Nuclear magnetic fluorescence bimodal imaging probe as well as preparation method and application thereof

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a nuclear magnetic fluorescence bimodal imaging probe and a preparation method and application thereof. The invention provides a nuclear magnetic fluorescent bimodal imaging probe which comprises superparamagnetic iron oxide particles, near-infrared fluorescent dye, a targeting ligand and a biocompatible polymer, and the superparamagnetic iron oxide particles, the near-infrared fluorescent dye and the targeting ligand are covalently coupled through the biocompatible polymer. And a polyphenol compound is used as a link to form metal-ligand coordination. The probe endows the material with stronger mechanical properties and stability through a covalent cross-linking and coordination cross-linking dual-network structure, so that the probe is not easy to depolymerize after entering a human body, it is ensured that the superparamagnetic iron oxide particles and the near-infrared fluorescent dye can be co-localized at a tumor, and the imaging accuracy is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a nuclear magnetic fluorescence dual-mode imaging probe and a preparation method and application thereof. BACKGROUND

[0002] Indocyanine green (ICG) is a traditional contrast agent that has been approved for clinical application. It can be taken up by the liver and retained in the liver tumor area for more than 7 days, so it is suitable for tumor imaging. In addition, indocyanine green is also an excellent intraoperative navigation contrast agent. Its targeting retention mechanism in liver tumors mainly depends on organic anion transporting polypeptide 1B3 (OATP1B3) and sodium-taurocholate cotransporting polypeptide (NTCP) in hepatocytes. In normal liver tissue, indocyanine green can be rapidly taken up and emit fluorescence under excitation light irradiation, and then gradually weaken as it is excreted through the biliary system. However, in the presence of liver tumors or cirrhotic nodules, the biliary excretion function is impaired, resulting in prolonged retention of indocyanine green in the lesion tissue, and a delayed fading phenomenon occurs. Based on this characteristic, indocyanine green can be used for fluorescence imaging of hepatocellular carcinoma (HCC). In addition, since different differentiated hepatocellular carcinomas have different uptake abilities of indocyanine green, this imaging method can also be used to preliminarily evaluate the differentiation degree of the tumor. At the same time, necrotic tumor tissue cannot take up indocyanine green, so it can also be used to judge the survival status of the tumor.

[0003] However, indocyanine green still faces many challenges in its further application in cancer diagnosis and treatment. First, due to the limited tissue penetration ability of indocyanine green fluorescence imaging, its application is usually limited to intraoperative imaging, making it difficult to implement preoperative imaging. This limitation makes it difficult to evaluate the survival status and differentiation degree of the tumor before treatment, thereby affecting the judgment of treatment effect and the determination of the operation time. In addition, indocyanine green also has the problems of non-specific binding with proteins and off-target retention, which can easily lead to false positive results. Although the indocyanine green clearance test is the only real-time dynamic liver function quantitative detection method widely used in clinical practice, it still has difficulties in regional liver function evaluation. In addition, indocyanine green is only suitable for biological imaging scenarios with high signal-to-noise ratio, and effective fluorescence imaging of some tissues is difficult to achieve. These problems together restrict the preoperative precise planning of hepatocellular carcinoma treatment programs and increase the difficulty of clinical decision-making.

[0004] Magnetic resonance imaging (MRI) is an effective means for locating tumors, but its application also has shortcomings. The specificity of MRI is limited, the false positive rate is high, and traditional imaging methods are difficult to quantitatively evaluate regional liver function reserve. In addition, MRI probes still face challenges in biocompatibility and targeting: for example, magnetic nanoparticles easily interact with plasma proteins in the physiological environment, leading to an increase in particle size and severe aggregation; the innate immune system recognizes these particles as foreign bodies, which are quickly phagocytosed and cleared by macrophages, severely affecting the efficiency of diagnosis and treatment. Traditional ferroferric oxide nanoparticles lack tumor targeting as MRI contrast agents, making it difficult to achieve precise imaging of liver tumors. These defects limit the widespread application of MRI in precise diagnosis and treatment of liver cancer.

[0005] Therefore, how to effectively integrate the advantages of MRI and fluorescence imaging, and overcome the technical bottlenecks of each other, to achieve precise imaging of liver tumors and liver function evaluation, is a technical problem to be solved in the field of biomedical imaging at present. SUMMARY

[0006] Based on this, an embodiment of the present application provides a nuclear magnetic fluorescence dual-mode imaging probe and a preparation method and application thereof.

[0007] Another aspect of the present application provides a nuclear magnetic fluorescence dual-mode imaging probe, comprising: ultra-small superparamagnetic iron oxide, near-infrared fluorescent dye, biocompatible polymer, targeting ligand and polyphenolic compound.

[0008] Among them, the ultra-small superparamagnetic iron oxide, the near-infrared fluorescent dye and the targeting ligand are covalently coupled through the biocompatible polymer, and form a metal-ligand coordination bond through the polyphenolic compound.

[0009] In some embodiments, the near-infrared fluorescent dye includes indocyanine green.

[0010] In some embodiments, the targeting ligand includes folic acid.

[0011] In some embodiments, the biocompatible polymer includes polyethylene glycol; the weight average molecular weight of the polyethylene glycol is 2000.

[0012] In some embodiments, the polyphenolic compound includes dopamine or a derivative thereof.

[0013] Another aspect of the present application provides a preparation method of the nuclear magnetic fluorescence dual-mode imaging probe, comprising:

[0014] Mixing the near-infrared fluorescent dye, the biocompatible polymer and the targeting ligand to prepare a composite; and,

[0015] The polyphenolic compound, the complex and the ultra-small superparamagnetic iron oxide are mixed and reacted to prepare a nuclear magnetic fluorescence bimodal imaging probe.

[0016] In some embodiments, the ultra-small superparamagnetic iron oxide comprises oleic acid-modified magnetic iron oxide particles.

[0017] In some embodiments, the method for preparing the oleic acid-modified magnetic iron oxide particles comprises:

[0018] The iron source is dissolved in a mixture of dibenzyl ether and oleylamine, and heated to prepare the oleic acid-modified ultra-small superparamagnetic iron oxide. The heating includes a first heating at a temperature of 100-120°C for 0.5-1h, and a second heating at a temperature of 280-320°C for 2-2.5h.

[0019] In some embodiments, the step of preparing the complex comprises:

[0020] The targeting ligand, L-glutamic acid-1-methyl ester and dimethyl sulfoxide are mixed and catalytically reacted to prepare a methyl-esterified targeting ligand.

[0021] The methyl-esterified targeting ligand and the diamino biocompatible polymer are reacted in N,N-dimethylformamide under the condition of a condensing agent and an organic base to prepare a targeting ligand-biocompatible polymer; and,

[0022] The targeting ligand-biocompatible polymer and indocyanine green are reacted in dichloromethane under the condition of a condensing agent and an organic base to prepare a complex.

[0023] In some embodiments, the diamino biocompatible polymer comprises NH2-polyethylene glycol-NH2.

[0024] In some embodiments, the catalyst in the catalytic reaction comprises tetramethylguanidine.

[0025] In some embodiments, the condensing agent comprises benzotriazol-1-yl-oxytris-pyrrolidino-phosphonium hexafluorophosphate; and,

[0026] In some embodiments, the organic base comprises triethylamine.

[0027] In some embodiments, the reacting of the polyphenolic compound, the complex and the ultra-small superparamagnetic iron oxide comprises:

[0028] dissolving N-hydroxysuccinimide, N,N'-dicyclohexyl carbodiimide and dopamine hydrochloride in a mixed solvent containing chloroform, N,N-dimethylformamide and anhydrous sodium carbonate, stirring to prepare a mixed solution;

[0029] mixing the mixed solution with the complex to prepare a mixture;

[0030] adding ultra-small superparamagnetic iron oxide to the mixture, reacting under an inert gas; and collecting the nuclear magnetic fluorescence dual-modality imaging probe by precipitation and magnetic separation;

[0031] In some embodiments, the inert gas comprises nitrogen.

[0032] In some embodiments, the temperature of stirring is 24-26°C, and the time is 1.5-2.5h; and,

[0033] In some embodiments, the magnetic separation comprises using a permanent magnet.

[0034] Another aspect of the present application provides the use of the nuclear magnetic fluorescence dual-modality imaging probe in the preparation of a magnetic resonance-near infrared fluorescence dual-modality imaging reagent.

[0035] In some embodiments, the magnetic resonance-near infrared fluorescence dual-modality imaging reagent comprises a liver cancer magnetic resonance-near infrared fluorescence dual-modality imaging reagent.

[0036] Another aspect of the present application provides a magnetic resonance-near infrared fluorescence dual-modality imaging reagent comprising the nuclear magnetic fluorescence dual-modality imaging probe.

[0037] The present application provides a nuclear magnetic fluorescence dual-modality imaging probe, comprising ultra-small superparamagnetic iron oxide, near infrared fluorescent dye, targeting ligand and biocompatible polymer, wherein the ultra-small superparamagnetic iron oxide, near infrared fluorescent dye and targeting ligand are covalently coupled by the biocompatible polymer and form metal-ligand coordination through polyphenolic compounds as a link. The probe has a double network structure coexisting with covalent cross-linking and coordination cross-linking, which endows the material with stronger mechanical properties and stability, so that the probe is not easy to depolymerize after entering the body, ensuring that the superparamagnetic iron oxide particles and the near infrared fluorescent dye can be co-localized at the tumor, improving the imaging accuracy. At the same time, the introduction of the targeting ligand enhances the specific recognition ability of the probe to the tumor tissue, solving the problem of non-specific binding of indocyanine green, and the modification of the biocompatible polymer improves the biocompatibility and blood circulation time of the probe, reducing the recognition and removal of the immune system. This dual-modality imaging probe can not only realize the preoperative determination of tumor viability and tumor differentiation degree by nuclear magnetic resonance, but also can be used for precise navigation during surgery through fluorescence imaging, providing a new technical means for precise diagnosis and treatment of hepatocellular carcinoma. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 Preparation and characterization of a nanocomposite material provided in an embodiment of this application; Figure 1 A in the image is a TEM image of ultra-small superparamagnetic iron oxide; Figure 1 B in the image is a TEM image of an ultra-small superparamagnetic iron oxide-indocyanine green-polyethylene glycol-folic acid.

[0040] Figure 2 This application provides a synthetic route for indocyanine green-polyethylene glycol-folic acid according to one embodiment.

[0041] Figure 3 Characterization of the parameters of a dual-modal nuclear magnetic fluorescence imaging probe provided in an embodiment of this application; Figure 3 In this context, A represents the Zeta potential of the ultra-small superparamagnetic iron oxide-indocyanine green-polyethylene glycol-folic acid. Figure 3 In this context, B represents the hydrated particle size of ultra-small superparamagnetic iron oxide-indocyanine green-polyethylene glycol-folic acid.

[0042] Figure 4 A dual-modal imaging probe for nuclear magnetic resonance fluorescence (NMR) provided in one embodiment of this application is used for in vivo NMR imaging of hepatocellular carcinoma in Balb / c mice.

[0043] Figure 5 The nuclear magnetic resonance fluorescence dual-modal imaging probe provided in one embodiment of this application is used for fluorescence imaging in Balb / c mice with hepatocellular carcinoma in situ. Detailed Implementation

[0044] The application will be described in further detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are only used to explain the present application and not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the disclosure of the present application more thoroughly and comprehensively understood. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or changes without departing from the spirit and scope of the present application, and the equivalent forms obtained by the modifications or changes also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0046] The term

[0047] Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings:

[0048] The term "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, including any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0049] In the present application, "multiple", "various", "multiple times", "multiple" and the like are used without specific limitation, which means greater than or equal to two in number. For example, "one or more" means one or more than two.

[0050] As used herein, "combinations thereof", "any combination thereof", "any combination", and the like include all suitable combinations of any two or more of the recited items.

[0051] As used herein, "suitable", "suitable manner", "any suitable manner", and the like refer to the ability to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0052] In the present application, "further", "still further", "in particular", and the like are used for the purpose of description and represent differences in content, but should not be understood as limiting the scope of protection of the present application.

[0053] In the present application, "optionally", "optional", and "optional" mean that it can or can not be present, i.e., it is selected from either of the two parallel schemes "has" or "has not". If there are multiple "optional" in a technical solution, and there is no contradictory or mutually restrictive relationship, each "optional" is independent.

[0054] In the present application, the technical features described in an open manner include both closed technical solutions consisting of the listed features and open technical solutions including the listed features.

[0055] In the present application, with respect to a numerical interval (i.e., a numerical range), if not otherwise specified, the optional numerical distribution within the above numerical interval is considered to be continuous, and includes both numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range and every numerical value between the two numerical endpoints. If not otherwise specified, when a numerical interval refers only to integers within the numerical interval, including both endpoint integers of the numerical range and every integer between the two endpoints, in the present application, it is equivalent to directly listing each integer, such as t being an integer selected from 1 to 10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0056] In the present application, the temperature parameter, if not otherwise specified, allows for constant temperature treatment and also allows for fluctuations within a certain temperature interval. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0057] In the present application, both (w / w) and wt% represent weight percentage, (v / v) represents volume percentage, and (w / v) represents mass volume percentage.

[0058] All the documents mentioned in the present application are incorporated by reference in the present application as if each document is incorporated by reference individually. Unless and to the extent conflicting with the purpose and / or technical solution of the present application, the documents referred to in the present application are incorporated by reference in their entirety. When the present application refers to the documents, the definitions of the relevant technical features, terms, names, phrases, etc. in the documents are also incorporated by reference. When the present application refers to the documents, the examples, preferred modes of the relevant technical features are also incorporated by reference into the present application, but are limited to the implementation of the present application. It should be understood that when the content of the reference conflicts with the description in the present application, the present application is given priority or is modified adaptively according to the description in the present application.

[0059] In view of the shortcomings of indocyanine green, the present application modifies it into a nuclear magnetic visualization, proposes a fluorescence-nuclear magnetic-targeting three-in-one probe design, and solves the clinical treatment pain points by solving the shortcomings of indocyanine green.

[0060] The present application provides a nuclear magnetic fluorescence dual-mode imaging probe, comprising: superparamagnetic iron oxide particles, near-infrared fluorescent dye, biocompatible polymer, targeting ligand and polyphenol compound.

[0061] The superparamagnetic iron oxide particles, the near-infrared fluorescent dye and the targeting ligand are covalently coupled through the biocompatible polymer, and a metal-ligand coordination bond is formed through the polyphenol compound.

[0062] The present application covalently couples superparamagnetic iron oxide, indocyanine green and folic acid together through polyethylene glycol to prepare a nuclear magnetic fluorescence dual imaging probe with high biocompatibility and direct targeting of liver tumors. The covalent bond connects the superparamagnetic iron oxide, indocyanine green, polyethylene glycol and folic acid together, and forms a metal-ligand coordination through dopamine as a linker. This double network structure of covalent crosslinking and coordination crosslinking can endow the material with stronger mechanical properties and improve the stability of the material. Therefore, compared with ordinary packaging, the probe is not easy to depolymerize after entering the body, so that the superparamagnetic iron oxide and indocyanine green can be co-located at the tumor, improving the imaging accuracy.

[0063] Due to the co-localization characteristics of indocyanine green superparamagnetic iron oxide, the present application can observe the metabolism of the probe by nuclear magnetic resonance, and can realize the determination of the tumor viability and the differentiation degree of the tumor before operation, and also has the potential to realize regional liver function evaluation. Folic acid-polyethylene glycol modified nanoparticles are an effective platform for targeted drug delivery. The incorporation of folic acid enhances the specificity of drug delivery to folic acid receptor-expressing cells, thereby promoting the improvement of imaging accuracy and solving the problem of nonspecific binding of indocyanine green.

[0064] In some embodiments, the superparamagnetic iron oxide particles comprise superparamagnetic iron oxide nanoparticles. Superparamagnetic iron oxide nanoparticles have a longer blood circulation time, are safe to use even for patients with chronic kidney disease, and can be used to obtain high-quality images in high-field magnetic resonance systems, and tissues that are difficult to image using indocyanine green fluorescence imaging or that do not produce clear images can be imaged using magnetic resonance.

[0065] In some embodiments, the near-infrared fluorescent dye comprises indocyanine green. Indocyanine green is a clinically approved contrast agent with good fluorescence properties and biological safety, can be taken up by the liver and accumulated at liver tumors, and is suitable for tumor imaging.

[0066] In some embodiments, the targeting ligand comprises folate. Folate receptors are highly expressed on the surface of a variety of tumor cells, and the incorporation of folate enhances the specificity of drug delivery to cells expressing folate receptors, thereby improving the accuracy of imaging and addressing the problem of non-specific binding of indocyanine green.

[0067] In some embodiments, the biocompatible polymer comprises polyethylene glycol; polyethylene glycol modification can significantly improve the biocompatibility of the nanoparticles, reduce the adsorption of plasma proteins and the recognition and phagocytosis of macrophages, prolong the blood circulation time, and improve the enrichment of the probe at the tumor site.

[0068] In some embodiments, the weight average molecular weight of the polyethylene glycol is 2000-4000. For example, the weight average molecular weight of the polyethylene glycol is 2000, 3000, or 4000, and any value therebetween.

[0069] In some embodiments, the polyphenolic compound comprises dopamine or a derivative thereof.

[0070] The present application demonstrates the potential of the probe in precise visualization of tumor invasion boundaries, regional liver function reserve assessment, and preoperative tumor viability determination by characterizing the physical and chemical properties of the probe and detecting the imaging effect in vivo and in vitro. The probe is helpful for achieving precise preoperative planning and precise clinical decision-making.

[0071] The present application covalently couples ultra-small superparamagnetic iron oxide, indocyanine green and folic acid together by polyethylene glycol to prepare a nuclear magnetic fluorescence dual imaging probe with high biocompatibility and direct targeting liver tumor, the covalent bond links ultra-small superparamagnetic iron oxide, indocyanine green, polyethylene glycol and folic acid together, and forms metal-ligand coordination through dopamine as a linker, the double network structure of covalent crosslinking and coordination crosslinking can endow the material with stronger mechanical properties and improve the stability of the material. Therefore, the probe is less likely to depolymerize compared with ordinary packaging after entering the body, so that the ultra-small superparamagnetic iron oxide and indocyanine green can be co-located at the tumor, improving the imaging accuracy.

[0072] Another aspect of the present application provides a preparation method of the nuclear magnetic fluorescence dual modality imaging probe, comprising: mixing the near-infrared fluorescent dye, the biocompatible polymer and the targeting ligand to prepare a complex; and reacting the polyphenolic compound, the complex and the superparamagnetic iron oxide particles to prepare the nuclear magnetic fluorescence dual modality imaging probe. The method prepares a dual modality imaging probe with high stability and good targeting through the dual effects of covalent coupling and coordination crosslinking, and has simple process, good repeatability and suitability for large-scale production.

[0073] In some embodiments, the superparamagnetic iron oxide particles include oleic acid modified magnetic iron oxide particles.

[0074] In some embodiments, the preparation method of the oleic acid modified magnetic iron oxide particles comprises:

[0075] The iron source is dissolved in a mixture of dibenzyl ether and oleylamine, and a heating reaction is performed to prepare the oleic acid modified superparamagnetic iron oxide particles.

[0076] In some embodiments, the conditions of the heating reaction include: first heating and second heating; the temperature of the first heating is 100-120℃, and the time is 0.5-1.5h; for example, the temperature is 100℃, 101℃, 102℃, 103℃, 104℃, 105℃, 106℃, 107℃, 108℃, 109℃, 110℃, 111℃, 112℃, 113℃, 114℃, 115℃, 116℃, 117℃, 118℃, 119℃ or 120℃, and the reaction time is 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, or 1.5h, and any value between them.

[0077] The second heating temperature is 280°C to 320°C, and the time is 1.5 hours to 2.5 hours. For example, the temperature is 280°C, 285°C, 290°C, 295°C, 300°C, 305°C, 310°C, 315°C, or 320°C, and any value in between. The time is 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2.0 hours, 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, or 2.5 hours, and any value in between.

[0078] In some embodiments, the step of preparing the complex comprises: dissolving the targeting ligand and L-glutamic acid-1-methyl ester in dimethyl sulfoxide, performing a catalytic reaction, and preparing a methyl esterified targeting ligand;

[0079] reacting the methyl esterified targeting ligand with a bis-amino biocompatible polymer in N,N-dimethylformamide under a condition of a condensing agent and an organic base, and preparing a targeting ligand-biocompatible polymer; and,

[0080] reacting the targeting ligand-biocompatible polymer with indocyanine green in dichloromethane under a condition of a condensing agent and an organic base, and preparing the complex.

[0081] In some embodiments, the bis-amino biocompatible polymer comprises NH2-polyethylene glycol-NH2. For example, the bis-amino biocompatible polymer comprises NH2-PEG2000-NH2.

[0082] In some embodiments, the catalyst in the catalytic reaction comprises tetramethylguanidine.

[0083] In some embodiments, the condensing agent comprises benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate.

[0084] In some embodiments, the organic base comprises triethylamine.

[0085] In some embodiments, the reacting the polyphenolic compound, the complex, and the superparamagnetic iron oxide particles comprises:

[0086] dissolving N-hydroxysuccinimide, N,N'-dicyclohexyl carbodiimide, and dopamine hydrochloride in a mixed solvent consisting of chloroform, N,N-dimethylformamide, and anhydrous sodium carbonate, stirring, and preparing a mixed solution;

[0087] mixing the mixed solution with the complex, and preparing a mixture;

[0088] adding superparamagnetic iron oxide particles to the mixture, performing a reaction under an inert gas; and, collecting a nuclear magnetic fluorescence bimodal imaging probe by precipitation and magnetic separation.

[0089] The polyphenol compound is used as a link to form a metal-ligand coordination with the superparamagnetic iron oxide particles, and to form a covalent bond with the near-infrared fluorescent dye-biocompatible polymer-targeting ligand complex. The double network structure of the covalent cross-linking and the coordination cross-linking greatly improves the stability of the probe.

[0090] In some embodiments, the inert gas comprises nitrogen.

[0091] In some embodiments, the temperature of the stirring is 24-26°C, and the time is 1.5-2.5h. For example, the temperature is 24°C, 25°C or 26°C, and the time is 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2.0h, 2.1h, 2.2h, 2.3h, 2.4h or 2.5h, and any value in between.

[0092] In some embodiments, the magnetic separation comprises using a permanent magnet.

[0093] Another aspect of the present application provides use of the magnetic resonance and fluorescent dual-mode imaging probe in the preparation of a magnetic resonance-near infrared fluorescent dual-mode imaging reagent.

[0094] In some embodiments, the magnetic resonance-near infrared fluorescent dual-mode imaging reagent comprises a liver cancer magnetic resonance-near infrared fluorescent dual-mode imaging reagent.

[0095] Another aspect of the present application provides a magnetic resonance-near infrared fluorescent dual-mode imaging reagent comprising the magnetic resonance and fluorescent dual-mode imaging probe.

[0096] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the examples are only used to illustrate but not to limit the scope of the present application. The experimental methods in the following examples without specific conditions are preferred to refer to the instructions given in the present application, and can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0097] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not otherwise specified. The temperature and time parameters allow for acceptable deviations caused by the instrument testing accuracy or operation accuracy.

[0098] It should be understood that the order of the above-mentioned processes in various embodiments of the present application does not mean the order of execution, and the execution order of the processes should be determined according to their functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0099] Example 1

[0100] The embodiment provides a nuclear magnetic fluorescence dual-mode imaging probe and a preparation method thereof.

[0101] The preparation raw material of the nuclear magnetic fluorescence dual-mode imaging probe comprises: ultra-small superparamagnetic iron oxide, indocyanine green (ICG) (medchemexpress, HY-W088089), folic acid (FA (medchemexpress, HY-16637), and polyethylene glycol (PEG2000) (medchemexpress, HY-Y0873B).

[0102] The preparation method of the nuclear magnetic fluorescence dual-mode imaging probe comprises the following steps.

[0103] S1, preparing ultra-small superparamagnetic iron oxide.

[0104] 2 mmol Fe(acac)3 is dissolved in a mixture of 10 mL dibenzyl ether and 10 mL oleylamine. The mixture is placed in a 110°C oil bath for 1 h, quickly heated to 300°C for 2 h, and then 50 mL of ethanol is added after cooling to room temperature. Finally, the precipitate is collected by centrifugation at 8000 rpm, washed with ethanol for 3 times, and stored at 4°C for standby, Figure 1 A in the figure is a TEM image of the ultra-small superparamagnetic iron oxide.

[0105] S2, preparing indocyanine green-polyethylene glycol-folic acid.

[0106] Folic acid (10 mg) and L-glutamic acid-1-methyl ester (10 mg) are dissolved in 2 mL of DMSO, and an appropriate amount of tetramethyl guanidine is added as a catalyst, and the reaction is carried out at room temperature for 6 h to obtain methyl esterified folic acid. The methyl esterified folic acid and NH2-polyethylene glycol-NH2 (10 mg) are dissolved in 2 mL of DMF, and Pybop (1.5 eq) and TEA (3 eq) are added, and the reaction is carried out at room temperature for 30 min to obtain folic acid-polyethylene glycol. Folic acid-polyethylene glycol and indocyanine green (10 mg) are dissolved in 2 mL of DCM, and Pybop (1.5 eq) and TEA (3 eq) are added, and the reaction is carried out at room temperature for 30 min to obtain indocyanine green-PEG2000-folic acid. The synthesis path of indocyanine green-polyethylene glycol-folic acid is as shown in Figure 2 .

[0107] S3, preparing ultra-small superparamagnetic iron oxide-indocyanine green-PEG2000-folic acid.

[0108] NHS (2 mg), DCC (3 mg) and dopamine hydrochloride (1.27 mg) were dissolved in a mixed solvent containing CHCl3(2 mL), DMF (1 mL) and anhydrous Na2CO3(10 mg). 0.3 mL of the mixed solution was taken, and folic acid-polyethylene glycol-indocyanine green (10 mg) was added. After the mixture was stirred at room temperature for 2 h, ultra-small superparamagnetic iron oxide nanoparticles (3 mg) were added, and the mixture was stirred at room temperature under N2protection overnight. Dopamine forms a coordination bond with the ultra-small superparamagnetic iron oxide, and indocyanine green-PEG2000-folic acid replaces oleic acid oleylamine to covalently bind to the ultra-small superparamagnetic iron oxide. 5 ml of hexane was added to precipitate the ultra-small superparamagnetic iron oxide nanoparticles, which were collected by a permanent magnet and dried under N2. The particles were then dispersed in water or PBS. The product was dialyzed in 1-fold PBS or water for 24 h to remove excess surfactants and other salts, and any precipitates were removed by a 200 nm syringe filter, Figure 1 B in FIG. 2B is a TEM image of the ultra-small superparamagnetic iron oxide-indocyanine green-polyethylene glycol-folic acid.

[0109] Parameter characterization: Figure 3 A in FIG. 2A is the ultra-small superparamagnetic iron oxide - Zeta potential of indocyanine green-polyethylene glycol-folic acid; Figure 3 B in FIG. 2B is the hydrated particle size of the ultra-small superparamagnetic iron oxide-indocyanine green-polyethylene glycol-folic acid.

[0110] Example 2

[0111] This example provides the results of in vivo nuclear magnetic resonance imaging of a nuclear magnetic fluorescence dual-mode imaging probe in a hepatocarcinoma orthotopic tumor Balb / c mouse. The hepatocarcinoma orthotopic tumor Balb / c mouse was purchased from Vital River, and the mouse hepatocarcinoma cells were H22 cell lines. The hepatocarcinoma orthotopic model was established by tumor block transplantation technology.

[0112] In this example, 20-25 g of hepatocarcinoma orthotopic tumor Balb / c mice were injected with the nuclear magnetic fluorescence dual-mode imaging probe prepared in Example 1 through the tail vein, and the dosage was 1.5 mg / g of ultra-small superparamagnetic iron oxide. Nuclear magnetic resonance imaging was performed 24 h after administration. The MSME sequence of T2 was used for nuclear magnetic resonance imaging.

[0113] The results are shown in FIG. 2C, which shows that the probe is obviously enriched in the tumor site, and the nuclear magnetic resonance image clearly shows the tumor boundary, which can accurately determine the tumor location and size. Figure 4

[0114] Example 3

[0115] ​The present embodiment provides a fluorescence imaging result verification of a nuclear magnetic fluorescence bimodal imaging probe in a liver cancer orthotopic tumor Balb / c mouse in vivo.

[0116] In the experiment, 20g-25g liver cancer orthotopic tumor Balb / c mice were used, and the nuclear magnetic fluorescence bimodal imaging probe prepared in Example 1 was injected through the tail vein, the dosage was 1.5mg / g of ultra-small superparamagnetic iron oxide, and fluorescence imaging was performed 24h after administration. The excitation wavelength was 780nm, and the emission wavelength was 810nm.

[0117] The results show that, as shown in Figure 5 , the probe has obvious fluorescence signal at the tumor site, and can clearly show the tumor boundary, which is consistent with the nuclear magnetic resonance imaging result.

[0118] The above-described embodiments only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the scope of patent protection. It should be noted that, for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of protection of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various modifications or modifications to the present application, and the equivalent forms obtained are also within the scope of protection of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims of the present application. Therefore, the scope of protection of the patent of the present application should be based on the contents of the appended claims, and the description can be used to explain the contents of the claims.

Claims

1. A nuclear magnetic fluorescence bimodal imaging probe, characterized in that, include: Ultraminiature superparamagnetic iron oxide, near-infrared fluorescent dyes, biocompatible polymers, targeted ligands, and polyphenolic compounds; The ultra-miniature superparamagnetic iron oxide, the near-infrared fluorescent dye, and the targeting ligand are covalently coupled through the biocompatible polymer and form metal-ligand coordination bonds through the polyphenolic compound. 2.The nuclear magnetic fluorescence bimodal imaging probe according to claim 1, characterized in that, The ultra-small superparamagnetic iron oxide particles include ultra-small superparamagnetic iron oxide nanoparticles; and / or... The near-infrared fluorescent dye includes indocyanine green; and / or, The targeting ligand includes folic acid. 3.The nuclear magnetic fluorescence bimodal imaging probe according to claim 1 or 2, characterized in that, The biocompatible polymer includes polyethylene glycol; the weight-average molecular weight of the polyethylene glycol is 2000. 4.The nuclear magnetic fluorescence bimodal imaging probe according to claim 1, characterized in that, The polyphenolic compounds include dopamine or its derivatives.

5. The method for preparing the nuclear magnetic fluorescence bimodal imaging probe according to any one of claims 1 to 4, characterized in that, include: A complex is prepared by mixing the near-infrared fluorescent dye, the biocompatible polymer, and the targeting ligand; as well as, The polyphenolic compound, the complex, and the ultra-miniature superparamagnetic iron oxide were mixed and reacted to prepare a nuclear magnetic fluorescence dual-modal imaging probe. 6.The method of claim 5, wherein the method further comprises the step of: adding a contrast agent to the solution of step (a). The ultra-small superparamagnetic iron oxide includes oleic acid-modified ultra-small superparamagnetic iron oxide; Optionally, the preparation method of the ultra-miniature superparamagnetic iron oxide includes: The iron source was dissolved in a mixture of dibenzyl ether and oleylamine and heated to prepare the oleic acid-modified superparamagnetic iron oxide particles. The heating reaction included a first heating and a second heating. The temperature of the first heating was 100℃-120℃ and the time was 0.5h-1h. The temperature of the second heating was 280℃-320℃ and the time was 2h-2.5h.

7. The method of claim 5 or 6, wherein the method further comprises the step of: (d) adding the compound of formula (I) to the solution of step (c) to form the probe. The steps for preparing the complex include: The targeted ligand, L-glutamic acid-1-methyl ester, and dimethyl sulfoxide were mixed and subjected to a catalytic reaction to prepare the methylated targeted ligand; The methylated targeting ligand was reacted with a diamino biocompatible polymer in N,N-dimethylformamide under condensing agent and organic base conditions to prepare a targeting ligand-biocompatible polymer; and, The targeted ligand-biocompatible polymer was reacted with indocyanine green in dichloromethane under the conditions of a condensing agent and an organic base to prepare a complex. Optionally, the method satisfies one or more of the following conditions: (1) The diamino biocompatible polymer includes NH2-polyethylene glycol-NH2; (2) The catalyst in the catalytic reaction includes tetramethylguanidine; (3) The condensing agent comprises benzotriazol-1-yl-oxytripyrrolidinium hexafluorophosphate; and, (4) The organic base includes triethylamine. 8.The method of claim 7, wherein the method further comprises the step of: reacting the compound of formula (I) with the compound of formula (II) in the presence of a base to obtain the compound of formula (III). The reaction of the polyphenolic compound, the complex, and ultra-miniature superparamagnetic iron oxide includes: N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide and dopamine hydrochloride were dissolved in a mixed solvent containing chloroform, N,N-dimethylformamide and anhydrous sodium carbonate, and stirred to prepare a mixed solution. The mixed solution is mixed with the complex to prepare a mixture; Ultra-miniature superparamagnetic iron oxide was added to the mixture, and the reaction was carried out under an inert gas atmosphere; and the nuclear magnetic fluorescence dual-modal imaging probe was collected by precipitation and magnetic separation. Optionally, the method satisfies one or more of the following conditions: (1) the inert gas comprises nitrogen; (2) the temperature of the stirring is 24-26℃, and the time is 1.5-2.5h; and (3) the magnetic separation comprises using a permanent magnet.

9. Use of the nuclear magnetic fluorescence dual-mode imaging probe according to any one of claims 1-4 in the preparation of a magnetic resonance-near infrared fluorescence dual-mode imaging reagent. Optionally, the magnetic resonance-near infrared fluorescence dual-mode imaging reagent comprises a liver cancer magnetic resonance-near infrared fluorescence dual-mode imaging reagent.

10. A magnetic resonance-near infrared fluorescence dual-mode imaging reagent comprising the nuclear magnetic fluorescence dual-mode imaging probe according to any one of claims 1-4.

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