A tumor-targeted imaging nanoassembly and its preparation method and application

Abstract

This application discloses a tumor-targeted imaging nanoassembly, its preparation method, and application, relating to the field of medical technology. The nanoassembly includes an active targeting unit, an imaging signal unit, and a metal ion. The active targeting unit coordinates with the metal ion, and the nanoassembly is formed by coordination self-assembly of a mixture containing the active targeting unit, the imaging signal unit, and the metal ion. This application solves the problems of low signal intensity and lack of responsiveness of existing tumor imaging probes. The tumor-targeted imaging nanoassembly provided by this application has a simple preparation process, high imaging sensitivity, and tumor microenvironment responsiveness, enabling deep tissue tumor imaging and diagnosis and treatment.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a tumor-targeted imaging nanoassembly and a preparation method and application thereof. Background Art

[0002] Malignant tumors in deep tissues lack obvious symptoms in the early stages, and the survival rate of patients is generally low, posing a serious threat to human health. 1 H magnetic resonance imaging is a widely used method for tumor screening and detection in clinical practice. It has excellent imaging depth and non-radiation characteristics. However, due to the presence of a large number of hydrogen protons in the human body, 1 H MRI has high background noise, and there is no obvious difference between lesion tissue and normal tissue. 19 F MRI is developed without the interference of endogenous fluoride. 19 F-fluorine magnetic resonance imaging can effectively reduce background interference and achieve high signal-to-noise ratio fluorine magnetic resonance imaging.

[0003] In recent years, various 19 Nanoprobes for F MRI have been reported one after another, including various nanoemulsions based on perfluorinated compounds, fluorinated polymers, etc. However, these 19 F MRI materials are either expensive, require lengthy and complex synthesis processes, and are difficult to manufacture industrially. Alternatively, they have a low fluorine atom ratio, resulting in low contrast agent signal intensity. Alternatively, the prepared contrast agent's signal remains permanently on and unresponsive, making it impossible to achieve selective fluorine MRI of lesions. Therefore, developing a tumor-targeted imaging material with a simple preparation process, high imaging sensitivity, and tumor microenvironment-responsive properties is crucial for deep-tissue tumor imaging and diagnosis and treatment. Summary of the Invention

[0004] In view of the shortcomings of the above-mentioned technologies, the present application solves the problems of low signal intensity and non-responsiveness of existing tumor imaging probes by providing a tumor-targeted imaging nanoassembly and its preparation method and application. The tumor-targeted imaging nanoassembly provided in the present application has a simple preparation process, high imaging sensitivity and tumor microenvironment responsiveness, and can realize deep tissue tumor imaging and diagnosis and treatment.

[0005] To achieve the above objectives, this application mainly provides the following technical solutions:

[0006] The present application provides a tumor-targeting imaging nanoassembly, which includes an active targeting unit, an imaging signal unit and a metal ion. The active targeting unit is coordinated with the metal ion, and the nanoassembly is formed by coordination self-assembly of a mixture containing the active targeting unit, the imaging signal unit and the metal ion.

[0007] Preferably, the nanoassembly has at least one of the following structures:

[0008] (1) The imaging signal unit is chemically coupled to the active targeting unit;

[0009] (2) The imaging signal unit is loaded in the nanoassembly.

[0010] Preferably, the active targeting unit is selected from at least one of amino acids or their derivatives and polypeptides.

[0011] Preferably, the amino acid or its derivative is selected from cysteine, leucine, isoleucine, histidine, arginine, glycine, alanine, valine, phenylalanine, aspartic acid or derivatives of the above amino acids.

[0012] Preferably, the amino acid derivative is a derivative formed by replacing the hydrogen on the amino group in the amino acid with a fluorenylmethyloxycarbonyl group, a tert-butyloxycarbonyl group or a trityl group.

[0013] Preferably, the polypeptide is selected from at least one of glutathione, tripeptide, phenylalanine dipeptide, and arginine-glycine-aspartic acid tripeptide.

[0014] Preferably, the active targeting unit is selected from at least one of cysteine, histidine, arginine, phenylalanine, derivatives formed by replacing the hydrogen on the amino group of the above amino acids with fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl, glutathione, and tripeptide.

[0015] Preferably, the imaging signal unit is selected from at least one of a magnetic resonance imaging signal molecule, a computerized tomography imaging signal molecule, and a fluorescence imaging signal molecule.

[0016] Preferably, the magnetic resonance imaging signal molecule is selected from 19 F group or compound; the electronic computer tomography imaging signal molecule is selected from 18 The fluorescent imaging molecule is selected from at least one of indocyanine green, rhodamine-6G, Ce6, and IR780.

[0017] Preferably, the magnetic resonance imaging signal molecule is selected from Groups or compounds containing the above groups;

[0018] The electronic computed tomography imaging signal molecule is selected from Group or a compound containing the above group.

[0019] Preferably, the metal ions are transition metal ions or rare earth metal ions.

[0020] Preferably, the transition metal ions are selected from copper ions, zinc ions, and iron ions, and the rare earth metal ions are selected from europium ions.

[0021] Preferably, the nanoassembly further comprises an anti-tumor drug, and the anti-tumor drug is loaded in the nanoassembly.

[0022] Preferably, the nanoassembly is formed by coordination self-assembly of a mixture comprising an active targeting unit, an imaging signal unit, an anti-tumor drug and metal ions; the anti-tumor drug is selected from at least one of doxorubicin hydrochloride, paclitaxel, docetaxel, gemcitabine, camptothecin, cisplatin, carboplatin, oxaliplatin, etoposide and etoposide.

[0023] Preferably, the nanoassembly is formed by coordination self-assembly of a mixture comprising the following active targeting units, imaging signal units and metal ions:

[0024] The active targeting unit is selected from at least one of cysteine, histidine, arginine, phenylalanine, derivatives formed by replacing the hydrogen on the amino group of the above amino acids with fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl, glutathione, and tripeptide;

[0025] The imaging signal unit is selected from At least one of indocyanine green, rhodamine-6G, Ce6, and IR780;

[0026] The metal ions are selected from copper ions, zinc ions or iron ions.

[0027] Preferably, the nanoassembly further comprises an anti-tumor drug; the nanoassembly is formed by coordination self-assembly of a mixture comprising the active targeting unit, the imaging signal unit, the anti-tumor drug and the metal ion; the anti-tumor drug is loaded in the nanoassembly;

[0028] The anti-tumor drug is selected from at least one of doxorubicin hydrochloride, paclitaxel, docetaxel, gemcitabine, camptothecin and cisplatin.

[0029] Preferably, the nanoassembly is formed by coordination self-assembly of a mixture comprising the following active targeting units and metal ions, or is formed by coordination self-assembly of a mixture comprising the following active targeting units, imaging signal units and metal ions:

[0030] The active targeting unit is selected from at least one of cysteine, histidine, arginine, phenylalanine, derivatives formed by replacing the hydrogen on the amino group of the above amino acids with fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl, glutathione, and tripeptides, and at least one hydrogen on the amino group of the above amino acids or peptides is replaced by a fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl group. 19 F or 18F is substituted by a group;

[0031] The said 19 The group of F is:

[0032] The said 18 The group of F is:

[0033] The imaging signal unit is selected from At least one of indocyanine green, rhodamine-6G, Ce6, and IR780;

[0034] The metal ions are selected from copper ions, zinc ions or iron ions.

[0035] Preferably, the nanoassembly further comprises an anti-tumor drug; the nanoassembly is formed by coordination self-assembly of a mixture comprising the active targeting unit, the anti-tumor drug and metal ions, or is formed by coordination self-assembly of a mixture comprising the active targeting unit, the imaging signal unit, the anti-tumor drug and metal ions; the anti-tumor drug is loaded in the nanoassembly;

[0036] The anti-tumor drug is selected from at least one of doxorubicin hydrochloride, paclitaxel, docetaxel, gemcitabine, camptothecin and cisplatin.

[0037] The present application also provides a method for preparing the above-mentioned tumor-targeting imaging nanoassembly, which at least comprises: reacting a mixture containing an active targeting unit, an imaging signal unit and metal ions to obtain the nanoassembly.

[0038] Preferably, in the mixture, the molar ratio of the active targeting unit to the metal ion is 1:1-2:1.

[0039] Preferably, the mixture further comprises a stabilizer SH-PEG.

[0040] The present application also provides the use of the above-mentioned tumor-targeting imaging nanoassembly in tumor imaging, tumor diagnosis or tumor treatment.

[0041] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0042] The nanoassembly provided in the present application uses amino acids or their derivatives or polypeptides as active targeting units, which can penetrate deeply into the tumor tissue microenvironment. The nanoassembly formed by coordination self-assembly of the active targeting unit, the imaging signal unit and the metal ion has pH response characteristics and can be disassembled in the specific acidic environment of the tumor tissue, thereby releasing the imaging signal in the tumor tissue and realizing the specific response of tumor tissue targeted imaging.

[0043] The nanoassembly provided in the present application contains transition metal ions or rare earth metal ions. After coordination, these metal ions have a stronger ability to catalyze hydrogen peroxide to produce hydroxyl radicals than free metal ions. Tumor sites generally have a higher content of hydrogen peroxide. After the nanoassembly enters the tumor tissue, it can catalyze these hydrogen peroxide to produce hydroxyl radicals with stronger oxidizing ability and can kill tumor cells, thus having a good chemokinetic therapeutic effect.

[0044] The nanoassembly provided in the present application can release imaging signals and anti-tumor drugs simultaneously in the tumor tissue microenvironment through self-assembly of imaging signal units and anti-tumor drugs, thereby realizing the integration of tumor tissue imaging, chemodynamic therapy and chemotherapy, and has excellent imaging-guided chemodynamic / chemotherapy combined treatment effects.

[0045] The nanoassembly provided in the present application uses amino acids or their derivatives or polypeptides as ligands, which coordinate with metal ions to form the skeleton structure of the nanoassembly, and fluorine-containing signal molecules are assembled therein, so that the nanoassembly has good biocompatibility and no obvious toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a diagram showing the penetration effect of the tumor-targeting imaging nanoassembly prepared in Examples 1 and 2 of the present application on three-dimensional tumor spheres;

[0047] Figure 2 This is a physical picture of the tumor-targeting imaging nanoassembly prepared in Example 2 of the present application in buffer solutions of different pH values;

[0048] Figure 3 The tumor targeting imaging nanoassembly prepared in Example 2 of the present application is in buffer solutions of different pH values. 19 F MRI signal data graph;

[0049] Figure 4 This is a graph showing changes in particle size and surface potential of the tumor-targeted imaging nanoassembly solution prepared in Example 2 of the present application after storage for 28 days;

[0050] Figure 5 This is a physical picture of the tumor-targeting imaging nanoassembly prepared in Example 3 of the present application in buffer solutions of different pH values;

[0051] Figure 6 The tumor targeting imaging nanoassembly prepared in Example 3 of the present application is in buffer solutions of different pH values. 19 F MRI signal data graph;

[0052] Figure 7This is an electron microscope photograph of the tumor-targeting imaging nanoassembly prepared in Example 3 of the present application;

[0053] Figure 8 The electron microscope photograph and particle size distribution diagram of the tumor-targeting imaging nanoassembly prepared in Example 4 of the present application;

[0054] Figure 9 The tumor-targeted imaging nanoassembly prepared in Example 4 of this application is used in the in situ tumor of mouse liver 19 F MRI;

[0055] Figure 10 The electron microscope photograph and particle size distribution diagram of the tumor-targeting imaging nanoassembly prepared in Example 5 of the present application;

[0056] Figure 11 This is a near-infrared fluorescence imaging image of the tumor-targeting imaging nanoassembly prepared in Example 5 of the present application in a subcutaneous tumor in a mouse;

[0057] Figure 12 The tumor targeting imaging nanoassembly prepared in Example 5 of this application is 19 F MRI contrast images;

[0058] Figure 13 This is a diagram showing the growth inhibition effect of the tumor-targeting imaging nanoassembly prepared in Examples 4, 5 and 6 of the present application on subcutaneous tumors in mice. DETAILED DESCRIPTION

[0059] In order to further illustrate the technical means and effects adopted by this application to achieve the intended application purpose, the following is a detailed description of the specific implementation methods, structures, features and effects of this application in combination with the accompanying drawings and preferred embodiments.

[0060] It should be noted that different "one embodiment" or "embodiment" in this application do not necessarily refer to the same embodiment. In addition, specific features, structures, or characteristics in one or more embodiments may be combined in any suitable form. It should be understood that the embodiments of this application are intended to explain the solutions of this application and are not intended to limit the scope of protection of this application.

[0061] In this application, ranges may be expressed as from "about" one particular value and / or to "about" another particular value. When expressing such ranges, examples include from a particular value and / or to another particular value. Similarly, when the antecedent "about" is used to indicate that a value is approximate, it should be understood that the specific value constitutes another aspect. It should also be understood that the endpoints of each range are meaningful both in relation to the other endpoint and independently of the other endpoint.

[0062] Unless otherwise expressly stated, it is not intended that any method described herein be construed as requiring that its steps be performed in a specific order. Therefore, when a method claim does not actually recite that its steps follow a certain order or when it does not specifically indicate in any other way in the claims or description that the steps are limited to a specific order, it is not intended to imply any particular order.

[0063] Although the transitional term "comprising" may be used to disclose various features, elements, or steps of a particular embodiment, it should be understood that this implicitly includes alternative embodiments that may be described using the transitional term "consisting of" or "consisting essentially of." Thus, for example, implicit alternative embodiments for a method comprising A+B+C include an embodiment in which the method consists of A+B+C and an embodiment in which the method consists essentially of A+B+C.

[0064] This application solves the problems of low signal intensity and non-responsiveness of existing tumor imaging probes by providing a tumor-targeted imaging nanoassembly and its preparation method and application. The tumor-targeted imaging nanoassembly provided by this application has a simple preparation process, high imaging sensitivity and tumor microenvironment responsiveness, and can achieve deep tissue tumor imaging and diagnosis and treatment.

[0065] The technical solution in the embodiments of the present application is to solve the above problems, and the overall idea is as follows:

[0066] An embodiment of the present application provides a tumor-targeting imaging nanoassembly, which includes an active targeting unit, an imaging signal unit and a metal ion. The active targeting unit is coordinated with the metal ion, and the nanoassembly is formed by coordination self-assembly of a mixture containing the active targeting unit, the imaging signal unit and the metal ion.

[0067] The active targeting unit contained in the nanoassembly provided by the present application can penetrate deep into the tumor tissue microenvironment, and the nanoassembly formed by the active targeting unit, the imaging signal unit and the metal ion through coordination self-assembly has a pH response characteristic and can be disassembled in the specific acidic environment of the tumor tissue, thereby releasing the imaging signal in the tumor tissue and achieving a specific response of tumor tissue targeted imaging. In a non-acidic environment, the nanoassembly formed by coordination self-assembly has a high stability, and its imaging signal cannot be released, that is, the imaging signal cannot be detected by the imaging instrument. When in an environment with a specific pH of the tumor tissue, the nanoassembly can change its coordination stability and disassemble, thereby releasing the imaging signal.

[0068] The nanoassembly provided in this application has tumor targeting properties for superficial tumors and deep tissue tumors such as in situ liver tumors, in situ bladder tumors, lung metastatic tumors, etc., and is suitable for use as a target for these tumor tissues. 19Imaging materials for F magnetic resonance imaging, fluorescence imaging, and computed tomography imaging, etc.

[0069] Specifically, the nanoassembly has at least one of the following structures:

[0070] (1) The imaging signal unit is chemically coupled to the active targeting unit;

[0071] (2) The imaging signal unit is loaded in the nanoassembly.

[0072] The nanoassembly provided in the present application uses the active targeting unit as a ligand to coordinate with the metal ion. The imaging signal unit can be chemically coupled to the active targeting unit or loaded into the nanoassembly, which can realize the assembly of imaging signal units of various imaging forms in the nanoassembly, such as 19 F magnetic resonance imaging signal molecules, fluorescence imaging functional imaging signal molecules, and electronic computed tomography imaging signal molecules.

[0073] Specifically, the active targeting unit is selected from at least one of an amino acid or its derivatives, or a polypeptide. The present application uses amino acids or their derivatives, or polypeptides, as active targeting units, enabling the nanoassembly to effectively penetrate tumor tissue, thereby delivering imaging signals to the tumor tissue, improving the targeting and imaging intensity of tumor imaging.

[0074] In the present application, amino acids or their derivatives can be selected from cysteine, leucine, isoleucine, histidine, arginine, glycine, alanine, valine, phenylalanine, aspartic acid, or derivatives of the above amino acids, wherein the amino acid derivatives are preferably derivatives formed by replacing the hydrogen on the amino group of these amino acids with fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), or trityl (Trt). The above amino acids or their derivatives have a sulfhydryl group, two amine groups, an imidazole group, or a hydrophobic benzene ring. Amino acids containing these groups are easily self-assembled to form nanoparticles through coordination with metal ions, hydrophobic-hydrophobic interactions, and π-π interactions between amino acids.

[0075] Specifically, amino acid derivatives substituted with fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), or trityl (Trt) groups refer to derivatives formed by replacing the hydrogen on the amino group in the amino acid with fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), or trityl (Trt), respectively. For example, the structural formula of histidine substituted with fluorenylmethyloxycarbonyl is The structural formula of tert-butyloxycarbonyl-substituted histidine is The structural formula of trityl-substituted histidine is

[0076] In the present application, the polypeptide can be selected from at least one of glutathione, tripeptide, phenylalanine dipeptide (FF), and arginine-glycine-aspartic acid tripeptide. The above polypeptides contain sulfhydryl groups, imidazole groups, benzene rings, or multiple amine groups. Polypeptides containing these groups are easily self-assembled to form nanoparticles through coordination with metal ions, hydrophobic-hydrophobic interactions between amino acids, and π-π interactions.

[0077] In the present application, preferably, the active targeting unit is selected from at least one of cysteine, histidine, arginine, phenylalanine, derivatives of the above amino acids substituted with fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc), glutathione, and tripeptides. The above amino acids or polypeptides have stronger coordination with metal ions and more suitable hydrophilicity and hydrophobicity. Nanoparticles formed by self-assembly of these amino acids or polypeptides have better stability and stronger pH responsiveness.

[0078] In the present application, the above-mentioned imaging signal unit can be selected from one or more of magnetic resonance imaging signal molecules, electronic computed tomography imaging signal molecules, and fluorescence imaging signal molecules.

[0079] Specifically, the magnetic resonance imaging molecule is a 19 F group or compound; the electron computed tomography imaging molecule is a 18 The group or compound of F; the fluorescent imaging molecule is selected from at least one of indocyanine green, rhodamine-6G, Ce6, and IR780.

[0080] In the present application, preferably, the magnetic resonance imaging signal molecule is selected from etc. 19 F small molecule, or etc. 19 F group or containing the above 19 Compounds containing the above 19 Compounds with F groups such as:

[0081] In the present application, preferably, the above-mentioned electronic computed tomography imaging signal molecule is selected from etc. 18 F group or containing the above 18 Compounds containing the above 18 The compound of group F may be the following:

[0082] In the above imaging signal unit, the above 19 F or 18The compounds containing F can be loaded into the nanoassembly, that is, when the amino acid or its derivative or polypeptide reacts with the metal ion, the above-mentioned 19 F or 18 The compound F is supported in the formed coordination assembly.

[0083] In the above imaging signal unit, the above 19 F or 18 The group of F is It can replace the hydrogen of the amino group in amino acid or its derivatives or polypeptide to form 19 F or 18 The amino acid or its derivative or polypeptide labeled with F forms an active targeting unit chemically coupled with an imaging signal unit, and the active targeting unit chemically coupled with an imaging signal unit can coordinate with metal ions to self-assemble to form the nanoassembly provided in the present application.

[0084] Specifically, the above 19 F or 18 F-labeled amino acids or their derivatives or polypeptides can be prepared by the following methods: 19 F or 18 The compound F undergoes amide condensation, aldehyde amine condensation or sulfhydryl-amino condensation reaction with the amino acid or its derivative or the amino group in the polypeptide to obtain:

[0085]

[0086] For example, 3,5-bis(trifluoromethyl) 19 F) Toluene formaldehyde and amino acid are reacted to prepare 19 F-labeled histidine is:

[0087] Amino acids, 3,5-bis(trifluoromethyl) 19 F) dissolving methylbenzaldehyde in an alcohol solvent and adding a water removing agent; stirring the reaction at 30-60°C for 6-12 hours and then cooling to room temperature; adding sodium borohydride in an ice bath in several portions; slowly adding acid to adjust the pH to neutral; centrifuging to remove the precipitate; recrystallizing and purifying to obtain 19 F-labeled amino acids.

[0088] For example, 3,5-bis(trifluoromethyl) 19 F) Preparation of peptides with methylbenzaldehyde by aldehyde-amine reaction 19 The F-labeled peptides are:

[0089] Peptide, 3,5-bis(trifluoromethyl) 19 F) dissolving methylbenzaldehyde in an alcohol solvent and adding a water removing agent; stirring the reaction at 30-60°C for 6-12 hours and then cooling to room temperature, and purifying to obtain a product with 19 F-labeled polypeptide.

[0090] In the above reaction, the alcohol solvent may be methanol or ethanol, the dehydrating agent may be anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous calcium chloride or molecular sieves; and the acid used to adjust the pH may be hydrochloric acid, sulfuric acid or acetic acid.

[0091] The above 19 F or 18 F-labeled amino acids or their derivatives or polypeptides can coordinate with metal ions to form nanoassemblies, or can form nanoassemblies with metal ions together with other imaging signal molecules. 19 F or 18 F-labeled amino acids or their derivatives or polypeptides, containing 19 F or 18 F compounds or fluorescent imaging molecules, and metal ions form nanoassemblies through coordination self-assembly, which contains 19 F or 18 The F compound or fluorescent imaging molecule is loaded into the nanoassembly.

[0092] In the above-mentioned imaging signal unit, the magnetic resonance imaging molecules and the electronic computed tomography imaging molecules can be loaded into the nanoassembly by chemically coupling to amino acids or their derivatives or polypeptides, or can be loaded into the nanoassembly formed by the coordination of amino acids or their derivatives or polypeptides with metal ions.

[0093] In the above imaging signal unit, the fluorescent imaging molecule is loaded in a nanoassembly formed by coordination of amino acids or their derivatives or polypeptides with metal ions.

[0094] It should be noted that, in addition to magnetic resonance imaging molecules, electronic computed tomography imaging molecules and fluorescent imaging molecules, other imaging signal units can also be loaded into the nanoassembly provided in this application.

[0095] In the present application, the metal ion can be selected from transition metal ions or rare earth metal ions. Preferred transition metal ions are copper, zinc, and iron, and preferred rare earth metal ions are europium. These transition metal and rare earth metal ions, when coordinated with amino acids, derivatives, or imaging signal molecules, have the advantages of high coordination stability and the ability to induce specific responses in the tumor microenvironment.

[0096] The nanoassembly provided in the present application contains transition metal ions or rare earth metal ions. After coordination, these metal ions have a stronger ability to catalyze hydrogen peroxide to produce hydroxyl radicals than free metal ions. Tumor sites generally have a higher content of hydrogen peroxide. After the nanoassembly enters the tumor tissue, it can catalyze these hydrogen peroxide to produce hydroxyl radicals with stronger oxidizing ability and can kill tumor cells, thus having a good chemokinetic therapeutic effect.

[0097] The nanoassembly provided in this application can realize tumor targeting imaging in various imaging forms, especially 19 F magnetic resonance imaging, fluorescence imaging, and electronic computed tomography imaging. These imaging signal molecules can be assembled into nanoassemblies through simple chemical synthesis and then released responsively in the tumor microenvironment.

[0098] The nanoassembly provided in the present application uses amino acids or their derivatives or polypeptides as ligands, which coordinate with metal ions to form the skeleton structure of the nanoassembly, and fluorine-containing signal molecules are assembled therein, so that the nanoassembly has good biocompatibility and no obvious toxic side effects.

[0099] The present application also provides another nanoassembly, which further comprises an anti-tumor drug on the basis of the above nanoassembly, wherein the anti-tumor drug is loaded in the nanoassembly.

[0100] Specifically, the nanoassembly is formed by a mixture comprising an active targeting unit, an imaging signal unit, an anti-tumor drug, and metal ions through coordination self-assembly. When the mixture undergoes coordination self-assembly, the anti-tumor drug can be loaded into the nanoassembly.

[0101] Specifically, the anti-tumor drug is selected from one or more of doxorubicin hydrochloride, paclitaxel, docetaxel, gemcitabine, camptothecin, cisplatin, carboplatin, oxaliplatin, etoposide, and etoposide.

[0102] The nanoassemblies including anti-tumor drugs formed through the coordination reaction process can simultaneously release imaging signals and anti-tumor drugs in the tumor tissue microenvironment, thereby realizing the integration of tumor tissue imaging, chemodynamic therapy and chemotherapy, and have excellent imaging-guided chemodynamic / chemotherapy combined treatment effects.

[0103] As a first preferred embodiment of the present application, the nanoassembly is formed by coordination self-assembly of a mixture comprising the following first active targeting unit, first imaging signal unit and first metal ion:

[0104] The first active targeting unit is selected from at least one of cysteine, histidine, arginine, phenylalanine, derivatives formed by replacing the hydrogen on the amino group of the above amino acids with fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl, glutathione, and tripeptide;

[0105] The first imaging signal unit is selected from At least one of indocyanine green, rhodamine-6G, Ce6, and IR780;

[0106] The first imaging signal unit is more preferably

[0107] The first metal ion is selected from copper ion, zinc ion or iron ion.

[0108] As a second preferred embodiment of the present application, based on the above-mentioned first preferred embodiment, the nanoassembly also includes an anti-tumor drug, that is, the nanoassembly is formed by coordination self-assembly of a mixture including the above-mentioned first active targeting unit, the first imaging signal unit, the anti-tumor drug and the first metal ion; wherein the anti-tumor drug is selected from doxorubicin hydrochloride, paclitaxel, docetaxel, gemcitabine, camptothecin, and cisplatin.

[0109] As a third preferred embodiment of the present application, the nanoassembly is formed by coordination self-assembly of a mixture comprising the following second active targeting unit and a second metal ion, or a mixture comprising the following second active targeting unit, a second imaging signal unit, and a second metal ion, which is formed by coordination self-assembly:

[0110] The second active targeting unit is selected from at least one of cysteine, histidine, arginine, phenylalanine, derivatives formed by replacing the hydrogen on the amino group of the above amino acids with fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl, glutathione, and tripeptides, and at least one hydrogen on the amino group of the above amino acids or peptides is replaced by a fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl group. 19 F or 18 F is substituted with a group; preferably, one hydrogen on the amino group in these amino acids or peptides is replaced with a group containing 19 F is substituted by a group containing 19 The group of F is:

[0111] More preferably 19 The group F is

[0112] Contains 18 The group of F is:

[0113] More preferably 18 The group F is

[0114] The second imaging signal unit is selected from At least one of indocyanine green, rhodamine-6G, Ce6, and IR780;

[0115] More preferably, the second imaging signal unit is At least one of indocyanine green, rhodamine-6G, Ce6, and IR780;

[0116] The second metal ion is selected from copper ion, zinc ion or iron ion.

[0117] As the fourth preferred embodiment of the present application, on the basis of the above-mentioned third preferred embodiment, the nanoassembly also includes an anti-tumor drug, that is, the nanoassembly is formed by coordination self-assembly of a mixture including the above-mentioned second active targeting unit, the anti-tumor drug and the second metal ion, or is formed by coordination self-assembly of a mixture including the above-mentioned second active targeting unit, the second imaging signal unit, the anti-tumor drug and the second metal ion; wherein the anti-tumor drug is selected from one or more of doxorubicin hydrochloride, paclitaxel, docetaxel, gemcitabine, camptothecin and cisplatin.

[0118] The present application also provides a method for preparing the above-mentioned tumor-targeting imaging nanoassembly, which at least includes: reacting a mixture containing an active targeting unit, an imaging signal unit and a metal ion to obtain a nanoassembly; or reacting a mixture containing an active targeting unit, an imaging signal unit, an anti-tumor drug and a metal ion to obtain a nanoassembly.

[0119] Specifically, in the above mixture, the molar ratio of active targeting units to metal ions is 1: 1-2: 1. By controlling the molar ratio of active targeting units to metal ions in the mixture within this range, the stability of the nanoassembly formed by coordination self-assembly can be ensured.

[0120] Specifically, the above mixture also includes a stabilizer SH-PEG to improve the stability of the formed nanoassembly in a non-responsive state.

[0121] The embodiments of the present application also provide applications of the above-mentioned tumor-targeting imaging nanoassembly in tumor imaging, tumor diagnosis, and tumor treatment.

[0122] Example 1

[0123] Preparation of tumor targeting imaging nanoassembly: 0.1 mmol of the following structural formula (1-1) 19 F-labeled histidine was dissolved in 100 mL of water, and 10 mg of SH-PEG, 0.1 mg / mL rhodamine-6G aqueous solution, and 1.0 mL of 0.05 mol / L zinc nitrate aqueous solution were added under magnetic stirring. The mixture was stirred at room temperature for 60 minutes, and the mixture was dispersed in water after centrifugation to obtain an aqueous solution of tumor-targeted imaging nanoassemblies.

[0124]

[0125] Example 2

[0126] Preparation of tumor targeting imaging nanoassembly: 0.1 mmol of the above structural formula (1-1) 19F-labeled histidine was dissolved in 100 mL of water, and 10 mg of SH-PEG, 0.1 mg / mL rhodamine-6G aqueous solution 0.1 mL, and 1.0 mL of 0.05 mol / L copper nitrate aqueous solution were added under magnetic stirring. The mixture was stirred at room temperature for 60 minutes, and the mixture was dispersed in water after centrifugation to obtain an aqueous solution of tumor-targeted imaging nanoassemblies.

[0127] Example 3

[0128] Preparation of tumor-targeted imaging nanoassembly: 0.1 mmol of histidine derivative with the following structural formula (1-2) was dissolved in 10 mL of water, and 1 mg of SH-PEG, 1 mL of 0.1 M NaOH aqueous solution, and 10 μL of bis(trifluoromethyl)propene with the following structural formula (1-3) were added under magnetic stirring. 19 F) Toluene formaldehyde and 1 mL of 10 mg / mL zinc nitrate hexahydrate solution were stirred at room temperature for 60 minutes, centrifuged, and then dispersed in water to obtain an aqueous solution of tumor-targeted imaging nanoassemblies.

[0129]

[0130] Example 4

[0131] Preparation of tumor targeting imaging nanoassembly: 1.0 mmol of the above structural formula (1-1) 19 F-labeled histidine was dissolved in 100 mL of water, and 10 mg of SH-PEG and 10 mL of a 0.05 mol / L copper nitrate aqueous solution were added under magnetic stirring. The mixture was stirred at room temperature for 60 minutes, and the mixture was dispersed in water after centrifugation to obtain an aqueous solution of tumor-targeted imaging nanoassemblies.

[0132] Example 5

[0133] Preparation of tumor targeting imaging nanoassembly: 1.0 mmol of the above structural formula (1-1) 19 F-labeled histidine was dissolved in 100 mL of water, and 10 mg of SH-PEG, 2.0 mg of indocyanine green, and 10 mL of a 0.05 mol / L copper nitrate aqueous solution were added under magnetic stirring. The mixture was stirred at room temperature for 60 minutes, and the mixture was dispersed in water after centrifugation to obtain an aqueous solution of tumor-targeted imaging nanoassemblies.

[0134] Example 6

[0135] Preparation of tumor targeting imaging nanoassembly: 1.0 mmol of the above structural formula (1-1) 19F-labeled histidine was dissolved in 100 mL of water, and 10 mg of SH-PEG, 1.0 mg of doxorubicin hydrochloride (Dox), and 10 mL of a 0.05 mol / L copper nitrate aqueous solution were added under magnetic stirring. The mixture was stirred at room temperature for 60 minutes, and the mixture was dispersed in water after centrifugation to obtain an aqueous solution of tumor-targeted imaging nanoassemblies.

[0136] Mouse 1 H and 19 The F MRI experiment is as follows:

[0137] All in vitro and in vivo magnetic resonance imaging (including 1 H and 19 F magnetic resonance imaging) were performed on a Bruker small animal MRI system (7.0T).

[0138] 19 The sequence used for F magnetic resonance imaging is: T1-RARE; parameter setting: repetition time T R is 3000ms, echo time T E The image acquisition time is 4.64ms; field of view: 50mm×50mm, slice thickness: 5mm; image acquisition time: 9min 30s.

[0139] 1 The sequence used for H magnetic resonance imaging was: T2-TurboRARE; parameter setting: repetition time T R is 3000ms, echo time T E The image acquisition time is 40 ms; the field of view is 50 mm × 50 mm, the slice thickness is 1 mm; and the image acquisition time is 1 min 15 s.

[0140] Fluorescence imaging experiments of cells and mice are as follows:

[0141] Cell and tumor sphere fluorescence imaging experiments were performed on a Leica laser confocal microscope imaging system; the excitation wavelengths were 405 nm and 488 nm, the concentration of the nanoprobe was 1 mg / mL, and the incubation time was 4 hours.

[0142] Mouse fluorescence imaging was performed on a Leica small animal in vivo imaging system. The nanoprobe concentration used was 20 mg / mL, and the administration method was tail vein injection with a dosage of 100 μL. The excitation wavelength was 745 nm, the signal acquisition wavelength was 820 nm, and the acquisition time was 24 hours after tail vein injection.

[0143] Tumor imaging and tumor treatment experiments in mice are as follows:

[0144] Six-week-old mice were purchased and adapted for 3 days. 4T1 (or Hep 1-6) cells were inoculated subcutaneously (or in the liver) at a cell concentration of 1.5×10 6 / mL, the injection volume was 100μL. Tail vein administration treatment was started 5 days later. The nano drug concentration was 20mg / mL, the administration method was tail vein injection, the dosage was 100μL, the administration cycle was once every two days, and the administration was continued for two weeks. The wavelength of light in the photothermal therapy group was 808nm, and the intensity was 1W / cm 2 , 5 min each time, light exposure once every two days, for two weeks. The blank group was injected with the same amount of normal saline under the same experimental conditions.

[0145] The above imaging and treatment experiments were conducted on the nanoassemblies in the examples, and the results are as follows:

[0146] (1) The nanoassembly solutions prepared in Examples 1 and 2 were co-cultured with the three-dimensional tumor spheres for 60 minutes, and the penetration of the nanoassembly into the three-dimensional tumor spheres was observed under a confocal fluorescence microscope. The results were as follows: Figure 1 shown.

[0147] from Figure 1 It can be seen that the tumor-targeting imaging nanoassemblies prepared in Examples 1 and 2 are effectively taken up by tumor cells, and the nanoassembly of Example 2 penetrates deeper than that of Example 1, indicating that the nanoassembly with copper ions as the coordination center has a better penetration effect on tumor spheres than zinc ions. The reason is that copper ions can generate active oxygen anions, which can destroy the integrity of tumor spheres, thereby enhancing the penetration performance of the nanoassembly.

[0148] (2) The nanoassembly prepared in Example 2 was added to buffer solutions of different pH values to obtain Figure 2 The actual change diagram shown in the figure is tested separately. 19 F MRI signal, obtained as Figure 3 shown 19 F NMR signal-to-noise ratio diagram; The nanoassembly solution prepared in Example 2 was stored for 28 days, and the changes in its particle size and surface potential were detected, as shown in FIG. Figure 4 shown.

[0149] from Figure 2 It can be seen that the nanoassembly provided in Example 2 did not undergo any color change under the condition of pH 7.4, indicating that no disassembly occurred. As the pH gradually decreased, the degree of disassembly of the nanoassembly increased, resulting in a change in its color. Figure 3 It can be seen that the nanoassembly provided in Example 2 has a strong 19 F NMR signal, where pH is 5.5 19 The signal-to-noise ratio of the F NMR signal reaches the maximum, while there is almost no signal when the pH exceeds 7, indicating that the nanoassembly provided in Example 2 releases the19 F nuclear magnetic resonance imaging signal, thus having imaging responsiveness to acidic environment; Figure 4 It can be seen that after the aqueous solution of the nanoassembly provided in Example 2 was stored for 28 days, its particle size and surface potential changed little, indicating that the nanoassembly provided in Example 2 has good stability.

[0150] (3) The nanoassembly prepared in Example 3 was added to buffer solutions of different pH values to obtain Figure 5 The actual change diagram shown in the figure is tested separately. 19 F MRI signal, obtained as Figure 6 shown 19 F NMR signal-to-noise ratio diagram; The nanoparticle morphology of the nanoassembly solution prepared in Example 3 was characterized, and the results were as follows Figure 7 shown.

[0151] from Figure 5 It can be seen that the nanoassembly of Example 3 changes from milky white to transparent in an acidic environment, indicating that it undergoes disassembly in an acidic environment; Figure 6 It can be seen that the nanoassembly of Example 3 has 19 F nuclear magnetic resonance imaging responsiveness. The morphology of the nanoassembly of Example 3 was characterized, and its particle size was found to be 50.31±14.58 nm, indicating that the nanoassembly had a small particle size, which was beneficial for its penetration and accumulation in tumor tissues.

[0152] (4) The tumor-targeted imaging nanoassembly solution prepared in Example 4 was subjected to nanoparticle morphology characterization, and the nanoassembly solution was injected into a mouse model with liver in situ tumor via the tail vein. 19 F magnetic resonance imaging, the results are as follows Figure 8 and Figure 9 shown.

[0153] from Figure 8 It can be seen that the particle size of the tumor-targeting imaging nanoassemblies prepared in Example 4 is 98.5±41.5 nm, indicating that the particle size of the nanoassemblies is small, which is conducive to the penetration and enrichment of the nanoassemblies in tumor tissues; Figure 9 It can be seen that the tumor-targeted imaging nanoassembly prepared in Example 4 has good 19 F MRI effect.

[0154] (5) The tumor-targeted imaging nanoassembly solution prepared in Example 5 was subjected to nanoparticle morphology characterization, and the results were as follows: Figure 10 As shown; the nanoassembly solution prepared in Example 5 was injected into a subcutaneous tumor mouse model, and the mice were subjected to near-infrared fluorescence imaging. The results were as follows Figure 11 As shown; one side of the mouse leg was inoculated with a subcutaneous tumor, and the other side was normal. The nanoassembly prepared in Example 5 was injected into both sides at the same time, and then 19 F magnetic resonance imaging, imaging results such as Figure 12 shown.

[0155] from Figure 10 It can be seen that the particle size of the tumor-targeting imaging nanoassemblies prepared in Example 5 is 36.0±13.4 nm, indicating that the particle size of the nanoassemblies is small, which is conducive to the penetration and enrichment of the nanoassemblies in tumor tissues; Figure 11 It can be seen that the tumor-targeted imaging nanoassembly prepared in Example 5 has a good near-infrared fluorescence imaging effect on the subcutaneous tumor of mice. Figure 12 Comparison of MRI images of tumor tissue and normal tissue in 1 In H MRI mode, the imaging effect of tumor tissue and normal tissue is not significantly different. 19 Under F magnetic resonance imaging, tumor tissue has obvious 19 F MRI signal, but normal tissue has no 19 F magnetic resonance imaging signal, indicating that the nanoassembly of Example 5 is 19 F is a contrast agent for magnetic resonance imaging that responds specifically to tumors but not to normal tissues.

[0156] (6) The nanoassembly solutions prepared in Examples 4, 5, and 6 were respectively injected into the subcutaneous tumor mouse model via the tail vein, and the mice were treated with chemotherapy. When the nanoassembly of Example 5 was used for treatment, near-infrared laser (808 nm) irradiation was also performed. The results are shown in FIG. Figure 13 shown.

[0157] from Figure 13 It can be seen that the tumor growth inhibition rate of the blank group is 0, and the tumor growth inhibition rate of the nanoassembly of Example 4 is 77.3%. The nanoassembly of Example 6 is loaded with the anti-tumor drug Dox on the basis of Example 4, and its tumor growth inhibition rate is 86.8%. The nanoassembly of Example 5 is loaded with indocyanine green (ICG) on the basis of Example 4, and its tumor growth inhibition rate is 77.4%. When the nanoassembly of Example 5 is combined with near-infrared laser (808 nanometers) irradiation, its tumor growth inhibition rate reaches 94%. The above explanation shows that the nanoassembly provided in the embodiment of the present application not only has the chemodynamic therapeutic effect of tumors, but also can deliver anti-tumor drugs, realize chemodynamic therapy and chemical drug therapy as one, and improve the anti-tumor effect. In addition, the nanoassembly provided in the embodiment of the present application can also be combined with photothermal therapy to realize chemodynamic and photothermal therapy as one, and the anti-tumor effect is very significant.

[0158] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present application, which should be included in the scope of the claims of the present application.

Claims

1. A tumor-targeting imaging nanoassembly, characterized in that: The nanoassembly comprises an active targeting unit, an imaging signal unit and a metal ion, wherein the active targeting unit coordinates with the metal ion, and the nanoassembly is formed by a mixture comprising the active targeting unit, the imaging signal unit and the metal ion through coordination self-assembly; The imaging signal unit is chemically coupled to the active targeting unit; The active targeting unit is selected from at least one of cysteine, histidine, arginine, phenylalanine, derivatives formed by replacing hydrogen on the amino group of the above amino acids with fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl, glutathione, and tripeptides, and at least one hydrogen on the amino group of the above amino acids or peptides is replaced by the imaging signal unit, and the imaging signal unit is 19 F or 18 F group; The said 19 The group of F is: The said 18 The group of F is:

2. The tumor-targeting imaging nanoassembly according to claim 1, characterized in that: The metal ions are transition metal ions or rare earth metal ions.

3. The tumor-targeting imaging nanoassembly according to claim 2, characterized in that: The transition metal ions are selected from copper ions, zinc ions, and iron ions, and the rare earth metal ions are selected from europium ions.

4. The tumor-targeting imaging nanoassembly according to claim 1, characterized in that: The nanoassembly further includes an anti-tumor drug, which is loaded in the nanoassembly.

5. The tumor-targeting imaging nanoassembly according to claim 4, characterized in that: The nanoassembly is formed by coordination self-assembly of a mixture comprising an active targeting unit, an imaging signal unit, an anti-tumor drug and metal ions; the anti-tumor drug is selected from at least one of doxorubicin hydrochloride, paclitaxel, docetaxel, gemcitabine, camptothecin, cisplatin, carboplatin, oxaliplatin, etoposide and etoposide.

6. The method for preparing the tumor-targeting imaging nanoassembly according to any one of claims 1 to 3, characterized in that: The preparation method at least comprises: reacting a mixture containing an active targeting unit, an imaging signal unit and metal ions to obtain the nanoassembly.

7. The method for preparing the tumor-targeting imaging nanoassembly according to claim 6, characterized in that: In the mixture, the molar ratio of the active targeting unit to the metal ion is 1:1-2:

1.

8. Use of the tumor-targeting imaging nanoassembly according to any one of claims 1 to 5 in the preparation of tumor imaging materials, tumor diagnostic materials or tumor therapeutic agents.

Images