A hypoxia-responsive T2-T1 switchable MRI contrast agent, its preparation method and application

Abstract

This invention relates to a hypoxia-responsive T2-T1 switchable MRI contrast agent, its preparation method, and its application. The hypoxia-responsive T2-T1 switchable MRI contrast agent comprises iron oxide nanoclusters and polyethylene glycolamine and / or ethanolamine modified on the iron oxide nanoclusters. The iron oxide nanoclusters are obtained by cross-linking iron oxide nanoparticles via hypoxia-sensitive azo bonds. The MRI contrast agent of this invention can specifically respond to the hypoxia environment of the tumor microenvironment, thereby achieving a change from aggregation to dispersion of extremely small iron oxide nanoclusters, thus enabling the switching from T2-type to T1-type contrast signals. Furthermore, after dissociation in response to the hypoxia environment, the re-formed extremely small iron oxide nanoparticles are more readily metabolized, increasing biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of MRI diagnostic contrast agent technology, and relates to a novel MRI diagnostic contrast agent, specifically a hypoxia-responsive T2-T1 switchable MRI contrast agent, its preparation method, and its application. Background Technology

[0002] Cancer seriously endangers human health, and early diagnosis and clinical treatment of tumors can effectively improve patient survival rates, which is of great significance to cancer treatment. Therefore, early diagnosis of tumors is particularly important.

[0003] Currently, some common imaging techniques include computed tomography (CT), positron emission tomography (PET), single photonemission computed tomography (SPECT), optical imaging (OI), ultrasound (US), and magnetic resonance imaging (MRI). Among these, MRI, with its extremely high spatial resolution and tissue contrast, provides anatomical images of soft tissues and is considered one of the most important diagnostic methods for imaging the brain, cartilage, heart, blood vessels, and detecting tumors.

[0004] While MRI offers numerous advantages, its relatively low contrast is a drawback in clinical diagnosis, particularly for early-stage tumors, impacting diagnostic accuracy. Therefore, MRI contrast agents are used clinically to address this limitation. Currently, gadolinium-based small-molecule chelate contrast agents are commonly used to enhance tissue contrast. However, these agents lack specificity and selectivity for tumor tissues and are rapidly metabolized by the kidneys. Consequently, with the advancement of tumor tissue research, responsive contrast agents have been increasingly reported in recent years. The signal intensity of these contrast agents changes with specific parameters in the physiological environment, "activating" the contrast signal at the tumor site, thus achieving specificity and selectivity for tumor tissues.

[0005] Extremely small iron oxide nanoparticles (<4 nm) are a novel type of T1 contrast agent developed in recent years, while aggregated iron oxide nanoclusters become a type of T2 contrast agent due to magnetic coupling effects. This transition between dispersed and aggregated states allows for the mutual conversion between T1 bright signals and T2 dark signals. Currently, researchers have developed various intelligent MRI-switching nanoprobes responsive to the specific physiological parameters of the tumor microenvironment, such as slightly acidic environment, high glutathione (GSH) content, high reactive oxygen species (ROS) content, and hypoxia, achieving tumor-specific imaging.

[0006] For example, researchers have modified the surface of tiny iron oxide nanoparticles with GSH-sensitive polymer molecules, thereby achieving a transition from T1-weighted MRI to T2-weighted MRI signals under the influence of high GSH concentrations at tumor sites. This tumor-specific T1-T2 switching can reduce background signal interference and increase specificity. However, this T1-T2 signal switching is a change from a bright signal to a dark signal, which is often susceptible to artifacts caused by the natural aggregation of iron oxide and internal bleeding, leading to diagnostic errors. Moreover, in clinical practice, brighter signals are more favored by clinicians. Currently reported pH- or GSH-responsive aggregates are mostly synthesized using methods such as ATRP or RAFT, which also suffer from drawbacks such as cumbersome procedures and low yields.

[0007] Therefore, it is essential to develop a novel MRI contrast agent that is highly specific, has high diagnostic accuracy, and is biosafety-free. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the purpose of this invention is to provide a novel MRI diagnostic contrast agent, specifically a hypoxia-responsive T2-T1 switchable MRI contrast agent, its preparation method, and its application.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] On one hand, the present invention provides a hypoxia-responsive T2-T1 switchable MRI contrast agent, wherein the hypoxia-responsive T2-T1 switchable MRI contrast agent comprises iron oxide nanoclusters and polyethylene glycolamine and / or ethanolamine modified on the iron oxide nanoclusters; the iron oxide nanoclusters are obtained by crosslinking iron oxide nanoparticles through hypoxia-sensitive azo bonds.

[0011] The MRI contrast agent of this invention uses monodisperse, extremely small iron oxide nanoparticles (ESIONPs) as its core. These nanoparticles are themselves a T1-type contrast agent, then linked into nanoclusters by hypoxia-responsive azo bonds. Due to magnetic coupling, the contrast agent becomes a T2-type contrast agent. Finally, it is modified with polyethylene glycolamine and / or ethanolamine to ensure good hydrophilicity and biocompatibility, and also helps the molecules escape recognition by the reticuloendothelial system, leading to a reduced plasma clearance rate, which helps improve imaging results. The MRI contrast agent of this invention can specifically respond to the hypoxic environment of the tumor microenvironment, thereby achieving a change from aggregation to dispersion of the extremely small iron oxide nanoclusters, thus achieving a switch from T2-type to T1-type contrast signals, a signal switching method highly favored in clinical practice. Furthermore, this strategy of reducing size, at the beginning of injection, due to the high permeability and retention effect of solid tumors, further increases the accumulation of nanoparticles in the tumor region, prolonging the imaging window time. After dissociating in response to a hypoxic environment, it reverts to extremely small iron oxide nanoparticles, which facilitates the metabolism of the nanoparticles and increases biosafety.

[0012] Experiments revealed that the hypoxia-responsive T2-T1 switching MRI contrast agent involved in this invention exhibits a longitudinal relaxation rate r1 of 7.86 mM before and after the response. -1 s -1 It became 8.30mM -1 s -1 The lateral relaxation rate r2 is 116.85 mM -1 s -1 It became 13.34mM -1 s -1 The r2 / r1 ratio also changed from 14.86 to 1.61, which is the gold standard for determining the type of contrast agent. This indicates that the contrast agent responded to the hypoxic environment, changing from a T2-type contrast agent to a T1-type contrast agent. Furthermore, this contrast agent can effectively reduce background signal interference during tumor diagnosis, thereby improving its specificity and selectivity. In addition, this contrast agent exhibits good biocompatibility and extremely low biotoxicity.

[0013] Preferably, the iron oxide nanoparticles have a particle size of less than 4 nm, such as 3.8 nm, 3.6 nm, 3.2 nm, 3 nm, 2.8 nm, 2.6 nm, 2.2 nm, 2 nm, etc.

[0014] Preferably, the size of the iron oxide nanoclusters is no greater than 200 nm, such as 180 nm, 160 nm, 150 nm, 120 nm, 100 nm, 800 nm, 60 nm, etc.

[0015] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0016] The size of iron oxide nanoclusters, no larger than 200 nm, is highly advantageous for contrast agents to reach the tumor site through the high permeability and retention effect (EPR effect) of solid tumors. Once at the tumor site, the nanocluster contrast agent dissociates in response to the hypoxic environment, specifically switching from T2 to T1 contrast signals at the tumor site. This switching from dark to bright signals has very high specificity, avoiding artifacts caused by the natural aggregation of iron oxide nanoparticles and internal bleeding, which could lead to diagnostic errors. Furthermore, after dissociation in response to hypoxia, the nanocluster contrast agent reverts to monodisperse ESIONPs, which can be rapidly metabolized by the kidneys, thus exhibiting higher biocompatibility.

[0017] In a second aspect, the present invention provides a method for preparing a hypoxia-responsive T2-T1 switchable MRI contrast agent according to the first aspect, the preparation method comprising:

[0018] (1) Preparation of monodisperse iron oxide nanoparticles with fatty acid-modified surface;

[0019] (2) The active ester is modified on the surface of the product in step (1) and activated to obtain the activated product;

[0020] (3) The activated product was coupled with an azo bond coupling agent to obtain iron oxide nanoclusters;

[0021] (4) Modify the iron oxide nanoclusters of step (3) with polyethylene glycolamine and / or ethanolamine to obtain the hypoxia-responsive T2-T1 switchable MRI contrast agent.

[0022] Compared to existing technologies, the contrast agent preparation method of this invention is simple, convenient, and low-cost. The prepared contrast agent can reach the tumor site through the tumor's high permeability and retention effect (EPR effect), and responds to the hypoxic environment of the tumor microenvironment, achieving the MRI signal transition from T2 to T1 (dark signal to bright signal), which can be used for precise tumor diagnosis. Since hypoxia is a significant characteristic of many solid tumors and other common diseases such as atherosclerosis, the contrast agent prepared in this invention, with its highly sensitive response to hypoxia, offers the possibility of application in various disease models, and is a universal hypoxia-responsive T2-T1 switching MRI contrast agent.

[0023] Regarding step (2) of the preparation method, the present invention also creatively provides a method for modifying iron oxide nanoparticles by surface ligand exchange with active lipids, which endows the iron oxide nanoparticles with higher surface activity, thereby improving the reaction rate of subsequent modification reactions, etc. Furthermore, the product of step (1) is dispersed in n-hexane, while the iron oxide nanoparticles obtained by surface ligand exchange modification with active lipids can be very well dispersed in dimethyl sulfoxide, which is a widely used organic solvent. This undoubtedly further broadens the application potential of extremely small iron oxide nanoparticles.

[0024] Preferably, step (1) specifically includes: first, reacting iron salt with fatty acid salt to obtain a fatty acid iron complex precursor, and then subjecting it to a thermal decomposition reaction to obtain the product.

[0025] Preferably, the molar ratio of the iron salt to the fatty acid salt is 1:(2-4), such as 1:2, 1:2.5, 1:3, 1:3.5, 1:4, etc.

[0026] Preferably, the reaction between the iron salt and the fatty acid salt is carried out at 60-80℃ (e.g., 60℃, 65℃, 70℃, 75℃, 80℃, etc.) for 4-6 hours (e.g., 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, etc.).

[0027] Preferably, the obtained fatty acid iron complex precursor is further purified. Purification requires a separatory funnel and a vacuum drying oven.

[0028] Preferably, the thermal decomposition reaction is first subjected to vacuum treatment at 80-100℃ (e.g., 80℃, 85℃, 90℃, 95℃, 100℃, etc.) for 2-6 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, etc.), followed by high-temperature reaction at 240-260℃ (e.g., 240℃, 250℃, 255℃, 260℃, etc.) for 25-40 minutes (e.g., 25 minutes, 30 minutes, 35 minutes, 40 minutes, etc.). Temperature has a significant impact on the synthesized particle size.

[0029] Preferably, the thermal decomposition reaction is carried out in an inert gas atmosphere. Since the thermal decomposition reaction needs to be carried out at a high temperature of 240-260°C, diphenyl ether, which has a relatively high boiling point, is selected as the solvent.

[0030] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0031] Preferably, the active ester in step (2) includes DOPAC-PFP, which is prepared by mixing 3,4-dihydroxyphenylacetic acid and pentafluorophenol in an organic solvent, adding N,N'-dicyclohexylcarbodiimide solution dropwise to the system, and reacting at 15-40℃ (e.g., 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.) for 12-24h (e.g., 12h, 15h, 18h, 20h, 22h, 24h, etc.) to obtain the product.

[0032] Specifically, 3,4-dihydroxyphenylacetic acid (DOPAC) and pentafluorophenol (PFP) were dissolved in 1,4-dioxane. Then, 1,4-dioxane containing dissolved N,N'-dicyclohexylcarbodiimide (DCC) was slowly added dropwise to the above system, and the mixture was stirred overnight under nitrogen protection at room temperature. After the reaction was completed, the active ester DOPAC-PFP was obtained by silica gel column chromatography.

[0033] Preferably, the molar ratio of 3,4-dihydroxyphenylacetic acid (DOPAC) to pentafluorophenol (PFP) is less than 1:1.

[0034] Preferably, the organic solvent comprises anhydrous 1,4-dioxane.

[0035] Preferably, the eluent used in the silica gel column chromatography is n-hexane / ethyl acetate (V:V = 3:1).

[0036] Preferably, step (2) specifically includes: mixing the active ester with iron oxide nanoparticles with fatty acid modified on the surface and dissolving them in an organic solvent, and refluxing them at 40-60℃ (e.g., 40℃, 45℃, 50℃, 55℃, 60℃, etc.) for 60-84h (e.g., 60h, 65h, 70h, 75h, 80h, 82h, 84h, etc.).

[0037] Specifically, the product from step (1) was weighed and dissolved in tetrahydrofuran (THF), and then the active ester was added. The mixture was refluxed at 40-60°C for 60-84 hours under nitrogen protection. After the reaction was completed, the product was concentrated by rotary evaporation to reduce the volume. The product was then dialyzed for several days to remove excess active ester. Finally, the product was dissolved in dimethyl sulfoxide (DMSO) for later use.

[0038] Preferably, the mass ratio of the active ester to the iron oxide nanoparticles with fatty acid surface modification is (5-15):1, for example, 5:1, 8:1, 10:1, 12:1, 13:1, 15:1, etc.

[0039] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0040] Preferably, the azo bond-containing coupling agent in step (3) includes diaminoazobenzene, 4,4'-azobenzoic acid chloride or 2,2'-azoaniline.

[0041] Preferably, the coupling reaction in step (3) is carried out at 15-40℃ (e.g., 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.) for 18-30h (e.g., 18h, 20h, 22h, 25h, 28h, 30h, etc.).

[0042] Specifically, the activation product from step (2) is dissolved in a dimethyl sulfoxide solution, and the azo bond coupling agent is dissolved in a dimethyl sulfoxide solution (the concentration should be as low as possible). The solution is then slowly added dropwise to the above system (the dropwise addition of the azo bond coupling agent should be as slow as possible, and it should be added dropwise under stirring). After the dropwise addition is complete, N,N-diisopropylethylamine (DIPEA) is added, and the reaction is stirred at 15-40°C for 18-30 h to finally obtain azo bond-linked tiny iron oxide nanoclusters.

[0043] Preferably, the mass ratio of the azo bond coupling agent to the activated product in step (3) is 1:(4-6), such as 1:4, 1:4.5, 1:5, 1:5.5, 1:6, etc.

[0044] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0045] Preferably, step (4) specifically includes: mixing iron oxide nanoclusters with polyethylene glycolamine at 15-40℃ (e.g., 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.) for 18-30h (e.g., 18h, 20h, 22h, 25h, 28h, 30h, etc.), and then mixing them with ethanolamine at 15-40℃ (e.g., 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.) for 18-30h (e.g., 18h, 20h, 22h, 25h, 28h, 30h, etc.) to obtain the final product.

[0046] Preferably, the polyethylene glycolamine in step (4) is a methyl-terminated polyethylene glycolamine.

[0047] Preferably, the amount of polyethylene glycolamine and / or ethanolamine used is more than 10 times the mass of the iron oxide nanoclusters, for example, 10 times, 12 times, 15 times, 18 times, 20 times, etc.

[0048] Preferably, after the reaction is completed, the following post-processing is performed: ether precipitation, water dissolution, dialysis purification using a dialysis bag, and then separation to obtain a product with a suitable particle size.

[0049] Regarding the separation operation to obtain extremely small iron oxide nanoclusters with suitable particle size, one method is to obtain them by magnetic separation followed by removal of larger nanoclusters by filtration membrane. Another method is to remove the uncoupled extremely small iron oxide nanoparticles from the supernatant by centrifugation, and then remove the excessively coupled iron oxide nanoclusters by filtration membrane.

[0050] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0051] Thirdly, the present invention provides the use of the hypoxia-responsive T2-T1 switchable MRI contrast agent according to the first aspect in the preparation of tumor diagnostic or detection reagents.

[0052] Fourthly, the present invention provides a contrast composition comprising the hypoxia-responsive T2-T1 switchable MRI contrast agent described in the first aspect and pharmaceutically acceptable excipients.

[0053] Preferably, the excipients include a diluent.

[0054] Fifthly, the present invention provides a pharmaceutical composition comprising the hypoxia-responsive T2-T1 switchable MRI contrast agent described in the first aspect, a drug, and a pharmaceutically acceptable pharmaceutical carrier.

[0055] The drug may be a therapeutic drug, a tracer molecule, or other drug component.

[0056] In a sixth aspect, the present invention provides a non-medical imaging method, the imaging method comprising: administering to the subject to be imaged a hypoxia-responsive T2-T1 switchable MRI contrast agent as described in the first aspect or an imaging composition as described in the fourth aspect, and performing imaging.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The MRI contrast agent of this invention uses monodisperse extremely small iron oxide nanoparticles (ESIONPs) as its core. These nanoparticles are themselves a T1-type contrast agent, then linked into nanoclusters by hypoxia-responsive azo bonds. Due to magnetic coupling, the contrast agent becomes a T2-type contrast agent. Finally, it is modified with polyethylene glycol amine and / or ethanolamine to ensure good hydrophilicity and biocompatibility, and also to help the molecules escape recognition by the reticuloendothelial system, leading to a decrease in plasma clearance, which helps improve imaging results. The MRI contrast agent of this invention can specifically respond to the hypoxic environment of the tumor microenvironment, thereby achieving a change from aggregation to dispersion of the extremely small iron oxide nanoclusters, thus achieving a switch from T2-type to T1-type contrast signals. This is a signal switching method that is very popular in clinical practice. A schematic diagram is shown below.Figure 1 As shown. Furthermore, this strategy of reducing the size of nanoparticles, at the start of injection, due to the high permeability and retention effect of solid tumors, increases the accumulation of nanoparticles in the tumor region, prolonging the imaging window. After dissociating in response to the hypoxic environment, they revert to extremely small iron oxide nanoparticles, which facilitates nanoparticle metabolism and increases biocompatibility.

[0059] Experiments revealed that the hypoxia-responsive T2-T1 switching MRI contrast agent involved in this invention exhibits a longitudinal relaxation rate r1 of 7.86 mM before and after the response. -1 s -1 It became 8.30mM -1 s -1 The lateral relaxation rate r2 is 116.85 mM -1 s -1 It became 13.34mM -1 s -1 The r2 / r1 ratio also changed from 14.86 to 1.61, which is the gold standard for determining the type of contrast agent. This indicates that the contrast agent responded to the hypoxic environment, changing from a T2-type contrast agent to a T1-type contrast agent. Furthermore, this contrast agent can effectively reduce background signal interference during tumor diagnosis, thereby improving its specificity and selectivity. In addition, this contrast agent exhibits good biocompatibility and extremely low biotoxicity.

[0060] Compared to existing technologies, the contrast agent preparation method of this invention is simple, convenient, and low-cost. The prepared contrast agent can reach the tumor site through the tumor's high permeability and retention effect (EPR effect), and responds to the hypoxic environment of the tumor microenvironment, achieving the MRI signal transition from T2 to T1 (dark signal to bright signal), which can be used for precise tumor diagnosis. Since hypoxia is a significant characteristic of many solid tumors and other common diseases such as atherosclerosis, the contrast agent prepared in this invention, with its highly sensitive response to hypoxia, offers the possibility of application in various disease models, and is a universal hypoxia-responsive T2-T1 switching MRI contrast agent.

[0061] Compared to existing technologies, the contrast agent preparation method of this invention is simple, convenient, and low-cost. Specifically, the method of modifying iron oxide nanoparticles with active lipids via surface ligand exchange endows the iron oxide nanoparticles with higher surface activity, thereby increasing the reaction rate of subsequent modification reactions. Furthermore, the iron oxide nanoparticles obtained by surface ligand exchange modification with active lipids can be very well dispersed in dimethyl sulfoxide (DMSO), a widely used organic solvent, which undoubtedly further expands the application potential of extremely small iron oxide nanoparticles. Attached Figure Description

[0062] Figure 1 This is a schematic diagram illustrating the working principle of the hypoxia-responsive T2-T1 switching MRI contrast agent involved in this invention.

[0063] Figure 2 This is a transmission electron microscope image of the ESIONPs-OA nanoparticles in Example 1 of this invention.

[0064] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the synthesized material named DOPAC-PFP in Example 1 of this invention.

[0065] Figure 4 This is a transmission electron microscope image of the ESIONPs-PFP nanoparticles in Example 1 of this invention.

[0066] Figure 5 This is a transmission electron microscope image of the hypoxia-responsive MRI nanocluster contrast agent (EAmP) in Example 1 of the present invention.

[0067] Figure 6 This is a transmission electron microscope image of the non-hypoxia-responsive MRI nanocluster contrast agent (EBmP) in Comparative Example 1 of the present invention.

[0068] Figure 7a This is a comparison of the longitudinal relaxation rate (r1) of the MRI nanocluster contrast agent (EAmP) in Example 1 of the present invention before incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro and after 4 hours of incubation.

[0069] Figure 7b These are solution images of the MRI nanocluster contrast agent (EAmP) in Example 1 of this invention before and after incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro.

[0070] Figure 8a This is a comparison of the transverse relaxation rate (r2) of the MRI nanocluster contrast agent (EAmP) in Example 1 of the present invention before incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro and after 4 hours of incubation.

[0071] Figure 8b These are solution images of the MRI nanocluster contrast agent (EAmP) in Example 1 of this invention before and after incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro.

[0072] Figure 9aThis is a comparison of the longitudinal relaxation rate (r1) of the MRI nanocluster contrast agent (EBmP) in Comparative Example 1 of this invention before and after incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro.

[0073] Figure 9b This is a solution imaging image of the MRI nanocluster contrast agent (EBmP) in Comparative Example 1 of the present invention before and after incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro.

[0074] Figure 10a This is a comparison of the transverse relaxation rate (r2) of the MRI nanocluster contrast agent (EBmP) in Comparative Example 1 of the present invention before incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro and after 4 hours of incubation.

[0075] Figure 10b This is a solution imaging image of the MRI nanocluster contrast agent (EBmP) in Comparative Example 1 of the present invention before and after incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro.

[0076] Figure 11 This is a cytotoxicity test diagram of the MRI nanocluster contrast agent (EAmP) in mouse breast cancer cells (4T1) (a) and human umbilical vein endothelial cells (HUVEC) (b) in Example 1 of the present invention.

[0077] Figure 12 This is a tissue toxicology test diagram of the MRI nanocluster contrast agent (EAmP) in BALB / c mice in Example 1 of the present invention.

[0078] Figure 13 These are in vivo T1-weighted MRI images of the MRI nanocluster contrast agent (EAmP) in Example 1 and the MRI nanocluster contrast agent (EBmP) in Control Example 1 of the present invention in tumor-bearing mice transplanted with 4T1 cells.

[0079] Figure 14 This is a quantitative average signal intensity analysis of the MRI nanocluster contrast agent (EAmP) (a) in Example 1 of the present invention and the MRI nanocluster contrast agent (EBmP) (b) in Control Example 1 after in vivo T1-weighted MRI imaging in tumor-bearing mice transplanted with 4T1 cells. Detailed Implementation

[0080] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0081] Example 1

[0082] This embodiment provides a hypoxia-responsive T2-T1 switching MRI contrast agent, the preparation method of which is as follows:

[0083] (1) Synthesis of the ferric oleate complex precursor: Sodium oleate (18.3 g, 60 mmol) and ferric chloride hexahydrate (5.4 g, 20 mmol) were first dissolved in a mixed solvent of 30 mL water, 40 mL anhydrous ethanol, and 70 mL n-hexane. The mixture was then refluxed and stirred at 70 °C for 4 h. After the reaction was completed, the mixture was cooled to 20 °C and washed several times with water to remove impurities. The resulting dark red liquid was then rotary evaporated and vacuum dried to obtain the ferric oleate complex precursor. The ferric oleate complex precursor (1.8 g, 2 mmol) was then dissolved in a mixed solvent system of oleyl alcohol (1.61 g, 6 mmol), oleic acid (0.57 g, 2 mmol), and diphenyl ether (10 g, 58 mmol). The mixed solution was first evacuated at 90 °C for 2 h to remove excess impurities, and then reacted at 250 °C under an argon atmosphere for 0.5 h. After the solution was cooled to 20 °C, impurities were removed with a large amount of acetone. Finally, the precipitate was collected by centrifugation and dissolved in n-hexane to obtain ESIONPs-OA. Figure 2 The transmission electron microscope (TEM) image shows that the obtained ESIONPs-OA particles are uniform in size, with an average particle size of 3 nm.

[0084] (2) Dissolve 1.8 g (10 mmol) of 3,4-dihydroxyphenylacetic acid and 2.2 g (12 mmol) of pentafluorophenol in 60 mL of anhydrous 1,4-dioxane. Then, slowly add N,N'-dicyclohexylcarbodiimide (2.5 g (12 mmol)) dissolved in 10 mL of anhydrous 1,4-dioxane to the above system dropwise. Stir under nitrogen protection at 20 °C for 12 h. After the reaction is complete, filter off the precipitate, collect the filtrate, and concentrate it by rotary evaporation. Then, purify it by silica gel column chromatography using n-hexane / ethyl acetate (V:V = 3:1) as the eluent. The final active ester was obtained and named DOPAC-PFP. Figure 3 The NMR spectrum confirms the correct synthesis of DOPAC-PFP. The synthetic route can be represented by the following chemical equation:

[0085]

[0086] (3) 60 mg of ESIONPs-OA obtained in step (1) was quantitatively dried by rotary evaporation under vacuum and redissolved in 60 mL of tetrahydrofuran. Then, 700 mg of the active ester DOPAC-PFP obtained in step (2) was weighed and added to the solution to dissolve it. The mixture was then refluxed at 50 °C for 72 h under nitrogen protection. After the reaction, the mixture was concentrated by rotary evaporation to reduce the volume, and dialyzed for several days to remove excess DOPAC-PFP from the product. The final product was named ESIONPs-PFP and dissolved in DMSO for later use. Figure 4 As shown in the transmission electron microscopy (TEM) images, the obtained ESIONPs-PFP particles are uniform in size. Compared with ESIONPs-OA, the particle size and dispersibility remain unchanged, and it still exhibits good monodispersity. The synthetic route can be represented by the following chemical equation:

[0087]

[0088] (4) Quantitatively dissolve 5 mg of ESIONPs-PFP in 20 mL of dimethyl sulfoxide solution. Then, weigh 1 mg of the coupling agent diaminoazobenzene and dissolve it in 5 mL of dimethyl sulfoxide solution. Slowly add the diaminoazobenzene solution dropwise to the ESIONPs-PFP solution using a syringe. After the addition is complete, add 50 μL of N,N-diisopropylethylamine (DIPEA) and stir at 20 °C for 24 hours to obtain azo-linked tiny iron oxide nanoclusters. Then, add 50 mg of mPEG10000-NH2 to the above solution and continue stirring for 24 hours. After the reaction is complete, add 20 μL of ethanolamine and continue the reaction for 24 hours to increase its water solubility. After the reaction is complete, precipitate the precipitate with a large amount of anhydrous diethyl ether. Collect the precipitate, dissolve it in a small amount of water, and then dialyze it using a dialysis bag with a molecular weight cutoff of 35,000 for several days to purify it. Then, through separation (specifically, firstly, centrifugation to remove uncoupled tiny iron oxide nanoparticles from the supernatant, and then filtration to remove excessively coupled tiny iron oxide nanoclusters), a contrast agent EAMP with suitable particle size and excellent water solubility, modified with polyethylene glycol and ethanolamine, is obtained. Figure 5 As shown, the synthesized hypoxia-responsive nanoclusters of contrast agents have a particle size primarily between 100-200 nm and exhibit excellent solubility in water. The synthetic route can be represented by the following chemical equation:

[0089]

[0090] Comparative Example 1

[0091] This comparative example provides an MRI contrast agent whose preparation method differs from that of Example 1 only in step (4) by replacing 1 mg of diaminoazobenzene with 0.868 mg of benzidine. The final product is a non-hypoxia-responsive minimal iron oxide nanocluster contrast agent, named EBmP. Figure 6 Transmission electron microscopy (TEM) images show that EBmP has a similar morphology to EAmP, consisting of nanoclusters ranging from 100 to 200 nm, and exhibits excellent solubility in water. The synthetic route can be represented by the following chemical equation:

[0092]

[0093] Performance Test 1

[0094] This test used rat microsomes as a substitute for azo reductase for in vitro simulation experiments. The longitudinal relaxation time T1 and T1-weighted imaging of the MRI contrast agent product (35 μg Fe) obtained in Example 1 of this invention, reduced nicotinamide adenine dinucleotide phosphate (NADPH) (50 μL, 0.5 mg), and rat microsomes (20 μL, 20 mg / mL) under simulated hypoxia conditions were measured before and after 4 hours of incubation on a 0.5T MRI scanner. The operation method included:

[0095] Samples with iron concentrations of 0.11 mM, 0.17 mM, 0.25 mM, 0.37 mM, and 0.56 mM were prepared respectively. After testing on a 0.5T MRI scanner, the transverse relaxation rates of the MRI contrast agent of this invention, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and rat microsomes before and after incubation under simulated hypoxia conditions were found to be 7.86 mM. -1 ·s -1 and 8.30mM -1 ·s -1 (like Figure 7a As shown in the figure, the longitudinal relaxation rate of the contrast agent of the present invention, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and rat microsomes increased slightly after incubation under simulated hypoxic conditions in vitro.

[0096] like Figure 7b As shown, T1-weighted imaging at different concentrations reveals that the T1 contrast effect of the contrast agent obtained in Example 1 of this invention is significantly darker before incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro than after 4 hours of incubation.

[0097] Note: T1 weighted imaging parameters are set as follows: echo time (TE) = 134ms, repetition time (TR) = 500ms, number of scans (NS) = 1.

[0098] Performance Test 2

[0099] This test was conducted on a 0.5T MRI scanner to measure the transverse relaxation time T2 and T2-weighted imaging of the MRI contrast agent product (35 μg Fe) obtained in Example 1 of this invention, reduced nicotinamide adenine dinucleotide phosphate (NADPH) (50 μL, 0.5 mg), and rat microsomes (20 μL, 20 mg / mL) before and after incubation under simulated hypoxia conditions for 4 hours. The procedure included:

[0100] Samples with iron concentrations of 0.11 mM, 0.17 mM, 0.25 mM, 0.37 mM, and 0.56 mM were prepared respectively. After testing on a 0.5T MRI scanner, the transverse relaxation rates of the MRI contrast agent of this invention, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and rat microsomes before and after incubation under simulated hypoxia conditions were found to be 116.85 mM. -1 ·s -1 and 13.34mM -1 ·s -1 (like Figure 8a As shown in the figure, the contrast agent of the present invention, after being incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro, exhibited a significant decrease in its transverse relaxation rate. Furthermore, the ratio of transverse relaxation rate to longitudinal relaxation rate (r2 / r1) is the gold standard for determining the type of contrast agent. Before incubation, the ratio was 14.86, and after incubation, it was 1.61. This change in ratio indicates that the contrast agent obtained in this embodiment can respond under simulated hypoxia conditions, transforming from a T2-type contrast agent to a T1-type contrast agent.

[0101] like Figure 8b As shown, T2-weighted imaging at different concentrations reveals that the T2 contrast agent obtained in Example 1 of this invention is significantly darker before incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro than after 4 hours of incubation.

[0102] Note: T2 weighted imaging parameters are set as follows: echo time (TE) = 334ms, repetition time (TR) = 2000ms, number of scans (NS) = 1.

[0103] Performance Test 3

[0104] This test was conducted on a 0.5T MRI scanner to measure the longitudinal relaxation time T1 and T1-weighted imaging of the MRI contrast agent product (35 μg Fe) obtained in Comparative Example 1 of this invention, along with reduced nicotinamide adenine dinucleotide phosphate (NADPH) (50 μL, 0.5 mg) and rat microsomes (20 μL, 20 mg / mL) under simulated hypoxia conditions before and after 4 hours of incubation. The procedure included:

[0105] Samples with iron concentrations of 0.11 mM, 0.17 mM, 0.25 mM, 0.37 mM, and 0.56 mM were prepared respectively. After testing on a 0.5T MRI scanner, linear fitting was performed with iron ion concentration (mM) as the abscissa and the reciprocal of the longitudinal relaxation time as the ordinate. The longitudinal relaxation rates of the MRI contrast agent of this invention, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and rat microsomes before and after incubation under simulated hypoxia conditions were 5.72 mM. -1 ·s -1 and 3.55mM -1 ·s -1 (like Figure 9a As shown in the figure, the longitudinal relaxation rate of the contrast agent in Comparative Example 1, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and rat microsomes decreased after incubation under simulated hypoxia conditions in vitro.

[0106] like Figure 9b As shown, T1-weighted imaging at different concentrations reveals that the T1 contrast effect of the contrast agent obtained in Comparative Example 1 was slightly brighter before incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro than after 4 hours of incubation.

[0107] Note: T1 weighted imaging parameters are set as follows: echo time (TE) = 134ms, repetition time (TR) = 500ms, number of scans (NS) = 1.

[0108] Performance Test 4

[0109] This test was conducted on a 0.5T MRI scanner to measure the transverse relaxation time T2 and T2-weighted imaging of the MRI contrast agent product (35 μg Fe) obtained in Comparative Example 1 of this invention, along with reduced nicotinamide adenine dinucleotide phosphate (NADPH) (50 μL, 0.5 mg) and rat microsomes (20 μL, 20 mg / mL) under simulated hypoxia conditions before and after 4 hours of incubation. The procedure included:

[0110] Samples with iron concentrations of 0.11 mM, 0.17 mM, 0.25 mM, 0.37 mM, and 0.56 mM were prepared respectively. After testing on a 0.5T MRI scanner, the transverse relaxation rates of the MRI contrast agent of this invention, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and rat microsomes before and after incubation under simulated hypoxia conditions were found to be 39.31 mM. -1 ·s -1 and 42.89mM -1 ·s -1 (like Figure 10a As shown in the figure, it can be seen that the contrast agent of Comparative Example 1, after being incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro, exhibited a slight increase in transverse relaxation rate. Furthermore, the ratio of transverse relaxation rate to longitudinal relaxation rate (r2 / r1) is the gold standard for determining the type of contrast agent. The ratio before incubation was 7.50, and after incubation, it was 11.07. These increases may be due to the influence of some biological enzymes (rat microsomes), leading to some minor aggregation. However, the ratio indicates that the contrast agent obtained in Comparative Example 1 cannot respond under simulated hypoxia conditions, failing to transform from a T2-type contrast agent to a T1-type contrast agent.

[0111] like Figure 10b As shown, T2-weighted imaging at different concentrations reveals that the T2 contrast-enhancing effect of the contrast agent obtained in Comparative Example 1 was brighter before incubation with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rat microsomes under simulated hypoxia conditions in vitro than after 4 hours of incubation. This is also due to the slight influence of biological enzymes.

[0112] Note: T2 weighted imaging parameters are set as follows: echo time (TE) = 334ms, repetition time (TR) = 2000ms, number of scans (NS) = 1.

[0113] Performance Test 5

[0114] This test investigates the toxicity of the contrast agent obtained in Example 1 to normal cells and cancer cells. The procedure includes using CCK-8 to determine the cytotoxicity of the contrast agent in mouse breast cancer cells (4T1) and human umbilical vein endothelial cells (HUVEC).

[0115] The following explanation uses 4T1 cells as an example. 100 μL of 4T1 cells were seeded into each well of a 96-well plate at a density of 5000 cells per well. The 96-well plate was then placed in a CO2 incubator and cultured at 37°C for 24 h. Next, a contrast agent was dissolved in complete culture medium and serially diluted to multiple concentrations (eight different concentrations ranging from 0.06 to 1.00 mM were used in this invention). This solution was added to the wells of the 96-well plate and co-incubated with the cells for 24 h. Then, 100 μL of 10% CCK-8 solution was added to each well, and the plate was incubated for 3 h. The absorbance (OD) at 450 nm was measured using a microplate reader. 450nm Four replicates were performed for each contrast agent concentration (referred to as the experimental group) and the control group. The relative cell viability was calculated based on the absorbance values. The blank group consisted of complete culture medium without cells, and the control group consisted of cells cultured without any additional material.

[0116] The cytotoxicity test for HUVEC cells is basically the same as described above.

[0117] Relative cell viability (%) = 100 × (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group)

[0118] like Figure 11 As shown, both 4T1(a) and HUVEC(b) cells exhibited high levels of viability. Even at an iron concentration of 1 mM, the cell viability of both 4T1 cells and human umbilical vein endothelial cells (HUVECs) remained above 98%. These results indicate that the cytotoxicity of the contrast agent involved in this invention is negligible.

[0119] Performance Test 6

[0120] This test investigates the biological tissue toxicity of the MRI contrast agent obtained in Example 1. The procedure includes:

[0121] The tissue toxicity of contrast agents in normal BALB / c mice (4-6 weeks, 20g) was determined using the hematoxylin-eosin staining (H&E staining method).

[0122] Four-week-old BALB / c mice were divided into two groups: an experimental group and a control group.

[0123] The first group was injected via tail vein with 150 μL of physiological saline solution containing the contrast agent obtained in Example 1, with an iron ion concentration of 0.1 mM / kg; the second group was injected via tail vein with 150 μL of physiological saline as a control group. After being raised under normal conditions for 3 days, the animals were euthanized by cervical dislocation, and the heart, liver, spleen, lungs, and kidneys were collected for H&E section staining and microscopic observation.

[0124] like Figure 12As shown, compared with the saline control group mice, all organs of the contrast agent-treated group mice obtained in Example 1 of this invention exhibited similar cell morphology and structure. Specifically, the hepatocytes in the liver sections were relatively normal, with no signs of inflammation. No pulmonary fibrosis was observed in the lung sections either. This indicates that the MRI contrast agent involved in this invention does not produce any significant toxicity in vivo, and therefore has great potential for biomedical applications.

[0125] Performance Test 7

[0126] This test investigates the in vivo MRI imaging experiment of the contrast agent obtained in Example 1. The operation method includes:

[0127] A batch of SPF-condition BALB / c mice (4-6 weeks old, 20g) was purchased from the company. 4T1 cells were selected as subcutaneous tumor model cells. After culturing, the cells were uniformly dispersed in PBS solution at a density of 2 × 10⁻⁶. 6 The PBS solution was administered at a dose of 1 / mL, and then each Balb / c mouse was subcutaneously injected into the axilla. After culturing for 7-10 days, a tumor model was successfully established. The tumor-bearing mice obtained could be used for subsequent in vivo experiments. All animal experiments followed the protocol approved by the Institute of Animal Protection, Chinese Academy of Sciences.

[0128] The successfully constructed 4T1 tumor-bearing mice were divided into two groups: the hypoxia response experimental group (EAmP) and the non-hypoxia response control group (EBmP). First, 100 μL of 29% urethane solution was injected intraperitoneally. After both groups of mice were deeply anesthetized, a blank scan was performed before contrast agent injection. Then, the experimental group received EAMP via tail vein injection, and the control group received EBmP via tail vein injection. The iron ion dose for both groups was 0.1 mmol / kg. The mice were then fixed and placed in a 1.5T miniature magnetic resonance imaging (MRI) device, and T1-weighted imaging was performed at 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h post-injection.

[0129] like Figure 13 As shown, after injecting two groups of contrast agents into tumor-bearing mice via the tail vein at a dose of 0.1 mmol / kg, the two groups exhibited significantly different imaging enhancement effects over time. Furthermore, to more intuitively analyze the changes in the signal intensity ratio (signal intensity after injection versus before injection) of the tumor region in the MRI images, the signal intensity was represented by grayscale values ​​determined by ImageJ software. Specifically, the brightness of the MRI image of the tumor site in the EAMP group increased significantly over time, showing obvious T1 signal enhancement. In addition, the signal intensity ratio gradually increased, reaching 135 ± 9.6% at the 6-hour time point (e.g., ...). Figure 14(As shown in (a)). This indicates that the contrast agent described in Example 1 has a sensitive hypoxia response, resulting in dissociation and selective activation of T1 MRI signals at the tumor site. Switching from a T2-type contrast agent to a T1-type contrast agent improves the specificity and selectivity of tumor diagnosis. However, the brightness of the T1 images in the EBmP-injected control group began to darken from the first hour and gradually decreased over time. The relative signal intensity ratio also illustrates this fact, as shown in (a). Figure 14 As shown in (b), the brightness decreased to 85±5.6% at the 6th hour. This is because the nanoclusters EBmP are unresponsive to hypoxic environments and can only accumulate at the tumor site over time, continuously presenting a T2 contrast signal.

[0130] Therefore, the hypoxia-responsive T2-T1 switching MRI contrast agent of this invention exhibits high specificity and selectivity for tumor sites. It can selectively activate T1 MRI signals at tumor sites, switching from dark signals to bright signals, thereby improving the specificity and selectivity of tumor detection and achieving excellent imaging contrast performance. Furthermore, it has good biocompatibility and low toxicity, and due to its response to tumor hypoxia, the nanocluster contrast agent can dissociate and be rapidly metabolized in vivo.

[0131] Note: T2-weighted mouse imaging parameters were set as follows: TE = 60.86 ms, TR = 3000 ms, matrix = 512 × 256, slice thickness = 0.27 mm.

[0132] The applicant declares that this invention illustrates a hypoxia-responsive T2-T1 switchable MRI contrast agent, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials in the product, addition of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

[0133] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0134] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A hypoxia-responsive T2-T1 switchable MRI contrast agent, characterized in that, The hypoxia-responsive T2-T1 switchable MRI contrast agent comprises iron oxide nanoclusters and polyethylene glycolamine and / or ethanolamine modified on the iron oxide nanoclusters; the iron oxide nanoclusters are obtained by crosslinking iron oxide nanoparticles with hypoxia-sensitive azo bonds; The iron oxide nanoparticles have a particle size of less than 4 nm; The size of the iron oxide nanoclusters is no greater than 200 nm; The hypoxia-responsive T2-T1 switchable MRI contrast agent is prepared by the following method, the method comprising: (1) Preparation of monodisperse iron oxide nanoparticles with fatty acid-modified surface; (2) The active ester is modified on the surface of the product in step (1) to activate it, thereby obtaining the activated product; (3) The activated product was coupled with an azo bond coupling agent to obtain iron oxide nanoclusters; (4) Modify the iron oxide nanoclusters of step (3) with polyethylene glycolamine and / or ethanolamine to obtain the hypoxia-responsive T2-T1 switchable MRI contrast agent; The active ester in step (2) includes DOPAC-PFP, which is prepared by mixing 3,4-dihydroxyphenylacetic acid and pentafluorophenol in an organic solvent, adding N,N'-dicyclohexylcarbodiimide solution dropwise to the system, and reacting at 15-40℃ for 12-24h to obtain the product.

2. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 1, characterized in that, The preparation method includes: (1) Preparation of monodisperse iron oxide nanoparticles with fatty acid-modified surface; (2) The active ester is modified on the surface of the product in step (1) to activate it, thereby obtaining the activated product; (3) The activated product was coupled with an azo bond coupling agent to obtain iron oxide nanoclusters; (4) Modify the iron oxide nanoclusters of step (3) with polyethylene glycolamine and / or ethanolamine to obtain the hypoxia-responsive T2-T1 switchable MRI contrast agent; The active ester in step (2) includes DOPAC-PFP, which is prepared by mixing 3,4-dihydroxyphenylacetic acid and pentafluorophenol in an organic solvent, adding N,N'-dicyclohexylcarbodiimide solution dropwise to the system, and reacting at 15-40℃ for 12-24h to obtain the product.

3. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 2, characterized in that, Step (1) specifically includes: first, reacting iron salt with fatty acid salt to obtain fatty acid iron complex precursor, and then thermally decomposing it to obtain the product.

4. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 3, characterized in that, The molar ratio of the iron salt to the fatty acid salt is 1:(2-4).

5. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 3, characterized in that, The iron salt reacts with the fatty acid salt at 60-80°C for 4-6 hours.

6. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 3, characterized in that, The fatty acid iron complex precursor was then purified.

7. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 3, characterized in that, The thermal decomposition reaction is first subjected to vacuum treatment at 80-100℃ for 2-6 h, and then reacted at high temperature at 240-260℃ for 25-40 min.

8. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 2, characterized in that, The organic solvent includes anhydrous 1,4-dioxane.

9. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 2, characterized in that, Step (2) specifically includes: mixing the active ester with iron oxide nanoparticles with fatty acid modified on the surface and dissolving them in an organic solvent, and refluxing at 40-60℃ for 60-84 h.

10. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 9, characterized in that, The mass ratio of the active ester to the iron oxide nanoparticles with fatty acid surface modification is (5-15):

1.

11. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 2, characterized in that, The azo bond coupling agent in step (3) includes diaminoazobenzene, 4,4'-azobenzoic acid chloride or 2,2'-azoaniline.

12. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 2, characterized in that, The coupling reaction described in step (3) is carried out at 15-40℃ for 18-30 h.

13. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 2, characterized in that, The mass ratio of the azo bond coupling agent to the activated product in step (3) is 1:(4-6).

14. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 2, characterized in that, Step (4) specifically includes: mixing iron oxide nanoclusters with polyethylene glycolamine and reacting them at 15-40℃ for 18-30 h, and then mixing them with ethanolamine and reacting them at 15-40℃ for 18-30 h to obtain the final product.

15. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 2, characterized in that, The polyethylene glycolamine mentioned in step (4) is a methyl-terminated polyethylene glycolamine.

16. The method for preparing the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 2, characterized in that, The amount of polyethylene glycolamine and / or ethanolamine used is more than 10 times the mass of the iron oxide nanoclusters.

17. The use of the hypoxia-responsive T2-T1 switchable MRI contrast agent according to claim 1 in the preparation of tumor diagnostic or detection reagents.

18. A contrast-enhancing composition, characterized in that, The contrast composition comprises the hypoxia-responsive T2-T1 switchable MRI contrast agent of claim 1 and pharmaceutically acceptable excipients.

19. The imaging composition according to claim 18, characterized in that, The excipients include a diluent.

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