MRET-effect-mediated ultra-sensitive ROS-responsive contrast agent and preparation method and application thereof

Abstract

The invention provides an MRET effect mediated ultra-sensitive ROS (reactive oxygen species) response type contrast agent as well as a preparation method and application thereof. The ultra-sensitive ROS response type contrast agent is prepared from two parts, namely a superparamagnetic quenching agent and a paramagnetic T1 reinforcing agent, wherein the superparamagnetic quenching agent is iron oxide nano particles modified by ferrocene; and the paramagnetic T1 reinforcing agent is beta-CD-DOTA (Gd) modified by beta-cyclodextrin. In an ROS environment, a host-guest assembly is formed based on the host-guest interaction of ferrocene and beta-cyclodextrin, and DOTA (Gd) is opened and released, so that the enhancement effect of a T1 signal under the mediation of an MRET effect is quenched, and the T1 signal is activated from OFF to ON. The contrast agent provided by the invention not only can respond when the ROS concentration is as low as 0.1 mM, but also can be applied to contrast diagnosis of diseases in other ROS environments, so that the application range of the contrast agent is greatly expanded.

Description

Technical Field

[0001] The present invention relates to the technical field of contrast agents, and in particular to an ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect, and a preparation method and application thereof. Background Art

[0002] Tumors are one of the most serious threats to human life and health. As an important branch of modern medicine, medical imaging plays an irreplaceable role in tumor diagnosis. With the continuous advancement of technology, medical imaging has provided strong support for the precise positioning and efficacy evaluation of tumors, and provided key information for clinical decision-making. The continuous development of imaging has significantly reduced the mortality rate of tumor patients. In particular, non-invasive imaging technology has played an important role in guiding the diagnosis and treatment of tumors. In the field of medical imaging, early diagnosis plays a vital role in improving patient survival. Magnetic resonance imaging (MRI or MR imaging) has significant advantages due to its non-ionizing radiation and strong penetration. MRI technology plays an increasingly important role in tumor diagnosis and treatment due to its non-invasiveness and superior penetration depth. However, more than 30% of MRI scans rely on the injection of contrast agents to enhance the detection signal. The MRI contrast agents widely used in clinical practice are complexes of the rare earth element gadolinium (Gd). Since the discovery in 2006 that free gadolinium ions may induce nephrogenic systemic fibrosis (NSF), the safety and stability of MRI contrast agents have attracted increasing attention.

[0003] On the other hand, there are still many problems in the application of MRI technology in the early diagnosis of diseases, such as the low signal-to-noise ratio (SNR) of MRI contrast agents. During magnetic resonance imaging, the use of MRI contrast agents reduces the ratio between the signal intensity of the image and the background noise, thereby affecting the quality of the image and the accuracy of the diagnosis. In order to overcome the limitations of the low signal-to-noise ratio of traditional MRI contrast agents, contrast agents that activate MRI contrast agents to respond to lesions have been widely studied. For example, Chinese invention patent CN118022009A discloses a hypoxia-responsive T2-T1 switchable MRI contrast agent and its preparation method and application; the MRI contrast agent involved in the patent can specifically respond to the hypoxic environment of the tumor microenvironment, thereby realizing the change from aggregation to dispersion of extremely small iron oxide nanoclusters, and then realizing the switching from T2-type contrast signal to T1-type contrast signal, and after dissociating in response to the hypoxic environment, the extremely small iron oxide nanoparticles that are transformed again are more conducive to metabolism, increasing biosafety. The transformation of the two contrast signals is obtained by using extremely small iron oxide nanoparticles to respond to changes in the microenvironment, which increases the specificity of the tumor area. Monodisperse extremely small iron oxide nanoparticles are a T1 contrast agent, while clusters of small iron oxide nanoparticles after aggregation are a T2 contrast agent. Such a transition between dispersion and aggregation states can realize the mutual conversion of T1 signals and T2 signals. The contrast agent can effectively reduce the interference of background signals during tumor diagnosis, thereby improving the specificity and selectivity of the contrast agent. For another example, Jingxiang Wang et al. publicly reported in Chemical Science Issue 13, 2019 a ROS-responsive magnetic resonance imaging probe composed of three components: ferromagnetic nanoparticles, hyaluronic acid and Gd-DTPA, which is used for high-sensitivity imaging of ROS in vivo and diagnosis of sepsis. Paramagnetic contrast agents greatly reduce the longitudinal relaxation time (T1) of hydrogen nuclei in surrounding water molecules, thereby enhancing the T1 signal of surrounding water. However, when the paramagnetic contrast agent is close to superparamagnetic magnetic beads (several nanometers), the enhancement effect of the contrast agent on the T1 signal of water will be quenched, that is, the MRET effect. The probe provided by the above technical solution is based on the MRET principle, combined with the contrast agent Gd-DTPA, and connected to the ferromagnetic nanoparticles modified by hyaluronic acid. Under normal conditions, since Gd-DTPA and ferromagnetic nanoparticles are very close, the T1 signal of the surrounding water will not be affected. In the presence of ROS, the above technical solution can act as a free radical inhibitor on ROS based on hyaluronic acid to degrade ROS, resulting in the detachment of Gd-DTPA from the surface of ferromagnetic nanoparticles, so that the T1 signal is greatly enhanced. However, the response marker in the above scheme still requires a certain magnitude, and how to achieve more sensitive MRI signal activation is one of the problems that those skilled in the art need to solve.

[0004] Based on the prior art, the present invention provides an ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect, using ferrocene-modified iron oxide ferromagnetic nanoparticles as a superparamagnetic quencher and β-cyclodextrin-modified Gd-DOTA as a paramagnetic T1 enhancer to achieve the MRET effect in a ROS environment. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect and a preparation method and application thereof, which has the characteristics of ultra-sensitive response and can respond to ROS at the level of 0.1mM, which is also a major breakthrough in the development of contrast agents.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides an ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect, which consists of two parts: a superparamagnetic quencher and a paramagnetic T1 enhancer; wherein the superparamagnetic quencher is ferrocene-modified iron oxide nanoparticles (INOPS-Fc); and the paramagnetic T1 enhancer is a small molecule β-CD-DOTA (Gd) modified with β-cyclodextrin.

[0007] Under normal circumstances, the superparamagnetic quencher and the paramagnetic T1 enhancer are tightly combined to form a host-guest assembly based on the host-guest interaction of ferrocene and β-cyclodextrin; under the ROS environment, the host-guest interaction is disconnected, the host-guest assembly opens and releases DOTA (Gd), so that the superparamagnetic quencher and the paramagnetic T1 enhancer are separated, the enhancement effect of the T1 signal mediated by the MRET effect is quenched, and the T1 signal is activated from OFF to ON.

[0008] Preferably, the superparamagnetic quencher is prepared by a ligand exchange reaction between oleic acid-modified iron oxide nanoparticles (INOPS-OA) and ferrocene, wherein the ferrocene Fc-modified iron oxide nanoparticles are dispersible in an aqueous phase.

[0009] Preferably, the particle size of the iron oxide nanoparticles is 10 to 15 nm.

[0010] Preferably, the grafting rate of ferrocene is 3-7%.

[0011] Preferably, in the ultrasensitive ROS-responsive contrast agent, the molar ratio of Fe / Gd is between (15 and 30) / 1.

[0012] Preferably, the molar ratio of ferrocene in the superparamagnetic quencher to β-cyclodextrin in the paramagnetic T1 enhancer is 1:3 to 1:9; the reaction process should ensure that cyclodextrin is in excess, and the unreacted cyclodextrin is finally removed by filtration.

[0013] Preferably, the concentration of ROS in the ROS environment is 0.1-1 mM.

[0014] As one of the purposes of the invention, the present invention also provides a method for preparing the above-mentioned MRET effect-mediated ultrasensitive ROS-responsive contrast agent, the specific steps of which include:

[0015] (1) Preparation of paramagnetic quencher INOPS-Fc;

[0016] INOPS-Fc was obtained by subjecting oleic acid-modified iron oxide nanoparticles INOPS-OA to a ligand exchange reaction;

[0017] The first ligand exchange reaction yielded INOPS-BF 4 Then a second ligand exchange was performed to finally obtain;

[0018] (2) Preparation of paramagnetic T1 enhancer β-CD-DOTA(Gd): alkynyl-DOTA(Gd) was subjected to a click reaction with 7-(6-azido-6-deoxy)-β-cyclodextrin to obtain β-CD-DOTA(Gd);

[0019] (3) mixing the INOPS-Fc prepared in step (1) and the β-CD-DOTA(Gd) prepared in step (2) to obtain the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect;

[0020] Step (1) and step (2) are performed in no particular order.

[0021] Preferably, in step (1), the ligand exchange reaction includes a first ligand exchange reaction and a second ligand exchange reaction.

[0022] More preferably, the ligand of the first ligand exchange reaction is nitrosoborate tetrafluoroborate; the process comprises dispersing INOPS-OA particles in toluene, dispersing nitrosoborate tetrafluoroborate in N,N-dimethylformamide (DMF), then adding the nitrosoborate tetrafluoroborate solution to the nanoparticle solution, and mixing them evenly for 5 to 10 minutes to obtain INOPS-OA. 4 .

[0023] More preferably, the ligand of the second ligand exchange reaction is polyacrylamine-ferrocene; comprising: INOPS-BF 4 Disperse in water, add polyacrylamine-ferrocene, stir and react for 24 hours, then dialyze to remove excess PAA-Fc to obtain INOPS-Fc.

[0024] Preferably, in step (2), β-CD-DOTA(Gd) is obtained by a click reaction between alkynyl-DOTA(Gd) and 7-(6-azido-6-deoxy)-β-cyclodextrin, wherein the ratio of the molar amount of the alkynyl group in β-CD-DOTA(Gd) to the molar amount of the azide group in 7-(6-azido-6-deoxy)-β-cyclodextrin is greater than 1:2.

[0025] As a preferred embodiment, the synthesis method of alkynyl-DOTA (Gd) comprises:

[0026] In the first step, cyclopentane and sodium bicarbonate are dispersed in acetonitrile, and then tert-butyl bromoacetate is added to carry out a substitution reaction, mixed, stirred evenly, reacted in an ice bath for 48 hours, filtered and purified to obtain a white solid, which is compound 1; wherein the molar ratio of tert-butyl bromoacetate / cyclopentane is 3.1-3.5:1, so as to ensure the smooth completion of the tri-substitution reaction to obtain the tri-substitution product;

[0027] The second step is to dissolve propargylamine in dichloromethane, then place a sodium hydroxide solution on the dichloromethane layer, carefully add bromoacetyl bromide to the organic layer (dichloromethane) using a syringe, gently stir for 5 to 10 minutes, produce a large amount of white precipitate, continue to vigorously stir the reaction for 2 to 5 hours, then use dichloromethane and water for liquid separation extraction to obtain an organic phase, continue to extract the aqueous phase with dichloromethane once, combine and collect the organic phases, continue to wash with sodium carbonate water, then dry over anhydrous magnesium sulfate, spin dry, and vacuum dry to obtain the compound 2alkynyl-Br;

[0028] The third step is to disperse compound 1 and compound 2 in anhydrous acetonitrile, then add potassium carbonate, and react the obtained mixed solution at 90° C. for 48 to 64 hours. The filtered solvent is dried and purified to obtain compound 3; the trisubstituted product reacts with alkynyl-Br to replace the last H of the cyclotengnin to obtain a tetrasubstituted product (compound 3), wherein the ratio of bromoacetyl bromide to propargylamine needs to be greater than 3 to ensure the smooth progress of the substitution reaction;

[0029] Step 4: dissolving compound 3 in formic acid, stirring and reacting at 60° C. overnight, removing the tert-butyl ester, and removing the solvent to obtain compound 4alkynyl-DOTA(COOH);

[0030] Step 5: Compound 4 and Gd 2 O 3 Disperse in 20 mL of water, stir and react at 100 °C for more than 24 hours to finally obtain alkynyl-DOTA(Gd). 2 O 3The chelation reaction chelates the Gd ions into DOTA and finally obtains alkynyl-DOTA(Gd); the reaction needs to be refluxed at high temperature for 12 hours to fully chelate.

[0031] As one of the purposes of the invention, the present invention also provides a contrast agent composition, comprising the above-mentioned ultrasensitive ROS-responsive contrast agent mediated by the MRET effect and a pharmaceutically acceptable adjuvant; the adjuvant is a diluent.

[0032] As one of the purposes of the invention, the present invention also provides the use of the above-mentioned MRET effect-mediated ultrasensitive ROS-responsive contrast agent in the imaging diagnosis of diseases with a ROS environment.

[0033] Furthermore, the present invention also provides the use of the above-mentioned MRET effect-mediated ultrasensitive ROS-responsive contrast agent in tumor imaging diagnosis.

[0034] Beneficial technical effects obtained by the present invention:

[0035] (1) The ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect provided by the technical solution of the present invention can respond ultra-sensitively to ROS environments as low as 0.1 mM concentration. When the contrast agent is exposed to the tumor ROS environment, the host-guest binding assembly Fc and β-CD in the contrast agent respond and break, resulting in the separation of the T1 enhancer and the quencher, thereby activating the T1 signal of DOTA (Gd) and presenting a T1 ON state, thereby achieving specific T1 contrast-enhanced imaging of the tumor site.

[0036] (2) The ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect provided by the present invention can be stably dispersed in a physiological environment, has good biocompatibility, and can specifically respond to release T1 signals in a ROS environment, thereby effectively improving sensitivity and reducing interference from background signals, and has specificity and selectivity for imaging tumor sites.

[0037] (3) The contrast agent provided by the present invention is based on the principle of ROS response, so that it can not only be used for magnetic resonance imaging detection and diagnosis of tumors, but also can be applied to the detection and diagnosis of other ROS environments, such as other inflammatory diseases, thereby achieving accurate diagnosis of various diseases.

[0038] (4) The ultrasensitive ROS-responsive contrast agent mediated by the MRET effect provided by the present invention has good biocompatibility and is very safe to use. Moreover, based on the MRET effect mediation and ROS response, it is easy to use and has ultrahigh sensitivity, and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1This is a schematic diagram of the working principle of the response of the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect of the present invention.

[0040] Figure 2 This is a transmission electron microscopy image of the INOPs-OA nanoparticles provided in Example 1 of the present invention.

[0041] Figure 3 This is a transmission electron microscopy image of the INOPs-BF4 nanoparticles provided in Example 1 of the present invention.

[0042] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the first step of PAA-Fc provided in Example 1 of the present invention.

[0043] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of the second step of PAA-Fc provided in Example 1 of the present invention.

[0044] Figure 6 This is the hydrogen nuclear magnetic resonance spectrum of compound 1 provided in Example 1 of the present invention.

[0045] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of compound 2 provided in Example 1 of the present invention.

[0046] Figure 8 This is the hydrogen nuclear magnetic resonance spectrum of compound 4 provided in Example 1 of the present invention.

[0047] Fig. 9 This is a transmission electron microscopy image of the INOPs-Fc nanoparticles provided in Example 1 of the present invention.

[0048] Fig.10 The longitudinal relaxation rate (r) of the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect provided in Example 1 of the present invention before and after ROS response under ROS simulation conditions in vitro 1 ) comparison chart.

[0049] Fig.11 This is a graph showing the cytotoxicity test of the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect provided in Example 1 of the present invention in human umbilical vein endothelial cells (HUVEC).

[0050] Fig.12 This is a graph showing the cytotoxicity test of the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect provided in Example 1 of the present invention in mouse breast cancer cells (4T1).

[0051] Fig.13 This is a T1-weighted MRI image of the contrast agent of Example 1 of the present invention in vivo in tumor-bearing mice transplanted with 4T1 cells. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The present invention provides an ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect, which can respond in a ROS environment to separate a T1 enhancer from a quencher, thereby activating the T1 signal of DOTA (Gd) and presenting a T1 ON state. Compared with the ROS-responsive contrast agent in the prior art, the ROS response can have an ultra-sensitive response at a ROS level of 0.1 mM.

[0054] One aspect of an embodiment of the present invention provides an ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect, which includes two parts: a superparamagnetic quencher and a paramagnetic T1 enhancer.

[0055] As a preferred embodiment, the superparamagnetic quencher is obtained by performing two ligand exchange reactions between oleic acid (OA)-modified iron oxide nanoparticles (INOPS-OA) and Fc-grafted PAA-Fc to obtain Fc-modified iron oxide nanoparticles (INOPS-Fc); the Fc-modified iron oxide nanoparticles can be dispersed in an aqueous phase.

[0056] As a preferred technical solution of the present invention, the superparamagnetic quencher is selected from iron oxide with an average particle size of 10-15nm, for example, it can be 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, etc., but it is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable; the particle size can shield the T1 signal at a close distance to the maximum extent, and release the T1 signal when the distance is dispersed after the response, thereby achieving a highly sensitive contrast signal response.

[0057] The synthesis process of INOPS-Fc requires two steps of ligand exchange reaction, wherein the grafting rate of Fc of PAA-Fc is selected to be 3-7% during the second ligand exchange, for example, 3%, 4%, 5%, 6%, 7%, etc., but not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0058] As a preferred embodiment, the preparation of the paramagnetic T1 enhancer is through a click reaction between alkynyl-DOTA(Gd) and 7-(6-azido-6-deoxy)-β-cyclodextrin to obtain a β-CD modified DOTA(Gd)T1 enhancer, namely β-CD-DOTA(Gd).

[0059] See also Figure 1, which is a schematic diagram of the working principle of the contrast agent of the present invention. It can be seen from the figure that the terminal of the superparamagnetic quencher is modified with Fc, and the terminal of the paramagnetic T1 enhancer is modified with β-CD. Under normal microenvironment, the superparamagnetic quencher and the paramagnetic T1 enhancer can realize close-range host-guest binding assembly through the strong host-guest binding effect between Fc and β-CD, so that the T1 signal of β-CD-DOTA (Gd) is shielded; when the contrast agent obtained by the host-guest binding assembly is in a ROS environment, the interaction force between Fc and β-CD becomes weak, and the host-guest binding assembly is opened, which eventually creates a long distance between INOPS-Fc and β-CD-DOTA (Gd), resulting in the superparamagnetic quencher being insufficient to shield the T1 signal of β-CD-DOTA (Gd), so that the contrast agent expresses a T1 bright signal in a ROS environment, and expresses a T1 off dark signal in a normal environment.

[0060] It is worth noting that when the contrast agent is in an environment with very low ROS content, for example, at a concentration of 0, 1 mM, the contrast agent can also achieve a sensitive response, ultimately achieving ultrasensitive diagnosis of the disease.

[0061] Furthermore, the present invention also provides a method for preparing the above-mentioned MRET effect-mediated ultrasensitive ROS-responsive contrast agent, comprising the following steps:

[0062] (1) Preparation of paramagnetic quencher INOPS-Fc: First, oleic acid-modified iron oxide nanoparticles (INOPS-OA) were prepared, and then the first ligand exchange reaction was performed to obtain INOPS-BF 4 Then a second ligand exchange was performed to finally obtain INOPS-Fc;

[0063] (2) Preparation of paramagnetic T1 enhancer: first prepare alkynyl-DOTA(Gd), then click-react it with 7-(6-azido-6-deoxy)-β-cyclodextrin to obtain β-CD-DOTA(Gd);

[0064] (3) mixing the paramagnetic quencher INOPS-Fc from step (1) and the paramagnetic T1 enhancer from step (2) to obtain the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect;

[0065] Step (1) and step (2) are performed in no particular order.

[0066] As a preferred embodiment, the oleic acid-modified iron oxide nanoparticles are hydrophobic nanoparticles; the preparation method thereof specifically comprises: mixing iron salt, fatty acid salt and solvent, and subjecting the mixture to vacuum treatment and thermal decomposition reaction to obtain the hydrophobic INOPS-OA nanoparticles.

[0067] Preferably, the molar ratio of the iron salt to the fatty acid salt is 1:(1.5-5), for example, 1:2.2, 1:2.8, 1:3, 1:3.7 or 1:4, etc., but not limited thereto.

[0068] Preferably, the temperature of the vacuum treatment is 80-100°C, for example, 80°C, 85°C, 85°C, 90°C, 95°C or 100°C, etc., but not limited thereto; the time of the vacuum treatment is 3-5h, for example, 3h, 3.5h, 4h, 4.5h, etc., but not limited thereto; the above-listed values ​​are not exhaustive, and other values ​​not listed within the numerical range are also applicable.

[0069] Preferably, the thermal decomposition reaction is carried out under a protective atmosphere, the reaction heating rate is 5-7°C / min, for example, 5°C / min, 6°C / min or 7°C / min, etc., the heating end point of the thermal decomposition reaction is 320-330°C, for example, 320°C, 322°C, 325°C, 327°C, or 330°C, etc., the thermal decomposition reaction time is 25-35min, for example, 26min, 28min, 30min, 32min or 34min, etc.; the above-listed values ​​are not exhaustive, and other values ​​not listed within the numerical range are equally applicable.

[0070] Preferably, the INOPS-OA nanoparticles formed by modifying INOPS with oleic acid are hydrophobic, and the particle size thereof is 10-15 nm.

[0071] As a preferred embodiment, the ligand for the first ligand exchange is nitroso tetrafluoroborate, which specifically includes: dispersing INOPS-OA particles in toluene, dispersing nitroso tetrafluoroborate in N,N-dimethylformamide (DMF), then adding the nitroso tetrafluoroborate solution to the nanoparticle solution, and mixing them evenly, mixing for 5 to 10 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes or 9 minutes, and then oscillating the reaction for 2-5 hours, for example, 2 hours, 3 hours, 3 hours, 4 hours or 5 hours, to obtain INOPS-OA. 4 .

[0072] Preferably, the concentration of the dispersion of INOPS-OA nanoparticles is 3-6 mg / mL.

[0073] Preferably, the concentration of the tetrafluoroborate nitrosoborate solution is 10 mg / mL.

[0074] As a preferred embodiment, the ligand for the second ligand exchange is PAA-Fc, and the specific preparation method comprises: 4Disperse in water, add PAA-Fc, stir and react for 24 hours, then dialyze to remove excess PAA-Fc, and finally obtain INOPS-Fc.

[0075] Preferably, in PAA-Fc, the grafting rate of Fc is 3% to 7% to ensure that the amount of Fc on the surface of INOPS is neither too much nor too little, thereby ensuring the shielding effect of the paramagnetic quencher.

[0076] Preferably, the dialysis bag can selectively retain molecules with a molecular weight of 14 kD.

[0077] The principle of the synthesis of the β-CD-DOTA (Gd) is as follows:

[0078]

[0079] As shown in the above reaction equation, the synthesis method of compound 1 includes: dispersing cyclopentane and sodium bicarbonate in acetonitrile, then adding tert-butyl bromoacetate, mixing, stirring evenly, reacting in an ice bath for 48 hours, and performing post-treatment to obtain compound 1.

[0080] Preferably, the molar ratio of cyclamenine to tert-butyl bromoacetate is 1:3.

[0081] Preferably, the post-treatment comprises filtering out the insoluble matter, then concentrating the filtrate by rotary evaporation, and purifying the remaining mixture by recrystallization from toluene to obtain a white solid, which is Compound 1.

[0082] Preferably, the synthesis of compound 2 specifically includes: dissolving propargylamine in dichloromethane, then placing a sodium hydroxide solution on the dichloromethane layer, carefully adding bromoacetyl bromide to the organic layer using a syringe, and gently stirring for 5 to 10 minutes. At this time, a large amount of white precipitate may be produced, and then continue to vigorously stir the reaction for 2 to 5 hours, then add a certain volume of dichloromethane and water, separate and extract to obtain an organic phase, continue to extract the aqueous phase with dichloromethane once, combine and collect the organic phases, continue to wash with sodium carbonate water, and then dry over anhydrous magnesium sulfate, spin dry, and vacuum dry to obtain compound 2.

[0083] Preferably, the molar ratio of propargylamine to bromoacetyl bromide is 1:3.

[0084] Preferably, the synthesis of compound 3 specifically includes: dispersing compound 1 and compound 2 in acetonitrile, then adding potassium carbonate, reacting the obtained mixed solution at 90° C. for 48 to 64 hours to generate an insoluble solid, and post-treating the solvent to obtain a light yellow solid, which is compound 3; the above reaction is carried out under anhydrous conditions.

[0085] Preferably, acetonitrile needs to be subjected to anhydrous treatment in advance.

[0086] Preferably, the molar ratio of compound 1 to compound 2 is 1:3.

[0087] Preferably, the post-treatment includes removing the insoluble solid by filtration, rotary evaporating the solvent to dryness, and purifying the obtained solid mixture by silica gel column to obtain compound 3.

[0088] Preferably, the column chromatography developing solvent used in the silica gel column purification is a combined solvent of chloroform and methanol in a ratio of 6:1.

[0089] Preferably, the synthesis of compound 4 specifically includes dissolving compound 3 in formic acid, stirring the reaction at 60°C overnight, then the reaction product forms an azeotrope with methanol to remove the solvent, the remaining brown solid is dissolved in methanol, and then precipitated in excess cold ether, and the precipitation process is repeated twice to obtain compound 4.

[0090] Preferably, the synthesis of compound 5 specifically comprises: reacting compound 4 with Gd 2 O 3 The product was dispersed in 20 mL of water and stirred at 100° C. for more than 24 h to finally obtain compound 5.

[0091] Preferably, the reaction time can be 24 h, 30 h, 35 h, 40 h or 45 h, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0092] Preferably, the synthesis of β-CD-DOTA (Gd) specifically comprises: dissolving compound 5 and 7-(6-azido-6-deoxy)-β-cyclodextrin in 10 mL of dimethyl sulfoxide, then adding pentamethyldiethylenetriamine and cuprous bromide, freezing and deoxygenating three times, then reacting at 50° C. for 48 hours under the protection of nitrogen, and then dialyzing for more than 48 hours for use;

[0093] Preferably, the ratio of the molar amount of the alkynyl groups in β-CD-DOTA(Gd) to the molar amount of the azide groups in 7-(6-azido-6-deoxy)-β-cyclodextrin should be controlled to be greater than 1:2.

[0094] Preferably, the dimethyl sulfoxide is anhydrous dimethyl sulfoxide.

[0095] Preferably, the dialysis bag selected for dialysis is 35KD.

[0096] Preferably, the dialysis time can be 48h, 50h, 52h, 55h or 60h, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0097] As a preferred embodiment, the mixed reaction of the paramagnetic quencher INOPS-Fc and the paramagnetic T1 enhancer includes: dispersing INOPS-Fc in 10 mL of water, then adding the paramagnetic T1 enhancer, reacting at room temperature for 24 hours, dialysis for more than 48 hours, and finally ultrafiltration and concentration to obtain the final contrast agent product;

[0098] Preferably, the molar ratio of the paramagnetic quencher INOPS-Fc to the paramagnetic T1 enhancer β-CD-DOTA (Gd) needs to be greater than 1:7 to ensure that the Fc group on the iron oxide can fully react with β-CD-DOTA (Gd).

[0099] Preferably, the dialysis time can be 48h, 50h, 52h, 55h or 60h, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0100] Preferably, the dialysis bag used for dialysis is 35 kD.

[0101] Preferably, the ultrafiltration tube selected for ultrafiltration concentration is 100KD.

[0102] As a preferred embodiment, the present invention further provides a contrast composition, which comprises a pharmaceutically acceptable adjuvant and an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect.

[0103] Preferably, the adjuvant comprises a diluent.

[0104] More preferably, the diluent is PBS solution.

[0105] The present invention also provides a non-medical imaging method, which comprises: administering the ultra-sensitive ROS-responsive contrast agent composition mediated by the MRET effect described in the first aspect and the second aspect to a subject to be imaged, and performing imaging.

[0106] The present invention also provides the use of the ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect prepared by the above method in tumor magnetic resonance imaging.

[0107] The present invention also provides the use of the ultra-sensitive ROS-responsive contrast agent mediated by the MRET effect prepared by the above method as a tumor detection agent.

[0108] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0109] Unless otherwise specified, the experimental materials used in the embodiments of the present invention can be purchased from conventional biochemical reagent companies.

[0110] Example 1

[0111] The present embodiment provides an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect, which is a mixture of a paramagnetic quencher INOPS-Fc and a paramagnetic T1 enhancer β-CD-DOTA (Gd); the paramagnetic quencher INOPS-Fc is an iron oxide nanoparticle modified with ferrocene Fc, and the paramagnetic T1 enhancer is a small molecule β-CD-DOTA (Gd) obtained by a click reaction between β-cyclodextrin and an alkynyl-macrocyclic ligand-gadolinium alkynyl-DOTA (Gd); wherein the molar ratio of Fc to β-CD is 1:3; and the molar ratio of Fe / Gd is 20:1.

[0112] The paramagnetic quencher INOPS-Fc is prepared by two ligand exchange reactions of oleic acid-modified iron oxide nanoparticles, wherein the grafting rate of Fc modification in INOPS-Fc is 5%, the particle size of the iron oxide nanoparticles is 12nm; and INOPS-Fc can be dispersed in an aqueous phase.

[0113] The paramagnetic T1 enhancer β-CD-DOTA(Gd) was obtained by click reaction of alkynyl-DOTA(Gd) and 7-(6-azido-6-deoxy)-β-cyclodextrin.

[0114] Specifically, the preparation method of the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect comprises the following steps:

[0115] (1) Preparation of paramagnetic quencher INOPS-Fc;

[0116] First, INOPS-OA nanoparticles were prepared; ferric chloride hexahydrate (20 mmol, 5.4 g), sodium oleate (60 mmol, 18.3 g), ethanol (40 mL), water (30 mL), and n-hexane (70 mL) were mixed, and after mixing, they were vacuum treated at 80°C for 3 h, and thermally decomposed under a nitrogen atmosphere at a reaction temperature of 320°C, a heating rate of 5°C / min, and a reaction time of 30 min. After the reaction, hydrophobic oleic acid-modified iron oxide nanoparticles (INOPS-OA) were obtained.

[0117] Then, INOPS-OA was subjected to the first ligand exchange reaction; the exchanged ligand was nitrosoborate tetrafluoroborate, specifically comprising: dispersing INOPS-OA particles in toluene at a concentration of 3 mg / mL; nitrosoborate tetrafluoroborate was dispersed in N,N-dimethylformamide (DMF) at a concentration of 10 mg / mL; then the nitrosoborate tetrafluoroborate solution was added to the nanoparticle solution and mixed evenly, shaken for 5 minutes, and then subjected to an oscillation reaction for 2 hours to obtain INOPS-BF 4 .

[0118] Finally, the second ligand exchange reaction was carried out; the exchange ligand was PAA-Fc, which specifically included: INOPS-BF 4 Disperse in water, then add PAA-Fc, INOPS-BF 4 The molar ratio of PAA-Fc to PAA-Fc was 1:2, and the reaction was stirred for 24 hours, and then the excess PAA-Fc was removed with a dialysis bag with a molecular weight cutoff of 14kD, and finally INOPS-Fc was obtained; among which, the Fc grafting rate of PAA-Fc was 5%.

[0119] (2) Preparation of paramagnetic T1 enhancer β-CD-DOTA(Gd);

[0120] First, cyclopentane and sodium bicarbonate in a feeding molar ratio of 1:3 are dispersed in acetonitrile, and then tert-butyl bromoacetate is added, mixed, and stirred evenly. After reacting for 48 hours in an ice bath, the insoluble matter is filtered out, and the filtrate is then concentrated by rotary evaporation. The remaining mixture is purified by recrystallization from toluene to obtain a white solid, which is compound 1.

[0121] Then, propargylamine is dissolved in dichloromethane, and then sodium hydroxide solution is placed on the dichloromethane layer, and bromoacetyl bromide is carefully added to the organic layer (dichloromethane) using a syringe, and gently stirred for 5 to 10 minutes to produce a large amount of white precipitate, and the reaction is continued to be vigorously stirred for 2 to 5 hours, and then dichloromethane and water are used for liquid separation and extraction to obtain an organic phase, and the aqueous phase is further extracted once with dichloromethane, and the organic phases are combined and collected, and then washed with sodium carbonate water, and then dried over anhydrous magnesium sulfate, spin-dried, and vacuum-dried to obtain compound 2; the molar ratio of propargylamine to bromoacetyl bromide is 1:3.

[0122] Third, compound 1 and compound 2 with a feeding molar ratio of 1:3 are dispersed in anhydrous acetonitrile, and then potassium carbonate is added. The obtained mixed solution is reacted at 90°C for 48 to 64 hours to generate an insoluble solid. After filtering the insoluble solid, the solvent is rotary evaporated to dryness to obtain a solid mixture, which is then purified by silica gel column to obtain compound 3; the above reaction is carried out under anhydrous conditions, and the column chromatography developing solvent used in the purification process is a combination solvent of chloroform and methanol in a ratio of 6:1.

[0123] Fourth, 1.23 mmol of compound 3 was dissolved in 60 mL of formic acid, and the reaction was stirred at 60°C overnight. The reaction product formed an azeotrope with methanol to remove the solvent. The remaining brown solid was dissolved in methanol and then precipitated in excess cold ether. The precipitation process was repeated twice to obtain compound 4.

[0124] Fifth, compound 4 was reacted with Gd 2 O 3 The product was dispersed in 20 mL of water and stirred at 100° C. for more than 24 h to finally obtain compound 5.

[0125] Finally, the synthesis of β-CD-DOTA (Gd) specifically includes: dissolving compound 5 and 7-(6-azido-6-deoxy)-β-cyclodextrin in 10 mL of anhydrous dimethyl sulfoxide, then adding pentamethyldiethylenetriamine and cuprous bromide, freezing and deoxygenating three times, and then reacting at 50° C. for 48 hours under the protection of nitrogen, and then dialyzing with a 35KD dialysis bag for more than 48 hours for use; wherein the ratio of the molar amount of the alkynyl group in compound 5 to the molar amount of the azide group in 7-(6-azido-6-deoxy)-β-cyclodextrin is 1:3.

[0126] (3) Preparation of ultrasensitive ROS-responsive contrast agents mediated by MRET effect;

[0127] 5 mg of the paramagnetic quencher INOPS-Fc prepared in step (1) was dispersed in 10 mL of deionized water, and then 12 mg of the paramagnetic T1 enhancer β-CD-DOTA (Gd) was added, and the reaction was stirred at room temperature for 24 h. A dialysis bag with a KD of 35,000 was selected, and the excess β-CD-DOTA (Gd) was removed by dialysis for more than 48 h. Finally, ultrafiltration and concentration were performed through a 100 KD ultrafiltration tube to obtain the final contrast agent; wherein the molar ratio of the Fc group in the paramagnetic quencher INOPS-Fc to the β-CD group in the paramagnetic T1 enhancer β-CD-DOTA (Gd) was 1:3, so as to ensure that the Fc group on the iron oxide could fully react with the β-CD-DOTA (Gd).

[0128] See also Figure 2 , is a transmission electron microscopy image of INOPs-OA nanoparticles in this embodiment; Figure 3 INOPs-BF in this embodiment 4 Transmission electron microscopy images of nanoparticles; it can be seen from the figure that INOPs-OA nanoparticles and INOPs-BF 4 The particle size and dispersion of the nanoparticles remained almost unchanged.

[0129] See also Figure 4 , which is INOPS-BF in this embodiment 4 H NMR spectrum of Figure 5 is the hydrogen nuclear magnetic resonance spectrum of INOPS-Fc in this example; Figure 6 is the hydrogen nuclear magnetic resonance spectrum of compound 1 in this example; Figure 7 is the hydrogen nuclear magnetic resonance spectrum of compound 2 in this example; Figure 8 is the hydrogen nuclear magnetic resonance spectrum of compound 4 in this example; Fig. 9 This is the hydrogen nuclear magnetic resonance spectrum of INOPs-Fc prepared in this example, which indicates that the products of each step of this example were successfully synthesized.

[0130] Example 2

[0131] This embodiment provides an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect. The preparation method is basically the same as that of Example 1, except that the molar ratio of the paramagnetic quencher INOPS-Fc to the paramagnetic T1 enhancer β-CD-DOTA(Gd)) is 1:6.

[0132] Example 3

[0133] This embodiment provides an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect. The preparation method is substantially the same as that of Example 1, except that the grafting rate in INOPS-Fc is 3%.

[0134] Example 4

[0135] This embodiment provides an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect. The preparation method is substantially the same as that of Example 1, except that the grafting rate in INOPS-Fc is 7%.

[0136] Comparative Example 1

[0137] This comparative example provides an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect, which is a mixture of a 10% grafted paramagnetic quencher INOPS-Fc and a paramagnetic T1 enhancer β-CD-DOTA (Gd) in which the molar ratio of Fc and β-CD groups in INOPS-Fc is 1:3.

[0138] 5 mg of paramagnetic quencher INOPS-Fc with a grafting rate of 10% of Fc was dispersed in 10 mL of deionized water, and 12 mg of paramagnetic T1 enhancer β-CD-DOTA (Gd) was added. The reaction was stirred at room temperature for 24 hours, and then the excess β-CD-DOTA (Gd) was removed by dialysis. The KD of the dialysis bag was 35000, and finally the contrast agent was obtained.

[0139] The contrast agent obtained in this comparative example was subjected to in vitro simulated ROS conditions (H 2 O 2=0.1 mM), a specific response occurred, but because the INOPs surface was connected to an excessive amount of Gd elements, the response effect was poor and the T1 contrast signal could not change from OFF to ON.

[0140] Comparative Example 2

[0141] This comparative example provides an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect, which is a mixture of a 15% grafted paramagnetic quencher INOPS-Fc and a paramagnetic T1 enhancer β-CD-DOTA (Gd) in which the molar ratio of Fc and β-CD groups in INOPS-Fc is 1:3.

[0142] 5 mg of paramagnetic quencher INOPS-Fc with a grafting rate of 15% of Fc was dispersed in 10 mL of deionized water, and then 18 mg of paramagnetic T1 enhancer β-CD-DOTA (Gd) was added. The reaction was stirred at room temperature for 24 hours, and then the excess β-CD-DOTA (Gd) was removed by dialysis. The KD of the dialysis bag was 35000, and finally the contrast agent was obtained.

[0143] The contrast agent obtained in this comparative example was subjected to in vitro simulated ROS conditions (H 2 O 2 =0.1 mM), a specific response occurred, but because the INOPs surface was connected to an excessive amount of Gd elements, the response effect was poor and the T1 contrast signal could not change from OFF to ON.

[0144] Comparative Example 3

[0145] This comparative example provides an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect, which is a mixture of 5% grafted paramagnetic quencher INOPS-Fc and paramagnetic T1 enhancer Ad-DOTA (Gd) with a molar ratio of Fc and Ad groups in INOPS-Fc being 1:3.

[0146] The preparation method of Ad-DOTA(Gd) is similar to that of β-CD-DOTA(Gd), and the specific steps include: dissolving adamantane amine (6.05 g, 40 mmol) in 180 mL of dichloromethane, and then placing 50 mL of sodium hydroxide solution (0.1 M) on the dichloromethane layer, and then carefully adding bromoacetyl bromide (10.5 mL, 120 mmol) into the organic layer by syringe, and stirring gently for 5 min under ice bath conditions. At this time, a large amount of white precipitate may be generated, and then continue to stir vigorously for 2.5 h, and continue to extract the aqueous phase with dichloromethane once, and combine and collect. The organic phase is collected, and then washed with sodium carbonate, dried over anhydrous magnesium sulfate, spin-dried, and vacuum-dried to obtain compound Ad-Br; compound 1 and compound Ad-Br are dispersed in anhydrous acetonitrile at a feed molar ratio of 1:3, and then potassium carbonate is added, and the obtained mixed solution is reacted at 90°C for 48 to 64 hours to generate an insoluble solid, and the solvent after filtering the insoluble solid is rotary evaporated to dryness to obtain a solid mixture, which is then purified by silica gel column to obtain compound 3'; the above reaction is carried out under anhydrous conditions, and the column chromatography developing solvent used in the purification process is a combination solvent of chloroform and methanol in a ratio of 6:1.

[0147] 1.23 mmol of compound 3' was dissolved in 60 mL of formic acid, and the reaction was stirred at 60°C overnight. The reaction product formed an azeotrope with methanol and the solvent was removed. The remaining brown solid was dissolved in methanol and then precipitated in excess cold ether. The precipitation process was repeated twice to obtain compound 4'.

[0148] Finally, compound 4' was reacted with Gd 2 O 3 The product was dispersed in 20 mL of water and stirred at 100°C for more than 24 h to finally obtain Ad-DOTA(Gd).

[0149] 5 mg of paramagnetic quencher INOPS-Fc with a grafting rate of 5% of Fc was dispersed in 10 mL of deionized water, and 6 mg of paramagnetic T1 enhancer Ad-DOTA (Gd) was added. The reaction was stirred at room temperature for 24 hours, and then the excess Ad-DOTA (Gd) was removed by dialysis. The KD of the dialysis bag was 35000, and finally the contrast agent was obtained.

[0150] The contrast agent obtained in this comparative example was subjected to in vitro simulated ROS conditions (H 2 O 2 =0.1 mM), no specific response occurred, and no transition of the T1 contrast signal from OFF to ON occurred.

[0151] Comparative Example 4

[0152] This comparative example provides an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect, which is a mixture of 5% grafted paramagnetic quencher INOPS-Fc and paramagnetic T1 enhancer Ad-DOTA (Gd) with a molar ratio of Fc and AD groups in INOPS-Fc being 1:3.

[0153] 5 mg of paramagnetic quencher INOPS-Fc with a grafting rate of 5% of Fc was dispersed in 10 mL of deionized water, and then 12 mg of paramagnetic T1 enhancer Ad-DOTA (Gd) was added. The reaction was stirred at room temperature for 24 hours, and then the excess Ad-DOTA (Gd) was removed by dialysis. The KD of the dialysis bag was 35000, and finally the contrast agent was obtained.

[0154] The contrast agent obtained in this comparative example was subjected to in vitro simulated ROS conditions (H 2 O 2 =0.1 mM), no specific response occurred, and no transition of the T1 contrast signal from OFF to ON occurred.

[0155] Comparative Example 5

[0156] This comparative example provides an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect, which is a mixture of a 15% grafted paramagnetic quencher INOPS-Fc and a paramagnetic T1 enhancer Ad-DOTA (Gd) with a molar ratio of Fc to AD groups in INOPS-Fc being 1:3.

[0157] 5 mg of paramagnetic quencher INOPS-Fc with a grafting rate of 15% of Fc was dispersed in 10 mL of deionized water, and then 18 mg of paramagnetic T1 enhancer Ad-DOTA (Gd) was added. The reaction was stirred at room temperature for 24 hours, and then the excess Ad-DOTA (Gd) was removed by dialysis. The KD of the dialysis bag was 35000, and finally the contrast agent was obtained.

[0158] The contrast agent obtained in this comparative example was subjected to in vitro simulated ROS conditions (H 2 O 2 =0.1 mM), no specific response occurred, and no transition of the T1 contrast signal from OFF to ON occurred.

[0159] Performance Test:

[0160] 1. Response performance test

[0161] The longitudinal relaxation time and transverse relaxation time of the product of Example 1 of the present invention were tested on a 0.5T MRI tester, and the longitudinal relaxation time and transverse relaxation time of the product of Example 1 of the present invention were tested on a 0.5T MRI tester. 2 O 2The concentration of 0.1 mM was used for incubation simulation experiments in vitro, and then the longitudinal relaxation time and transverse relaxation time of the contrast agent obtained in Example 1 of the present invention under the condition of simulating ROS in vitro were tested on a 0.5T MRI tester.

[0162] The specific test method includes: preparing contrast agent aqueous solution samples with iron concentrations of 0.175mM, 0.35mM, 0.7mM, and 1.4mM, respectively, and testing them on a 0.5T MRI tester, using the sum of the concentrations of iron ions and gadolinium ions (mM) as the horizontal coordinate and the reciprocal of the longitudinal relaxation time and the transverse relaxation time as the vertical coordinate to perform linear fitting to obtain the longitudinal relaxation rate (r 1 ), where r 1 and r 2 The unit is mM -1 ·s -1 .

[0163] The fitting results are as follows Fig.10 As shown, the longitudinal relaxation rate (r 1 ) value comparison chart; It can be seen from the figure that after ROS response, the r of Example 1 1 The value of changed from the initial 2.5 to 8.77, which was nearly 4 times stronger. 1 The significant increase in the value of can significantly illustrate the change of the T1 signal from OFF to ON.

[0164] 2. Cytotoxicity performance test

[0165] The toxicity test of normal cells and cancer cells was performed on the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect obtained in Example 1 of the present invention.

[0166] The operation method includes: using CCK-8 to determine the cytotoxicity of the contrast agent in mouse breast cancer cells (4T1 cells) and human umbilical vein endothelial cells (HUVEC cells); specifically including:

[0167] Take 100 μL of HUVEC cells and seed them into a 96-well plate at a density of 5000 cells per well. Place the 96-well plate in a CO 2Incubator, culture at 37℃ for 24h, then dissolve the contrast agent in complete culture medium and dilute to multiple concentrations. This test case made 8 different concentrations from 0.016 to 2.00mM, which are 0.06mM, 0.09mM, 0.13mM, 0.2mM, 0.3mM, 0.44mM, 0.67mM, 1mM; the diluted contrast agent solution was added to the wells of 96-well plate and incubated with cells for 24h, then 100μL of 10% CCK-8 solution was added to each well, placed in a cell culture incubator for 3h, and then the absorbance value OD at 450nm was measured by microplate reader 450mm Four parallel samples were prepared for each contrast agent, and the relative survival rate of cells was calculated based on the absorbance value.

[0168] The cytotoxicity test procedure for 4T1 cells was the same as that for HUVEC cells.

[0169] The blank group consisted of complete culture medium without cells, and the control group consisted of cells cultured without materials.

[0170] The relative cell survival rate was calculated as follows:

[0171] Relative cell survival rate (%) = 100 × (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group)

[0172] like Fig.11 and Fig.12 As shown, both HUVEC and 4T1 cell viability showed a high level of activity, and even at an iron concentration of 2 mM, the viability of both cells was above 98%. This indicates that the cytotoxicity of the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect provided by the present invention is negligible.

[0173] 3. MRI imaging experimental test

[0174] The contrast agent obtained in Example 1 of the present invention is subjected to an in vivo MRI imaging experiment, and the operation method thereof comprises:

[0175] BALB / c mice (4-6 weeks old, 20 g) that met the SPF breeding standards were selected, and 4T1 cells were used to establish a subcutaneous tumor model.

[0176] First, 4T1 cells were cultured and evenly suspended in PBS solution at a cell density of 2 × 10 6 cells / ml; then, 100 μL of PBS solution was injected subcutaneously into the axilla of each BALB / c mouse, and a tumor model was established after 7-10 days of culture, and 4T1 tumor-bearing mice were constructed and used for subsequent in vivo experiments.

[0177] The successfully constructed 4T1 tumor-bearing mice were first intraperitoneally injected with 100 μL of 29% urethane solution. After the mice were deeply anesthetized, a blank scan was performed before the injection of contrast agent. Then, the contrast agents of Example 1 and Comparative Example 1 (150 μL, the iron ion dose was 0.1 mmol / kg) were respectively injected through the tail vein. After that, the mice were fixed and placed in a 1.5T micro-magnetic resonance imaging instrument. T1-weighted imaging was performed at 2h, 4h, 6h, 8h, and 10h after the injection; wherein, the T1-weighted mouse imaging parameters were set as TE=60.86ms, TR=100ms, matrix=512×256, and slice thickness=0.27mm.

[0178] The results are as follows Fig.13 As shown, after the contrast agent of Example 1 was injected into the tumor-bearing mice through the tail vein at an injection volume of 0.0.1 mmol / kg, a significant imaging enhancement effect was shown over time, wherein the brightness of the MRI image of Example 1 at the tumor site increased significantly over time, showing a significant T1 signal enhancement. This may be due to the fact that the contrast agent was delivered to the tumor through the EPR effect, and then due to the high content of ROS in the tumor, the connection between the contrast agent paramagnetic quencher INOPS-Fc and the paramagnetic T1 enhancer was broken, causing the paramagnetic T1 enhancer to be released, achieving the transition of the T1 signal from OFF to ON, and lighting up the tumor.

[0179] The above test results show that the contrast agent provided by the present invention has an extremely low detection limit for ROS concentration, with the lowest concentration of 0.1 mM. When the contrast agent is used for tumor diagnosis, the tumor area is a dark signal at the beginning. As the binding force between ferrocene and β-cyclodextrin of the contrast agent is destroyed by the excessive ROS in the tumor environment, DOTA (Gd) is released, activating the T1 signal of the contrast agent, thereby realizing efficient and sensitive diagnosis of the T1 signal in the tumor area from OFF to ON.

[0180] It can be further directly inferred that the contrast agent provided by the present invention is not limited to the contrast diagnosis of tumors. As long as it is in a ROS environment, it can achieve efficient and sensitive diagnosis of T1 signals from OFF to ON through the MERT effect, such as inflammation and other lesion environments.

[0181] In summary, the dual-responsive MRET effect-mediated ultrasensitive ROS-responsive contrast agent provided by the present invention can selectively activate T1 MRI signals at the tumor site, switching from dark signals to bright signals, improving the specificity and high selectivity of tumor detection, and achieving excellent imaging contrast performance. In addition, the ultrasensitive ROS-responsive contrast agent also has good biocompatibility and low toxicity.

[0182] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0183] The various aspects, embodiments, features and examples of the present invention should be considered to be illustrative in all aspects and not limiting the present invention, the scope of the present invention being defined solely by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0184] The use of headings in the present invention is not meant to limit the present invention; both the Summary and Detailed Description sections may apply to any aspect, embodiment, or feature of the present invention.

[0185] Throughout this disclosure, where a composition is described as having, containing, or comprising particular components, or where a process is described as having, containing, or comprising particular process steps, it is contemplated that the compositions taught by the present invention also consist essentially of, or consist of, the recited components, and that the processes taught by the present invention also consist essentially of, or consist of, the recited process steps.

[0186] Unless specifically stated otherwise, use of the terms "include," "includes," "including," "have," "has," or "having" should generally be construed as open ended and non-limiting.

[0187] It should be understood that the order of the various steps or the order in which a particular action is performed is not very important, as long as the teachings of the present invention remain operable. In addition, two or more steps or actions may be performed simultaneously.

[0188] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or capable of achieving the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. An ultrasensitive ROS-responsive contrast agent mediated by the MRET effect, consisting of a superparamagnetic quencher and a paramagnetic T1 enhancer; wherein: The superparamagnetic quencher is ferrocene-modified iron oxide nanoparticles INOPS-Fc; The paramagnetic T1 enhancer is β-CD-DOTA(Gd) modified with β-cyclodextrin; Under normal conditions, the superparamagnetic quencher and the paramagnetic T1 enhancer form a host-guest assembly based on the host-guest binding of ferrocene and β-cyclodextrin; while under the ROS environment, the host-guest assembly opens and releases DOTA (Gd), activating the T1 signal from OFF to ON.

2. The ultrasensitive ROS-responsive contrast agent mediated by the MRET effect according to claim 1, characterized in that: The superparamagnetic quencher is dispersed in the water phase, and the grafting rate of ferrocene is 3-7%; the particle size of the iron oxide nanoparticles is 10-15nm; The molar ratio of the iron in the superparamagnetic quencher to the Gd in the paramagnetic T1 enhancer is 15 to 30:

1.

3. The ultrasensitive ROS-responsive contrast agent mediated by the MRET effect as claimed in claim 1 or 2, characterized in that: The molar ratio of ferrocene in the superparamagnetic quencher to β-cyclodextrin in the paramagnetic T1 enhancer is 1:3 to 1:9; And / or, the concentration of ROS in the ROS environment is 0.1-1 mM.

4. A method for preparing an ultrasensitive ROS-responsive contrast agent mediated by the MRET effect as described in any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of paramagnetic quencher INOPS-Fc; INOPS-Fc was obtained by subjecting oleic acid-modified iron oxide nanoparticles INOPS-OA to a ligand exchange reaction; The first ligand exchange reaction yielded INOPS-BF4, which was then subjected to a second ligand exchange reaction to finally obtain; (2) Preparation of paramagnetic T1 enhancer β-CD-DOTA(Gd): alkynyl-DOTA(Gd) was subjected to a click reaction with 7-(6-azido-6-deoxy)-β-cyclodextrin to obtain β-CD-DOTA(Gd); (3) mixing the INOPS-Fc prepared in step (1) and the β-CD-DOTA(Gd) prepared in step (2) to obtain the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect; Step (1) and step (2) are performed in no particular order.

5. The preparation method according to claim 4, characterized in that: In step (1), the ligand exchange reaction includes a first ligand exchange reaction and a second ligand exchange reaction; The ligand of the first ligand exchange reaction is nitroso tetrafluoroborate; the process includes dispersing INOPS-OA particles in toluene, dispersing nitroso tetrafluoroborate in N,N-dimethylformamide (DMF), then adding the nitroso tetrafluoroborate solution to the nanoparticle solution, and mixing them evenly for 5 to 10 minutes to obtain INOPS-BF4; The ligand of the second ligand exchange reaction is polyacrylamine-ferrocene; the method comprises: dispersing INOPS-BF4 in water, adding polyacrylamine-ferrocene, stirring and reacting for 24 hours, and then dialyzing to remove excess PAA-Fc to obtain INOPS-Fc.

6. The preparation method according to claim 4, characterized in that: In step (2), β-CD-DOTA(Gd) is obtained by a click reaction between alkynyl-DOTA(Gd) and 7-(6-azido-6-deoxy)-β-cyclodextrin; wherein the ratio of the molar amount of the alkynyl group in β-CD-DOTA(Gd) to the molar amount of the azide group in 7-(6-azido-6-deoxy)-β-cyclodextrin is greater than 1:

2.

7. The preparation method according to claim 6, characterized in that: The synthesis method of alkynyl-DOTA(Gd) includes: In the first step, cyclopentane and sodium bicarbonate are dispersed in acetonitrile, and then tert-butyl bromoacetate is added, mixed, stirred evenly, reacted in an ice bath for 48 hours, and filtered and purified to obtain a white solid, which is compound 1; The second step is to dissolve propargylamine in dichloromethane, then place a sodium hydroxide solution on the dichloromethane layer, carefully add bromoacetyl bromide to the organic layer (dichloromethane) using a syringe, gently stir for 5 to 10 minutes, produce a large amount of white precipitate, continue to vigorously stir the reaction for 2 to 5 hours, then use dichloromethane and water for liquid separation extraction to obtain an organic phase, the aqueous phase is further extracted once with dichloromethane, the organic phases are combined and collected, and then washed with sodium carbonate water, and then dried over anhydrous magnesium sulfate, spin-dried, and vacuum dried to obtain compound 2; The third step is to disperse compound 1 and compound 2 in anhydrous acetonitrile, then add potassium carbonate, and react the obtained mixed solution at 90° C. for 48 to 64 hours. The filtered solvent is dried and purified to obtain compound 3. Step 4, dissolving compound 3 in formic acid, stirring and reacting at 60° C. overnight, and removing the solvent to obtain compound 4; The fifth step is to disperse compound 4 and Gd2O3 in 20 mL of water, and stir the mixture at 100°C for more than 24 h to finally obtain alkynyl-DOTA (Gd).

8. A contrast agent composition, comprising the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect as described in any one of claims 1 to 3 and a pharmaceutically acceptable adjuvant; the adjuvant is a diluent. 9 . Use of the ultrasensitive ROS-responsive contrast agent mediated by the MRET effect as described in any one of claims 1 to 3 in the imaging diagnosis of diseases with a ROS environment.

10. Use of the ultrasensitive ROS-responsive imaging mediated by the MRET effect as described in any one of claims 1 to 3 in tumor imaging diagnosis.

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