Glutathione responsive signal switching MRI (Magnetic Resonance Imaging) nanoprobe as well as preparation method and application thereof

Abstract

The invention discloses a glutathione responsive signal switching MRI (Magnetic Resonance Imaging) nanoprobe as well as a preparation method and application thereof, and relates to the technical field of biomedical material preparation. The method comprises the following steps: firstly preparing extremely small iron oxide nanoparticles, then modifying the extremely small iron oxide nanoparticles into hydrophilic nanoparticles through tetrafluoroborate nitrite, and then carrying out crosslinking and self-assembly on the nanoparticles by utilizing selenium-rich polyethylene glycol to construct an iron oxide nano aggregate, thus obtaining the glutathione responsive signal switching MRI nano probe (contrast agent). The contrast agent prepared by the invention contains rich diselenide bonds, can present different conformations at parts with different GSH contents, further realizes conversion of an MRI signal from T2-T1, and has good biological safety. And the method is helpful for obtaining high-contrast-ratio liver magnetic resonance imaging, and is suitable for precise diagnosis of tiny liver cancer. The invention solves the problem of lack of a contrast agent capable of accurately diagnosing mHCC in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical material preparation, and particularly relates to a glutathione-responsive signal-switching MRI nanoprobe and a preparation method and application thereof. Background Art

[0002] Liver cancer (HCC) is one of the most common malignant tumors globally. The fatality rate of liver cancer is high, but for low blood supply liver cancer nodules with a diameter within 2 cm, they grow slowly, only undergo local infiltration, and are not prone to metastasis. Therefore, early and accurate diagnosis of micro-small hepatocellular carcinoma (MHCC) with a tumor diameter < 1 cm is a key factor affecting the clinical treatment effect. Although percutaneous liver biopsy is regarded as the gold standard for obtaining accurate pathological information, due to its invasiveness and related complications such as infection, bleeding, and pain, as well as potential sampling errors, it has certain limitations. This is especially true when frequent monitoring or diagnosis of small and ambiguous tumors is required.

[0003] Currently, clinical methods available for diagnosing MHCC include ultrasound imaging, CT, and MRI, etc. Clinical diagnosis tends to favor non-invasive imaging methods such as ultrasound and computed tomography (CT). These techniques show high sensitivity in diagnosing advanced HCC and can effectively evaluate the degree of lesions. However, for early tumors smaller than 1 cm, the limitations of these traditional imaging techniques become particularly obvious, and the diagnostic accuracy is significantly reduced. For example, ultrasound depends to a large extent on the experience and skills of the operator and has limitations in detecting nodules in overweight patients or nodules located deep in the liver and near the ribs; CT scans are fast, but their soft tissue resolution is low. When the density of the liver changes, the density of the lesion is similar to that of the liver, making it easy to miss the diagnosis, and CT has a relatively large ionizing radiation to the human body. In contrast, magnetic resonance imaging (MRI) shows great advantages in diagnosing mHCC. The main advantages of MRI technology include no ionizing radiation, high spatial and temporal resolution, infinite penetration depth, and excellent soft tissue contrast. By injecting contrast agents, MRI can provide detailed anatomical and biochemical information about the tumor and its surrounding tissues. Commonly used contrast agents include gadolinium-based (Gd-DTPA) T1 contrast agents and superparamagnetic iron oxide nanoparticle (SPION)-based T2 contrast agents.

[0004] Although traditional single - mode T1 contrast agents, such as Gd - DTPA, perform well in enhancing T1 relaxation rate, they usually produce the same phase difference in normal and tumor tissues, resulting in enhanced signal intensity in both, thus reducing the contrast and blurring the boundaries between tissues, making accurate diagnosis challenging. Therefore, the development of responsive magnetic resonance imaging contrast agents that can change signals under different conditions has become an urgent need for the accurate diagnosis of mHCC. Summary of the Invention

[0005] In order to solve the above - mentioned technical problems, the object of the present invention is to provide a glutathione - responsive signal - switching MRI nanoprobe and its preparation method and application, so as to solve the problem in the prior art of the lack of contrast agents capable of accurately diagnosing mHCC.

[0006] The technical solution of the present invention to solve the above - mentioned technical problems is as follows: Provide a preparation method of a glutathione - responsive signal - switching MRI nanoprobe, including the following steps:

[0007] (1) Synthesis of ultrasmall iron oxide nanoparticles: Mix iron oleate, oleic acid and oleyl alcohol, dissolve them in diphenyl ether to obtain a reaction solution, heat the reaction, cool to room temperature, then add acetone, centrifuge, and disperse in n - hexane to prepare an ultrasmall iron oxide nanoparticle solution;

[0008] (2) Surface modification of ultrasmall iron oxide nanoparticles: Disperse the ultrasmall iron oxide nanoparticle solution prepared in step (1) in toluene, add methanol containing nitrosyl tetrafluoroborate, then sonicate, stir, centrifuge, disperse in water, and filter through a membrane to prepare a hydrophilic ultrasmall iron oxide nanoparticle solution;

[0009] (3) Self - assembly of iron oxide nanoparticles: First disperse the hydrophilic ultrasmall iron oxide nanoparticle solution prepared in step (2) in a bis - selenium polyethylene glycol solution, stir for the first time, centrifuge for the first time, then disperse the obtained precipitate in the bis - selenium polyethylene glycol solution for the second time, stir for the second time, centrifuge for the second time, wash, centrifuge for the third time, and disperse in water to prepare a glutathione - responsive signal - switching MRI nanoprobe.

[0010] The beneficial effects of the present invention are as follows: The contrast agent prepared by the present invention contains abundant diselenide bonds, can present different conformations at sites with different GSH contents, and thus realizes the conversion of MRI signals from T2 to T1, and has good biological safety. It helps to obtain high - contrast liver magnetic resonance imaging and is suitable for the accurate diagnosis of small hepatocellular carcinoma.

[0011] On the basis of the above - mentioned technical solution, the present invention can also be improved as follows:

[0012] Further, in step (1), the mass-volume ratio of iron oleate, oleic acid, oleyl alcohol, diphenyl ether, and acetone is 1.5-2 g: 0.5-0.6 g: 1-2 g: 8-12 g: 45-55 mL.

[0013] Further, in step (1), the mass-volume ratio of iron oleate, oleic acid, oleyl alcohol, diphenyl ether, and acetone is 1.8 g: 0.57 g: 1.61 g: 10 g: 50 mL.

[0014] Further, in step (1), the mixing and dissolution are carried out at room temperature.

[0015] Further, in step (1), under the protection of an inert gas, it is heated and reacted at 250-280 °C for 30-40 min.

[0016] Further, in step (1), under the protection of an inert gas, it is heated and reacted at 250 °C for 30 min.

[0017] Further, in step (1), it is centrifuged at 12000-14000 rpm for 10-20 min.

[0018] Further, in step (1), it is centrifuged at 14000 rpm for 10 min.

[0019] Further, in step (1), the concentration of the ultrafine iron oxide nanoparticle solution is 0.5-2.5 mg / mL.

[0020] Further, in step (1), the concentration of the ultrafine iron oxide nanoparticle solution is 1.5 mg / mL.

[0021] Further, in step (2), the volume ratio of the ultrafine iron oxide nanoparticle solution, toluene, and methanol containing nitrosyl tetrafluoroborate is 0.5-2: 8-12: 4-6.

[0022] Further, in step (2), the volume ratio of the ultrafine iron oxide nanoparticle solution, toluene, and methanol containing nitrosyl tetrafluoroborate is 0.5: 10: 5.

[0023] Further, the concentration of nitrosyl tetrafluoroborate in the methanol containing nitrosyl tetrafluoroborate is 8-12 mg / mL.

[0024] Further, the concentration of nitrosyl tetrafluoroborate in the methanol containing nitrosyl tetrafluoroborate is 10 mg / mL.

[0025] Further, in step (2), it is ultrasonically treated for 5-10 min.

[0026] Further, in step (2), it is stirred at room temperature for 24-36 h.

[0027] Further, in step (2), centrifuge at 12000 - 14000 rpm for 10 - 20 min.

[0028] Further, in step (2), disperse in triple distilled water with a pH value of 8 - 10.

[0029] Further, in step (2), the pore size of the filter membrane is 0.2 - 0.3 μm.

[0030] Further, in step (2), the pore size of the filter membrane is 0.22 μm.

[0031] Further, in step (2), the concentration of the hydrophilic ultrafine iron oxide nanoparticle solution is 1 - 1.5 mg / mL.

[0032] Further, in step (2), the concentration of the hydrophilic ultrafine iron oxide nanoparticle solution is 1 mg / mL.

[0033] Further, in step (3), during the first dispersion, the volume ratio of the hydrophilic ultrafine iron oxide nanoparticle solution to the bis-selenium polyethylene glycol solution is 0.5 - 2:13 - 17.

[0034] Further, in step (3), during the first dispersion, the volume ratio of the hydrophilic ultrafine iron oxide nanoparticle solution to the bis-selenium polyethylene glycol solution is 0.5:15.

[0035] Further, in step (3), the bis-selenium polyethylene glycol solution is prepared by dissolving bis-selenium polyethylene glycol in phosphate buffer solution.

[0036] Further, the concentration of the phosphate buffer solution is 10 mmol / L and the pH value is 8.

[0037] Further, in step (3), the concentration of the bis-selenium polyethylene glycol solution is 4 - 6 mg / mL.

[0038] Further, in step (3), the concentration of the bis-selenium polyethylene glycol solution is 5 mg / mL.

[0039] Further, in step (3), during the second dispersion, the mass-volume ratio of the precipitate to the bis-selenium polyethylene glycol solution is 0.5 - 3 mg:8 - 12 mL.

[0040] The beneficial effects of adopting the above further technical solutions are:

[0041] Further, in step (3), during the second dispersion, the mass-volume ratio of the precipitate to the bis-selenium polyethylene glycol solution is 0.5 mg:10 mL.

[0042] Further, in step (3), the time for both the first stirring and the second stirring is 12 - 24 h.

[0043] Further, in step (3), the first centrifugation, the second centrifugation, and the third centrifugation are all carried out at 12,000 - 14,000 rpm for 10 - 20 min.

[0044] Further, in step (3), it is washed successively with triple distilled water and ethanol, and repeated 2 - 3 times.

[0045] Further, in step (3), it is dispersed in triple distilled water.

[0046] The present invention also provides a glutathione-responsive signal-switching MRI nanoprobe prepared by the preparation method of the above-mentioned glutathione-responsive signal-switching MRI nanoprobe.

[0047] The present invention also provides the application of the above-mentioned glutathione-responsive signal-switching MRI nanoprobe in the preparation of an MRI contrast agent.

[0048] The present invention has the following beneficial effects:

[0049] 1. The present invention first prepares extremely small iron oxide nanoparticles, then modifies them into hydrophilic nanoparticles by nitrite tetrafluoroborate, and then crosslinks the nanoparticles using diselenide polyethylene glycol (DSPE-PEG-Se-Se-PEG-DSPE) to self-assemble and construct iron oxide nanoparticle aggregates, that is, a glutathione-responsive signal-switching MRI nanoprobe (contrast agent) is obtained. The iron oxide nanoparticle aggregate contrast agent prepared by the present invention can target siderophilic hepatocytes and present T2 contrast; due to the rich glutathione (GSH) in liver cancer cells, the diselenide bond can be cut off, making it transform from an aggregated state to a monodispersed state, realizing the conversion of MRI signals from T2 to T1, and having good biosafety, which helps to obtain highly sensitive and accurate MRI images for diagnosing small liver cancers.

[0050] 2. Triggered by GSH in tumor tissues, the size of the extremely small iron oxide nanoparticle assembly of the glutathione-responsive signal-switching MRI nanoprobe of the present invention reversibly changes, realizing the switching of T2 dark contrast signals in normal tissues and T1 bright contrast signals in liver cancer tissues. Therefore, the extremely small iron oxide nanoparticle assembly maintains an aggregated state in the normal cell environment, while the extremely small iron oxide nanoparticle assembly in the tumor microenvironment lyses into a monodispersed state, so that MRI can clearly distinguish the tumor (bright) from the surrounding liver tissue (dark) images, significantly expanding the signal difference between normal and diseased tissues and improving the diagnostic accuracy of early liver cancer. Description of the Drawings

[0051] Figure 1 Schematic diagram for the preparation of a glutathione-responsive signal-switching MRI nanoprobe;

[0052] Figure 2 Transmission electron micrograph of the products at each stage of Example 1;

[0053] Figure 3 Hydrated particle size diagram of the products at each stage of Example 1;

[0054] Figure 4 Fitted bar graph of longitudinal relaxation rate before and after assembly and incubation of the product of Example 1;

[0055] Figure 5 Fitted bar graph of transverse relaxation rate before and after assembly and incubation of the product of Example 1;

[0056] Figure 6 Graph of cell safety determination of the nanoprobe prepared in Example 1;

[0057] Figure 7 MR imaging in vivo of a mouse model of in situ micro - hepatocellular carcinoma using the nanoprobe prepared in Example 1;

[0058] Figure 8 Tissue signal - to - noise ratio corresponding to the MR imaging in vivo of a mouse model of micro - hepatocellular carcinoma in Example 1. Detailed implementation manners

[0059] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained by commercial purchase.

[0060] Triple - distilled water is ultrapure water prepared by a three - distillation process.

[0061] The structural formula of bis - selenium polyethylene glycol is DSPE - PEG - Se - Se - PEG - DSPE, and the manufacturer is Shanghai Yuanye Bio - Technology Co., Ltd.

[0062] Example 1:

[0063] A glutathione - responsive signal - switching MRI nanoprobe, and its preparation method includes the following steps: (The preparation schematic diagram is shown in Figure 1 )

[0064] (1) Synthesis of ultrasmall iron oxide nanoparticles:

[0065] At room temperature, iron oleate, oleic acid and oleyl alcohol were mixed and dissolved in diphenyl ether to obtain a reaction solution. Under the protection of inert gas N2, the reaction was heated at 250 °C for 30 min, cooled to room temperature, then acetone was added for precipitation, and nanoparticles were collected by centrifugation at 14000 rpm for 10 min, and dispersed in n-hexane to prepare an extremely small iron oxide nanoparticle solution (concentration: 1.5 mg / mL); among them, the mass volumes of iron oleate, oleic acid, oleyl alcohol, diphenyl ether and acetone were 1.8 g, 0.57 g, 1.61 g, 10 g, 50 mL respectively;

[0066] (2) Surface modification of extremely small iron oxide nanoparticles:

[0067] 0.5 mL of the extremely small iron oxide nanoparticle solution prepared in step (1) was dispersed in 10 mL of toluene, 5 mL of methanol containing nitrosyl tetrafluoroborate (concentration of nitrosyl tetrafluoroborate: 10 mg / mL) was added, then ultrasonicated for 5 min, mechanically stirred at room temperature for 24 h, centrifuged at 14000 rpm for 10 min, the nanoparticles were dispersed in triple-distilled water with a pH value of 8, and filtered through a filter membrane with a pore size of 0.22 μm to prepare a hydrophilic extremely small iron oxide nanoparticle solution (concentration: 1 mg / mL, ESIONP);

[0068] (3) Self-assembly of iron oxide nanoparticles:

[0069] 0.5 mL of the hydrophilic extremely small iron oxide nanoparticle solution prepared in step (2) was first dispersed in 15 mL of a diselenide polyethylene glycol solution (concentration of diselenide polyethylene glycol solution: 5 mg / mL, prepared by dissolving diselenide polyethylene glycol in phosphate buffer solution, concentration of phosphate buffer solution: 10 mmol / L, pH value: 8), and first rotated and stirred at room temperature for 12 h, centrifuged at 14000 rpm for 10 min for the first time, then the obtained 0.5 mg precipitate was secondarily dispersed in 10 mL of a diselenide polyethylene glycol solution (concentration of diselenide polyethylene glycol solution: 5 mg / mL, prepared by dissolving diselenide polyethylene glycol in phosphate buffer solution, concentration of phosphate buffer solution: 10 mmol / L, pH value: 8), and secondarily rotated and stirred at room temperature for 12 h, centrifuged at 14000 rpm for 10 min for the second time, the precipitate was washed successively with triple-distilled water and ethanol, repeated 2 times, centrifuged at 14000 rpm for 10 min for the third time to collect the product, and dispersed in triple-distilled water to prepare a glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP).

[0070] Example 2:

[0071] A glutathione-responsive signal-switching MRI nanoprobe, and its preparation method includes the following steps:

[0072] (1) Synthesis of ultra-small iron oxide nanoparticles:

[0073] At room temperature, iron oleate, oleic acid, and oleyl alcohol were mixed and dissolved in diphenyl ether to obtain a reaction solution. Under the protection of an inert gas, the reaction was heated at 260 °C for 40 min, cooled to room temperature, and then acetone was added for precipitation. The nanoparticles were collected by centrifugation at 13,000 rpm for 12 min and dispersed in n-hexane to obtain an ultra-small iron oxide nanoparticle solution (concentration: 1 mg / mL). Among them, the mass volumes of iron oleate, oleic acid, oleyl alcohol, diphenyl ether, and acetone were 1.5 g, 0.57 g, 1.61 g, 10 g, and 50 mL, respectively.

[0074] (2) Surface modification of ultra-small iron oxide nanoparticles:

[0075] 1 mL of the ultra-small iron oxide nanoparticle solution prepared in step (1) was dispersed in 10 mL of toluene, 5 mL of methanol containing nitrosyl tetrafluoroborate (concentration of nitrosyl tetrafluoroborate: 10 mg / mL) was added, then sonicated for 8 min, mechanically stirred at room temperature for 24 h, centrifuged at 13,000 rpm for 12 min, the nanoparticles were dispersed in triple-distilled water with a pH value of 8, and filtered through a 0.22-μm pore size filter membrane to obtain a hydrophilic ultra-small iron oxide nanoparticle solution (concentration: 1 mg / mL).

[0076] (3) Self-assembly of iron oxide nanoparticles:

[0077] 0.5 mL of the hydrophilic ultra-small iron oxide nanoparticle solution prepared in step (2) was first dispersed in 15 mL of a bis-selenide polyethylene glycol solution (concentration of bis-selenide polyethylene glycol solution: 5 mg / mL, prepared by dissolving bis-selenide polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH value of 8), and first rotated and stirred at room temperature for 24 h, centrifuged at 13,000 rpm for 15 min for the first time. Then, the obtained 0.5 mg precipitate was secondarily dispersed in 10 mL of a bis-selenide polyethylene glycol solution (concentration of bis-selenide polyethylene glycol solution: 5 mg / mL, prepared by dissolving bis-selenide polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH value of 8), and second rotated and stirred at room temperature for 12 h, centrifuged at 13,000 rpm for 15 min for the second time. The precipitate was washed successively with triple-distilled water and ethanol, repeated 2 times, centrifuged at 13,000 rpm for 15 min for the third time to collect the product, and dispersed in triple-distilled water to obtain a glutathione-responsive signal-switching MRI nanoprobe.

[0078] Example 3:

[0079] A glutathione-responsive signal-switching MRI nanoprobe, and its preparation method comprises the following steps:

[0080] (1) Synthesis of ultrasmall iron oxide nanoparticles:

[0081] At room temperature, iron oleate, oleic acid, and oleyl alcohol are mixed and dissolved in diphenyl ether to obtain a reaction solution. Under the protection of an inert gas, the reaction solution is heated at 270 °C for 30 min, cooled to room temperature, and then acetone is added for precipitation. The nanoparticles are collected by centrifugation at 13000 rpm for 15 min and dispersed in n-hexane to obtain an ultrasmall iron oxide nanoparticle solution (concentration: 0.5 mg / mL). Among them, the mass-to-volume ratios of iron oleate, oleic acid, oleyl alcohol, diphenyl ether, and acetone are 1.8 g, 0.57 g, 1.61 g, 10 g, and 50 mL, respectively.

[0082] (2) Surface modification of ultrasmall iron oxide nanoparticles:

[0083] Disperse 2 mL of the ultrasmall iron oxide nanoparticle solution prepared in step (1) in 10 mL of toluene, add 5 mL of methanol containing nitrosyl tetrafluoroborate (concentration of nitrosyl tetrafluoroborate: 10 mg / mL), then ultrasonicate for 8 min, mechanically stir at room temperature for 36 h, centrifuge at 13000 rpm for 15 min, disperse the nanoparticles in triple-distilled water with a pH of 8, and filter through a 0.22-μm pore size filter membrane to obtain a hydrophilic ultrasmall iron oxide nanoparticle solution (concentration: 1 mg / mL).

[0084] (3) Self-assembly of iron oxide nanoparticles:

[0085] First, disperse 0.75 mL of the hydrophilic ultrasmall iron oxide nanoparticle solution prepared in step (2) in 15 mL of a bis-selenide polyethylene glycol solution (concentration of bis-selenide polyethylene glycol solution: 5 mg / mL, prepared by dissolving bis-selenide polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH of 8), and rotate and stir at room temperature for 24 h. Centrifuge for the first time at 12000 rpm for 20 min. Then, disperse the obtained 0.75 mg of precipitate in 10 mL of a bis-selenide polyethylene glycol solution (concentration of bis-selenide polyethylene glycol solution: 5 mg / mL, prepared by dissolving bis-selenide polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH of 8), rotate and stir at room temperature for 12 h, and centrifuge for the second time at 12000 rpm for 20 min. Wash the precipitate successively with triple-distilled water and ethanol, repeat 2 times, centrifuge for the third time at 12000 rpm for 20 min to collect the product, and disperse it in triple-distilled water to obtain a glutathione-responsive signal-switching MRI nanoprobe.

[0086] Example 4:

[0087] A glutathione-responsive signal-switching MRI nanoprobe, and its preparation method comprises the following steps:

[0088] (1) Synthesis of ultrasmall iron oxide nanoparticles:

[0089] At room temperature, iron oleate, oleic acid and oleyl alcohol are mixed and dissolved in diphenyl ether to obtain a reaction solution. Under the protection of an inert gas, the reaction solution is heated at 280 °C for 30 min, cooled to room temperature, and then acetone is added for precipitation. The nanoparticles are collected by centrifugation at 12,000 rpm for 20 min and dispersed in n-hexane to obtain an ultrasmall iron oxide nanoparticle solution (concentration: 2 mg / mL). Among them, the mass-to-volume ratios of iron oleate, oleic acid, oleyl alcohol, diphenyl ether and acetone are 2 g, 0.57 g, 1.61 g, 10 g, and 50 mL respectively;

[0090] (2) Surface modification of ultrasmall iron oxide nanoparticles:

[0091] Disperse 2 mL of the ultrasmall iron oxide nanoparticle solution prepared in step (1) in 10 mL of toluene, add 5 mL of methanol containing nitrosyl tetrafluoroborate (concentration of nitrosyl tetrafluoroborate: 10 mg / mL), then ultrasonicate for 8 min, mechanically stir at room temperature for 36 h, centrifuge at 12,000 rpm for 20 min, disperse the nanoparticles in triple-distilled water with a pH value of 8, and filter through a 0.22-μm pore size membrane to obtain a hydrophilic ultrasmall iron oxide nanoparticle solution (concentration: 1.5 mg / mL);

[0092] (3) Self-assembly of iron oxide nanoparticles:

[0093] First, disperse 2 mL of the hydrophilic ultrasmall iron oxide nanoparticle solution prepared in step (2) in 15 mL of a bis-seleno polyethylene glycol solution (concentration of bis-seleno polyethylene glycol solution: 5 mg / mL, prepared by dissolving bis-seleno polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH value of 8), rotate and stir at room temperature for 24 h for the first time, centrifuge at 12,000 rpm for 20 min for the first time, and then disperse the obtained 3 mg of precipitate in 10 mL of a bis-seleno polyethylene glycol solution (concentration of bis-seleno polyethylene glycol solution: 5 mg / mL, prepared by dissolving bis-seleno polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH value of 8), rotate and stir at room temperature for 12 h for the second time, centrifuge at 12,000 rpm for 20 min for the second time. Wash the precipitate with triple-distilled water and ethanol successively, repeat 2 - 3 times, centrifuge at 12,000 rpm for 20 min for the third time to collect the product, and disperse it in triple-distilled water to obtain a glutathione-responsive signal-switching MRI nanoprobe.

[0094] Example 5:

[0095] A glutathione-responsive signal-switching MRI nanoprobe, and its preparation method includes the following steps:

[0096] (1) Synthesis of ultrasmall iron oxide nanoparticles:

[0097] At room temperature, iron oleate, oleic acid, and oleyl alcohol are mixed and dissolved in diphenyl ether to obtain a reaction solution. Under the protection of an inert gas, the reaction solution is heated at 250 °C for 40 min, cooled to room temperature, and then acetone is added for precipitation. The nanoparticles are collected by centrifugation at 12000 rpm for 20 min and dispersed in n-hexane to obtain an ultrasmall iron oxide nanoparticle solution (concentration: 1.75 mg / mL). Among them, the mass / volume of iron oleate, oleic acid, oleyl alcohol, diphenyl ether, and acetone are 1.5 g, 0.5 g, 1 g, 8 g, and 45 mL respectively;

[0098] (2) Surface modification of ultrasmall iron oxide nanoparticles:

[0099] Disperse 2 mL of the ultrasmall iron oxide nanoparticle solution prepared in step (1) in 8 mL of toluene, add 6 mL of methanol containing nitrosyl tetrafluoroborate (concentration of nitrosyl tetrafluoroborate: 8 mg / mL), then ultrasonicate for 5 min, mechanically stir at room temperature for 24 h, centrifuge at 12000 rpm for 20 min, disperse the nanoparticles in triple-distilled water with a pH of 8, and filter through a 0.2-μm pore size filter membrane to obtain a hydrophilic ultrasmall iron oxide nanoparticle solution (concentration: 1 mg / mL);

[0100] (3) Self-assembly of iron oxide nanoparticles:

[0101] First, disperse 2 mL of the hydrophilic ultrasmall iron oxide nanoparticle solution prepared in step (2) in 13 mL of a bis-selenium polyethylene glycol solution (concentration of the bis-selenium polyethylene glycol solution: 4 mg / mL, prepared by dissolving bis-selenium polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH of 8), rotate and stir at room temperature for 12 h for the first time, centrifuge at 12000 rpm for 20 min for the first time, and then disperse the obtained 2 mg of precipitate in 8 mL of a bis-selenium polyethylene glycol solution (concentration of the bis-selenium polyethylene glycol solution: 4 mg / mL, prepared by dissolving bis-selenium polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH of 8), rotate and stir at room temperature for 12 h for the second time, centrifuge at 12000 rpm for 20 min for the second time. Wash the precipitate with triple-distilled water and ethanol successively for 3 times, centrifuge at 12000 rpm for 20 min for the third time to collect the product, and disperse it in triple-distilled water to obtain a glutathione-responsive signal-switching MRI nanoprobe.

[0102] Example 6:

[0103] A glutathione-responsive signal-switching MRI nanoprobe, and its preparation method includes the following steps:

[0104] (1) Synthesis of ultrasmall iron oxide nanoparticles:

[0105] At room temperature, iron oleate, oleic acid, and oleyl alcohol are mixed and dissolved in diphenyl ether to obtain a reaction solution. Under the protection of an inert gas, it is heated and reacted at 280 °C for 30 min, cooled to room temperature, then acetone is added for precipitation, and the nanoparticles are collected by centrifugation at 14000 rpm for 10 min and dispersed in n-hexane to prepare an ultrasmall iron oxide nanoparticle solution (concentration: 2.5 mg / mL); wherein, the mass-to-volume ratios of iron oleate, oleic acid, oleyl alcohol, diphenyl ether, and acetone are 2 g, 0.6 g, 2 g, 12 g, and 55 mL respectively;

[0106] (2) Surface modification of ultrasmall iron oxide nanoparticles:

[0107] Disperse 2 mL of the ultrasmall iron oxide nanoparticle solution prepared in step (1) in 12 mL of toluene, add 4 mL of methanol containing nitrosyl tetrafluoroborate (concentration of nitrosyl tetrafluoroborate: 12 mg / mL), then ultrasonicate for 10 min, mechanically stir at room temperature for 36 h, centrifuge at 14000 rpm for 10 min, disperse the nanoparticles in triple-distilled water with a pH value of 10, and filter through a filter membrane with a pore size of 0.3 μm to prepare a hydrophilic ultrasmall iron oxide nanoparticle solution (concentration: 1.5 mg / mL);

[0108] (3) Self-assembly of iron oxide nanoparticles:

[0109] Disperse 2 mL of the hydrophilic ultrafine iron oxide nanoparticle solution prepared in step (2) into 17 mL of a bis-selenium polyethylene glycol solution (the concentration of the bis-selenium polyethylene glycol solution is 6 mg / mL, prepared by dissolving bis-selenium polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH value of 8) for the first time, stir it by rotation at room temperature for 24 h for the first time, centrifuge it at 14000 rpm for 10 min for the first time, then disperse the obtained 3 mg of precipitate into 12 mL of a bis-selenium polyethylene glycol solution (prepared by dissolving bis-selenium polyethylene glycol in a phosphate buffer solution with a concentration of 10 mmol / L and a pH value of 8) for the second time, stir it by rotation at room temperature for 24 h for the second time, centrifuge it at 14000 rpm for 10 min for the second time, wash the precipitate successively with triple-distilled water and ethanol, repeat 2 times, centrifuge it at 14000 rpm for 10 min for the third time to collect the product, and disperse it in triple-distilled water to prepare a glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP).

[0110] Test Example

[0111] The products prepared in Examples 1-6 have basically the same performance. The following takes Example 1 as an example for detection.

[0112] I. Physical Property Determination

[0113] Respectively, the ultrafine iron oxide nanoparticles, hydrophilic ultrafine iron oxide nanoparticles (ESIONP), glutathione-responsive signal-switching MRI nanoprobes, and the cleavage conditions of the glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP) after incubation with 10 mmol / L GSH (glutathione) prepared in Example 1. This concentration is the concentration of glutathione in the aqueous solution of the nanoprobe. The morphology was detected by transmission electron microscopy (TEM) and Malvern particle size analyzer. The results are shown in Figures 2-3 ( Figure 2 In it, a is the ultrafine iron oxide nanoparticles, b is the hydrophilic ultrafine iron oxide nanoparticles, c is the glutathione-responsive signal-switching MRI nanoprobe, and d is the cleavage condition of the probe after incubation).

[0114] It can be seen from Figure 2 that the glutathione-responsive signal-switching MRI nanoprobe (contrast agent) prepared in this application can be cleaved in the GSH solution, thus realizing the conversion of T1 and T2 signals.

[0115] It can be seen from Figure 3 that the average hydrated particle size of the glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP) is about 108.2 nm, and the average hydrated particle size after cleavage by GSH incubation is about 8.8 nm.

[0116] II. Measurement of Signal Conversion Performance

[0117] The longitudinal relaxation rate and transverse relaxation rate of the hydrophilic ultra-small iron oxide nanoparticles (ESIONP) prepared in Example 1, the glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP), and the product obtained after incubation and cleavage of the glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP) with GSH were detected respectively. The parameters of the magnetic resonance detection sequence were as follows: T1 imaging parameters (TR / TE = 150, 300, 600, 1000, 2000, 4000, 8000 / 13 ms; FOV = 210×210 mm; Matrix size = 232×232; slice thickness = 3.0 mm (5 slices, gap = 0)). T2 imaging parameters (TR / TE = 5000 / 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ms; FOV = 210×210 mm; Matrix size = 232×232; slice thickness = 3.0 mm (5 slices, gap = 0)). The results are shown in Figures 4-5 .

[0118] As Figures 4-5 can be seen, the longitudinal relaxation rate of the monodisperse hydrophilic ultra-small iron oxide nanoparticles (ESIONP) is 5.04 mM -1 s -1 , and the transverse relaxation rate is 19.3 mM -1 s -1 ;

[0119] Before incubation with glutathione, the longitudinal relaxation rate of the glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP) is 1.36 mM -1 s -1 , and the transverse relaxation rate is 247.61 mM -1 s -1 .

[0120] After incubation with glutathione, the longitudinal relaxation rate of the contrast agent is 4.52 mM -1 s -1 , and the transverse relaxation rate is 24.2 mM -1 s -1 .

[0121] III. Cell Safety Measurement

[0122] The cell safety of the glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP) prepared in Example 1 was measured. The specific method was as follows: Respectively at 10 4For the density of cells / holes, 293T cells and THLE-3 cells were seeded in a 96-well plate and cultured with medium for 12 h. After 12 h, the cells were incubated with the MRI nanoprobe for 24 h. After 24 h, the medium was removed, and the cells were treated with 10% CCK-8 reagent. Finally, the absorbance was detected at a wavelength of 490 nm using a microplate reader. The results are shown in Figure 6 (The abscissa is the concentration of the MRI nanoprobe in the system).

[0123] As can be seen from Figure 6 , the CCK-8 results showed that the contrast agents at different concentrations did not show obvious cytotoxicity, and the survival rate exceeded 85%, indicating that the contrast agent of the present invention has good biocompatibility.

[0124] IV. Application of the Contrast Agent in the Diagnosis of Early Hepatocellular Carcinoma

[0125] The glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP) prepared in Example 1 was used for the detection of early hepatocellular carcinoma diagnosis. A monodisperse hydrophilic ultrasmall iron oxide nanoparticle (ESIONP) was used as a control group. The specific method was as follows: After injecting the contrast agent through the tail vein, in vivo MR imaging was performed on the mice with orthotopic micro-hepatocellular carcinoma, and magnetic resonance images of T1-WI and T2-WI were acquired at different time points. The results are shown in Figures 7-8 ( Figure 7 In, the upper figure is Cr-ESIONP, and the lower figure is ESIONP; Figure 8 In, a is Cr-ESIONP, and b is ESIONP).

[0126] As can be seen from Figures 7-8 , the tumor region of the mice changed from T2 to T1 signal, and the bright high T1 signal made it possible to clearly identify the tumor in the dark signal of normal tissues; while the control group of monodisperse ultrasmall iron oxide nanoparticles (ESIONP) lacked this response ability, and the T1 signals of both normal tissues and tumor tissues were enhanced, making it difficult to distinguish the lesion range. These results demonstrated the signal conversion performance of the glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP) of the contrast agent of the present invention under glutathione response, which greatly improved the sensitivity and specificity of tumor diagnosis.

[0127] According to the above analysis, the glutathione-responsive signal-switching MRI nanoprobe (Cr-ESIONP) prepared in the present invention can achieve early and accurate diagnosis of in-situ carcinoma of the liver.

[0128] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of a glutathione-responsive signal-switching MRI nanoprobe, characterized in that, It includes the following steps: (1) Synthesis of ultra-small iron oxide nanoparticles: Mix iron oleate, oleic acid, and oleyl alcohol, dissolve them in diphenyl ether to obtain a reaction solution, heat the reaction, cool to room temperature, then add acetone, centrifuge, and disperse in n-hexane to prepare an ultra-small iron oxide nanoparticle solution; (2) Surface modification of ultra-small iron oxide nanoparticles: Disperse the ultra-small iron oxide nanoparticle solution prepared in step (1) in toluene, add methanol containing nitrosyl tetrafluoroborate, then sonicate, stir, centrifuge, disperse in water, and filter through a membrane to prepare a hydrophilic ultra-small iron oxide nanoparticle solution; (3) Self-assembly of iron oxide nanoparticles: First disperse the hydrophilic ultra-small iron oxide nanoparticle solution prepared in step (2) in a bis-selenide polyethylene glycol solution, stir for the first time, centrifuge for the first time, then disperse the obtained precipitate in the bis-selenide polyethylene glycol solution for the second time, stir for the second time, centrifuge for the second time, wash, centrifuge for the third time, and disperse in water to prepare a glutathione-responsive signal-switching MRI nanoprobe.

2. The preparation method of the glutathione-responsive signal-switching MRI nanoprobe according to claim 1, wherein In step (1), the mass-volume ratio of iron oleate, oleic acid, oleyl alcohol, diphenyl ether, and acetone is 1.5 - 2 g : 0.5 - 0.6 g : 1 - 2 g : 8 - 12 g : 45 - 55 mL.

3. The preparation method of the glutathione-responsive signal-switching MRI nanoprobe according to claim 1, wherein, In step (1), under the protection of an inert gas, heat the reaction at 250 - 280 °C for 30 - 40 min.

4. The preparation method of the glutathione-responsive signal-switching MRI nanoprobe according to claim 1, characterized in that, In step (1), the concentration of the ultra-small iron oxide nanoparticle solution is 0.5 - 2.5 mg / mL.

5. The preparation method of the glutathione-responsive signal-switching MRI nanoprobe according to claim 1, characterized in that In step (2), the volume ratio of the ultra-small iron oxide nanoparticle solution, toluene, and methanol containing nitrosyl tetrafluoroborate is 0.5 - 2 : 8 - 12 : 4 - 6.

6. The preparation method of the glutathione-responsive signal-switching MRI nanoprobe according to claim 1, wherein In step (2), the concentration of the hydrophilic ultra-small iron oxide nanoparticle solution is 1 - 1.5 mg / mL.

7. The preparation method of the glutathione-responsive signal-switching MRI nanoprobe according to claim 1, wherein In step (3), during the first dispersion, the volume ratio of the hydrophilic ultra-small iron oxide nanoparticle solution and the bis-selenide polyethylene glycol solution is 0.5 - 2 : 13 - 17.

8. The preparation method of the glutathione-responsive signal-switching MRI nanoprobe according to claim 1, characterized in that, In step (3), the bis-selenide polyethylene glycol solution is prepared by dissolving bis-selenide polyethylene glycol in a phosphate buffer solution.

9. A glutathione-responsive signal-switching MRI nanoprobe prepared by the method for preparing a glutathione-responsive signal-switching MRI nanoprobe according to any one of claims 1 - 8.

10. Use of the glutathione-responsive signal-switching MRI nanoprobe according to claim 9 in the preparation of an MRI contrast agent.

Images