, In step (2), the stirring speed is 500-700 r / min.

4. The method for preparing BSA-biomimetic mineralized bimetal oxide nanoprobe according to claim 1, characterized in that, In step (2), the molar ratio of gadolinium to manganese in the mixed solution is 1-3:1-2.

5. The method for preparing BSA-biomimetic mineralized bimetal oxide nanoprobe according to claim 1, wherein, In step (2), the concentration of the bovine serum albumin is 10-75 mg / mL.

6. The method for preparing BSA-biomimetic mineralized bimetal oxide nanoprobe according to claim 1, wherein, In step (2), the pH is adjusted to 12 by adding a 1 M sodium hydroxide solution.

7. The method for preparing BSA-biomimetic mineralized bimetal oxide nanoprobe according to claim 1, wherein, In step (2), the mixed solution is purified by adding triple distilled water into a dialysis bag with a molecular weight cut-off value of 3400-3600 kda for 24 h, and the dialysate is replaced for 3-4 times.

8. The method for preparing BSA-biomimetic mineralized bimetal oxide nanoprobe according to claim 1, wherein, In step (2), during freeze-drying, the solution is frozen to solid state and then sublimed under vacuum.

9. The BSA biomimetic mineralized bimetal oxide nanoprobe prepared by the method of any one of claims 1-8.

10. The use of the BSA biomimetic mineralized bimetal oxide nanoprobe of claim 9 in the preparation of a blood vessel display and / or tumor imaging probe.

Images