A sequential dual - time MRI signal - switching contrast agent for targeted tumor localization, its preparation method and applications

Abstract

The present invention provides a sequential dual - time MRI signal - switching contrast agent for targeting tumors, its preparation method and application, belonging to the technical field of MRI diagnostic contrast agents. Using Angiopep - 2 peptide as a targeting peptide for crossing the BBB, and simultaneously utilizing two key biomarkers, weak acid and high GSH, in gliomas, it presents distinguishable MRI signal manifestations, namely T1 - T2 - T1 signal changes, which can effectively avoid the interference of endogenous stimuli, greatly improve the accuracy and reliability of glioma diagnosis, and is expected to achieve precise delineation of gliomas, providing strong technical support for the early diagnosis and precise treatment of gliomas.

Description

Technical Field

[0001] The present invention belongs to the technical field of MRI diagnostic contrast agents, and relates to a sequential dual - stage MRI signal - switching type contrast agent for targeted tumor localization, its preparation method and application. Background Art

[0002] Glioblastoma is the most common primary malignant tumor in the central nervous system. Its highly invasive and heterogeneous characteristics lead to extremely poor prognosis for patients. According to the World Health Organization classification standard, the median survival time of high - grade gliomas (such as glioblastoma multiforme, GBM) is only 12 - 15 months, and the 5 - year survival rate of low - grade gliomas is also less than 60%. Although the progress of MRI in neuroimaging technology has significantly improved the detection rate of brain tumors, the accurate diagnosis of gliomas still faces two core challenges: First, the traditional imaging has significant limitations in defining the tumor boundary. Since glioma cells often infiltrate along white matter fiber bundles, conventional T1 - and T2 - weighted enhanced MRI is difficult to accurately distinguish the tumor parenchyma from the surrounding edema zone, resulting in difficulty in precisely planning the surgical resection range. Second, the heterogeneity within the tumor (such as necrosis, angiogenesis, and cell density differences) also makes it difficult for traditional MRI to accurately diagnose it.

[0003] Regarding the precise delivery of glioblastoma, a huge challenge lies in the obstruction of the blood - brain barrier (BBB). The blood - brain barrier is a complex physiological barrier ultimately composed of brain microvascular endothelial cells, tight - junction proteins, pericytes, and astrocytes, which can limit more than 98% of macromolecular drugs and almost all nanoparticles from entering the central nervous system.

[0004] In recent years, the delivery strategies for crossing the BBB have mainly focused on two major directions: "active targeted transport penetration" and "local barrier regulation". The active targeted transport penetration strategy mainly relies on the surface modification of nanoparticles with targeting ligands, which can specifically recognize receptors overexpressed on the surface of BBB endothelial cells or glioma cells. By means of receptor - mediated endocytosis, it can efficiently achieve cross - BBB transport. The local barrier regulation strategy mainly uses technical means such as focused ultrasound, and uses the cavitation effect to disrupt the tight junctions between endothelial cells, temporarily opening the BBB barrier to enhance the permeability of nanoparticles. In comparison, the active targeted transport penetration strategy has significant advantages such as precise targeting, rapid enrichment, direct delivery, and no damage to the normal barrier structure and function compared with the local barrier regulation strategy. Therefore, the active targeted transport penetration strategy has been more widely applied in the field of brain delivery.

[0005] At present, in the practical application of the active targeting and transport penetration strategy, the Angiopep-2 peptide plays an important role. As a type of Kunitz-type protease inhibitor, the Angiopep-2 peptide has unique targeting recognition ability and can specifically recognize the receptor LRP-1 highly expressed on the surface of BBB endothelial cells and glioma cells. This property enables the Angiopep-2 peptide to be widely used in constructing a dual-targeted drug delivery system for glioma, which can not only cross the BBB but also further target glioma cells, greatly improving the delivery efficiency and accuracy of drugs or diagnostic reagents at the site of glioblastoma.

[0006] When extremely small iron oxide nanoparticles (<4 nm) convert between the dispersed and aggregated states, they exhibit the interesting property of switching the contrast signal between T1 bright signal and T2 dark signal. Based on this, T1-T2 or T2-T1 type contrast agents targeting the glioma environment have been developed, and the unique signal change ability in the glioma region greatly increases the specificity and sensitivity of the contrast agent.

[0007] However, these probes often respond to a single condition, such as pH, glutathione (GSH), or hypoxia. And single-response probes usually lack absolute specificity for pathological tissues. Therefore, further improving the specificity of the contrast agent is an urgent problem for those skilled in the art. Summary of the Invention

[0008] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a sequential dual-MRI signal switching type contrast agent for targeting tumors, its preparation method and application. The Angiopep-2 peptide is used as the targeting peptide to cross the BBB, and at the same time, two key biomarkers, weak acid and high GSH, in glioblastoma are utilized to present distinguishable MRI signal manifestations at different time scales.

[0009] To achieve the purpose of this invention, the following technical solutions are adopted:

[0010] In the first aspect, the present invention provides a sequential dual-MRI signal switching type contrast agent for targeting tumors, and the sequential dual-MRI signal switching type contrast agent is a mixture of a first contrast agent and a second contrast agent;

[0011] The first contrast agent includes, from the inside out, nanoparticles, a first responsive click bifunctional layer, a first responsive shielding bifunctional layer, and a first targeting layer. The first responsive click bifunctional layer and the first responsive shielding bifunctional layer are respectively independently connected to the nanoparticles, and the first responsive shielding bifunctional layer is connected to the first targeting layer;

[0012] The second contrast agent sequentially includes nanoparticles, a second responsive click bifunctional layer, a second responsive shielding bifunctional layer, and a second targeting layer from the inside to the outside. The second responsive click bifunctional layer and the second responsive shielding bifunctional layer are each independently connected to the nanoparticles, and the second responsive shielding bifunctional layer is connected to the second targeting layer;

[0013] The first responsive click bifunctional layer contains a GSH-responsive group and a first click group;

[0014] The second responsive click bifunctional layer contains a GSH-responsive group and a second click group;

[0015] Both the first responsive shielding bifunctional layer and the second responsive shielding bifunctional layer contain a pH-responsive group and a shielding group.

[0016] In the present invention, the targeted positioning of tumors refers to the targeted positioning of gliomas.

[0017] The sequential dual MRI signal switching contrast agent provided by the present invention uses Angiopep-2 peptide as a targeting peptide for crossing the BBB, and simultaneously utilizes two key biomarkers of gliomas, weak acid and high GSH, to present distinguishable MRI signal manifestations at different time scales. Based on the signal changes on the time scale, a feasible self-confirmation cross-validation mechanism is achieved.

[0018] It should be noted that the first contrast agent and the second contrast agent are themselves a T1-type contrast agent. Through the surface-modified Angiopep-2 peptide in the first contrast agent and the second contrast agent, the highly expressed receptor LRP-1 on the surface of BBB endothelial cells and glioma cells can be specifically recognized, enabling not only crossing the BBB but also further targeting glioma cells. Therefore, the active targeting effect enables the first contrast agent and the second contrast agent to be rapidly delivered to the glioma location and present a T1 contrast signal. Then, when it is in the weakly acidic microenvironment of the tumor, the pH-responsive group is gradually decomposed and broken, and then the shielding group falls off, exposing the internal click group. Under the action of click chemistry, it becomes an ESIONPs cluster, and at this time, the first contrast signal transformation is achieved (the T1 bright signal transforms into the T2 dark signal). Subsequently, the ESIONPs cluster is further endocytosed into glioma cells. Due to its large particle size, it can be enriched in cells for a long time. Under the action of high-concentration GSH, the GSH-responsive groups crosslinked inside the cluster are gradually broken, resulting in the dissociation of ESIONPs. At this time, the second contrast signal transformation is achieved (the T2 dark signal transforms into the T1 bright signal).

[0019] It should also be noted that through two signal changes, the interference of endogenous stimuli can be effectively avoided, greatly improving the accuracy and reliability of imaging. It is expected to achieve precise delineation of gliomas, providing strong technical support for the early diagnosis and precise treatment of gliomas. In addition, the first / second contrast agent has good biocompatibility and extremely low biological toxicity, which also broadens the way for further subsequent applications.

[0020] As a preferred technical solution of the present invention, the nanoparticles are iron(III) oxide with a modifying group connected to the surface.

[0021] Preferably, the modifying group includes pentafluorophenol ester (DOPAC-PFP).

[0022] Preferably, the average particle size of the iron(III) oxide is <4 nm, for example, it can be 3.8 nm, 3.6 nm, 3.2 nm, 3 nm, 2.8 nm, 2.6 nm, 2.2 nm or 2 nm, etc.

[0023] As a preferred technical solution of the present invention, the GSH-responsive group is an -S-S- functional group.

[0024] Preferably, the first click group is an N3 functional group.

[0025] Preferably, the second click group is a DBCO functional group.

[0026] Preferably, the pH-responsive group is a benzoic acid imide functional group.

[0027] Preferably, the shielding group includes an mPEGn functional group, where n is 9000 - 12000, for example, it can be 9200, 9500, 10000, 10500, 11000, 11500 or 11800, etc.

[0028] It should be noted that in the present invention, mPEG are all methoxy PEG.

[0029] As a preferred technical solution of the present invention, the preparation raw materials of the first responsive click bifunctional layer include a first polymer containing a GSH-responsive group and a first click group; the end of the first polymer has an amino group.

[0030] Preferably, the structural general formula of the first polymer is NH2-S-S-mPEGa-N3; where a is 1800 - 2200, for example, it can be 1820, 1850, 1880, 1900, 1920, 1950, 1980, 2000, 2050, 2100 or 2150, etc.

[0031] Preferably, an amide bond is formed between the amino group in the first polymer and the modifying group in the nanoparticle for connection.

[0032] Preferably, the raw materials for preparing the first response shielding bifunctional layer and the second response shielding bifunctional layer both include amino-terminated polyethylene glycol and a second polymer (Mal-mPEGn-CHO, where Mal is a maleimide group) with the structural general formula shown in formula (I).

[0033] Formula (I)

[0034] Wherein, n is 9000 - 12000.

[0035] Preferably, the structural general formula of the amino-terminated polyethylene glycol is NH2-mPEGa-NH2; wherein, a is 1800 - 2200.

[0036] Preferably, an amide bond is formed between the amino group at one end of the amino-terminated polyethylene glycol and the modifying group in the nanoparticle for connection, and a benzoic acid imine bond is formed between the amino group at the other end and the benzaldehyde group in the second polymer for connection.

[0037] It should be noted that under slightly acidic conditions (pH 4 - 6.8), the pH-responsive group in the response shielding bifunctional layer breaks, causing the shielding group to fall off, exposing the click group on the surface of the ESIONPs. Then, under the action of click chemistry, the ESIONPs aggregate, thereby realizing the first signal switching in the dual signal switching, that is, the switching of the contrast signal from T1 to T2.

[0038] Preferably, the raw materials for preparing the second response click bifunctional layer include a third polymer containing a GSH-responsive group and a second click group; the third polymer has an amino group at its end.

[0039] Preferably, the structural general formula of the third polymer is NH2-S-S-mPEGa-DBCO; wherein, a is 1800 - 2200.

[0040] It should be noted that in the environment of high concentration of GSH in glioma cells, the GSH-responsive group is slowly reduced and broken, causing the aggregated ESIONPs to dissociate, thereby realizing the second signal switching in the dual signal switching, that is, the change of the contrast signal from T2 to T1.

[0041] Preferably, an amide bond is formed between the amino group in the third polymer and the modifying group in the nanoparticle for connection.

[0042] Preferably, the raw materials for preparing the first targeting layer and the second targeting layer both include a targeting peptide, and the targeting peptide includes SH-Angiopep-2.

[0043] The maleimide group in the second polymer is connected by forming a thioether bond with the sulfhydryl group in the targeting peptide.

[0044] As a preferred technical solution of the present invention, the molar ratio of the first contrast agent to the second contrast agent is 1:(1 - 1.2), for example, it can be 1:1.02, 1:1.04, 1:1.06, 1:1.08, 1:1, 1:1.12, 1:1.14, 1:1.16 or 1:1.18, etc.

[0045] In a second aspect, the present invention provides a preparation method of the sequential dual - time MRI signal - switching contrast agent as described in the first aspect. The preparation method includes the following steps:

[0046] (1) Perform a first response click bifunctional layer connection reaction, a first response shielding bifunctional layer connection reaction, and a first targeting layer connection reaction on the nanoparticles in sequence to obtain the first contrast agent;

[0047] (2) Perform a second response click bifunctional layer connection reaction, a second response shielding bifunctional layer connection reaction, and a second targeting layer connection reaction on the nanoparticles in sequence to obtain the second contrast agent;

[0048] (3) Mix the first contrast agent obtained in step (1) and the second contrast agent obtained in step (2) to obtain the sequential dual - time MRI signal - switching contrast agent;

[0049] Steps (1) and (2) have no sequence.

[0050] The preparation method provided by the present invention has a simple and convenient process, low cost, and broad application prospects.

[0051] In the present invention, the preparation method of the nanoparticles in step (1) and step (2) includes: mixing hydrophobic Fe2O3 nanoparticles, modifying groups, and a solvent, and performing a ligand exchange reaction to obtain modified Fe2O3 nanoparticles.

[0052] In the present invention, the hydrophobic Fe2O3 nanoparticles include fatty acid - modified Fe2O3 nanoparticles (ESIONPs - OA).

[0053] In the present invention, the hydrophobic Fe2O3 nanoparticles can be prepared by conventional methods of the existing technology, and no specific limitation is made here. The preparation method of the hydrophobic Fe2O3 nanoparticles in the present invention specifically includes: mixing iron salts, fatty acid salts, and a mixed solvent, and performing vacuum treatment and thermal decomposition reaction to obtain the hydrophobic Fe2O3 nanoparticles.

[0054] In the present invention, the molar ratio of the iron salt to the fatty acid salt is 1:(2 - 4), for example, it can be 1:2.2, 1:2.5, 1:3, 1:3.5 or 1:3.8, etc. The temperature of the vacuum treatment is 70 - 90 °C, for example, it can be 72 °C, 75 °C, 78 °C, 80 °C, 85 °C or 87 °C, etc. The time of the vacuum treatment is 1 - 2 h, for example, it can be 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h or 1.9 h, etc. The thermal decomposition reaction is carried out under a protective atmosphere. The heating rate of the thermal decomposition reaction is 5 - 10 °C / min, for example, it can be 6 °C / min, 7 °C / min, 8 °C / min or 9 °C / min, etc. The heating end point of the thermal decomposition reaction is 245 - 260 °C, for example, it can be 246 °C, 248 °C, 250 °C, 252 °C, 254 °C, 256 °C or 258 °C, etc. The time of the thermal decomposition reaction is 25 - 35 min, for example, it can be 26 min, 28 min, 30 min, 32 min or 34 min, etc.

[0055] In the present invention, the modifying group includes pentafluorophenyl ester (DOPAC - PFP). The preparation method of the pentafluorophenyl ester specifically includes: dissolving 3,4 - dihydroxy phenylacetic acid (DOPAC) and pentafluorophenol (PFP) in 1,4 - dioxane, and then dropping 1,4 - dioxane dissolved with N,N'-dicyclohexylcarbodiimide (DCC) into the above solution, stirring for 20 - 30 h at room temperature under nitrogen protection. After the reaction, it is purified by silica gel column chromatography to obtain the pentafluorophenyl ester.

[0056] In the present invention, the 1,4 - dioxane needs to be anhydrously treated; the eluent used in the silica gel column chromatography is a mixed solution of n - hexane and ethyl acetate with a volume ratio of 3:1.

[0057] In the present invention, the mass ratio of the hydrophobic Fe2O3 nanoparticles to the modifying group is 1:(8 - 20), for example, it can be 1:9, 1:10, 1:11, 1:12, 1:13, 1:15, 1:16 or 1:18, etc. The solvent includes tetrahydrofuran (THF).

[0058] In the present invention, the ligand exchange reaction specifically includes: dissolving the hydrophobic Fe2O3 nanoparticles in the first solvent, and then adding the modifying group thereto, carrying out the ligand exchange reaction in a protective atmosphere. After the reaction, it is concentrated by rotary evaporation and dialyzed for several days to remove the excess modifying group in the product, and finally dispersed in tetrahydrofuran for standby.

[0059] In the present invention, the temperature of the ligand exchange reaction is 40 - 60 °C, for example, it can be 42 °C, 45 °C, 48 °C, 50 °C, 52 °C, 53 °C or 54 °C, etc., and the time of the ligand exchange reaction is 60 - 80 h, for example, it can be 62 h, 65 h, 68 h, 50 h, 52 h, 53 h or 54 h, etc.

[0060] As a preferred technical solution of the present invention, the method for the first responsive click bifunctional layer linking reaction in step (1) includes: mixing nanoparticles, a first polymer containing a GSH-responsive group and a first click group, and a first solvent, and performing a first reaction to obtain a first intermediate.

[0061] Preferably, the method for the first responsive shielding bifunctional layer linking reaction in step (1) includes: mixing the first intermediate, amino-terminated polyethylene glycol, and a second solvent, performing a second reaction, and then adding a second polymer thereto to perform a third reaction to obtain a second intermediate.

[0062] Preferably, the method for the first targeting layer linking reaction in step (1) includes: mixing the second intermediate and a targeting peptide, and performing a fourth reaction to obtain a first contrast agent.

[0063] Preferably, the method for the second responsive click bifunctional layer linking reaction in step (2) includes: mixing nanoparticles, a third polymer containing a GSH-responsive group and a second click group, and a second solvent, and performing a fifth reaction to obtain a third intermediate.

[0064] Preferably, the method for the second responsive shielding bifunctional layer linking reaction in step (2) includes: mixing the third intermediate, amino-terminated polyethylene glycol NH2-mPEGa-NH2, and a second solvent, performing a sixth reaction, and then adding a second polymer thereto to perform a seventh reaction to obtain a fourth intermediate.

[0065] Preferably, the method for the second targeting layer linking reaction in step (2) includes: mixing the fourth intermediate and a targeting peptide, and performing an eighth reaction to obtain a second contrast agent.

[0066] In the present invention, both the first reaction and the fifth reaction further include adding a catalyst, and the catalyst includes N,N-diisopropylethylamine (DIPEA). By adding the catalyst DIPEA, the reaction efficiency is accelerated.

[0067] In the present invention, both the third reaction and the seventh reaction are Schiff base reactions; both the fourth reaction and the eighth reaction are Michael addition reactions.

[0068] As a preferred technical solution of the present invention, the molar ratio of the first polymer to the amino-terminated polyethylene glycol is 1:(6 - 15), for example, it can be 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13 or 1:14, etc.

[0069] In the present invention, the dropping order of the first polymer / third polymer and the amino-terminated polyethylene glycol has an important influence on the contrast agent. If the amino-terminated polyethylene glycol is dropped first, since the amino-terminated polyethylene glycol is in excess, too many sites are occupied by the amino-terminated polyethylene glycol, resulting in a decrease in the modification rate of the click group.

[0070] Preferably, the molar ratio of the second polymer to the amino-terminated polyethylene glycol ≥ 2:1, for example, it can be 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, etc.

[0071] Preferably, the molar ratio of the third polymer to the amino-terminated polyethylene glycol is 1:(6 - 15), for example, it can be 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13 or 1:14, etc.

[0072] Preferably, the molar ratio of the targeting peptide to the second polymer is 1:(20 - 40), for example, it can be 1:22, 1:25, 1:26, 1:28, 1:30, 1:32, 1:35, 1:36 or 1:38, etc.

[0073] In the present invention, by controlling the molar ratio range of the targeting peptide to the second polymer, good dispersibility is ensured.

[0074] Preferably, both the first solvent and the second solvent include dimethyl sulfoxide (DMSO).

[0075] In the present invention, step (3) further includes a third solvent, and the third solvent includes physiological saline.

[0076] Preferably, the temperatures of the first reaction, the second reaction, the third reaction, the fourth reaction, the fifth reaction, the sixth reaction, the seventh reaction and the eighth reaction are all 20 - 30 °C, for example, it can be 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C or 28 °C, etc.

[0077] Preferably, the times of the first reaction, the second reaction, the third reaction, the fourth reaction, the fifth reaction, the sixth reaction, the seventh reaction and the eighth reaction are all ≥ 20 h, for example, it can be 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 28 h, 30 h, 32 h, 34 h, 35 h, 36 h, 37 h, 38 h or 40 h, etc.

[0078] Preferably, after the second reaction and the sixth reaction, first dialysis and pH adjustment to 8-9 are sequentially carried out.

[0079] In the present invention, the first dialysis is carried out using a dialysis bag for at least 1 day, and the molecular weight of the dialysis bag ≥ 30KDa. Excess mPEG is removed by the first dialysis.

[0080] In the present invention, the pH adjustment means adjusting the pH value of the solution to 8-9, for example, it can be 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8 or 8.9, etc.

[0081] Preferably, after the fourth reaction and the eighth reaction, precipitation treatment and second dialysis are sequentially carried out.

[0082] In the present invention, the precipitation treatment is to precipitate once in ice ether, and the collected precipitate is dissolved in a small amount of water.

[0083] In the present invention, the second dialysis is carried out using a dialysis bag for at least 1 day, and the molecular weight of the dialysis bag ≥ 30KDa. Excess molecules are removed by the second dialysis.

[0084] In the third aspect, the present invention provides a pharmaceutical composition, which includes a drug, a pharmaceutically acceptable pharmaceutical carrier, and the sequential dual MRI signal switching type contrast agent described in the first aspect.

[0085] In the present invention, the drug can be drug components such as therapeutic drugs and tracer molecules.

[0086] In the fourth aspect, the present invention provides an application of the sequential dual MRI signal switching type contrast agent described in the first aspect, and the sequential dual MRI signal switching type contrast agent is used in the preparation of tumor magnetic resonance imaging diagnostic or detection reagents.

[0087] The present invention also provides a contrast method for non-medical purposes, and the contrast method includes: administering the sequential dual MRI signal switching type contrast agent described in the first aspect to the object to be contrasted, and performing contrast.

[0088] The numerical ranges described in the present invention not only include the above-listed point values, but also include any point values between the above numerical ranges not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the range.

[0089] Compared with the prior art, the present invention has the following beneficial effects:

[0090] (1) The sequential dual - MRI signal - switching contrast agent provided by the present invention can rapidly deliver the contrast agent to the glioma region through the dual effects of Angiopep - 2 crossing the BBB and targeting glioma cells, and presents a T1 contrast signal. When it is in the weakly acidic microenvironment of the tumor, the pH - responsive group is gradually decomposed and broken, causing the shielding group to fall off, exposing the internal click group. Under the action of click chemistry, it becomes ESIONPs clusters. At this time, the first contrast signal transformation is achieved, that is, from T1 to T2; the ESIONPs clusters are further endocytosed into glioma cells. Due to their large particle size, they can be enriched in cells for a long time. Under the action of high - concentration GSH, the GSH - responsive groups cross - linked inside the clusters are gradually broken, resulting in the dissociation of ESIONPs. At this time, the second contrast signal transformation is achieved, that is, from T2 to T1;

[0091] (2) The sequential dual - MRI signal - switching contrast agent provided by the present invention uses Angiopep - 2 peptide as the targeting peptide to cross the BBB, and at the same time utilizes two key biomarkers, the weak acid of glioblastoma and high GSH, to present distinguishable MRI signal manifestations at different time scales, which can effectively avoid the interference of endogenous stimuli, greatly improve the accuracy and reliability of imaging, and is expected to achieve precise delineation of glioblastoma, providing strong technical support for the early diagnosis and precise treatment of glioblastoma. Brief Description of the Drawings

[0092] Figure 1 is the transmission electron microscopy image of ESIONPs - OA nanoparticles provided in the specific embodiment of the present invention.

[0093] Figure 2 is the transmission electron microscopy image of ESIONPs - PFP nanoparticles provided in the specific embodiment of the present invention.

[0094] Figure 3 is the nuclear magnetic resonance hydrogen spectrum of Mal - mPEG10000 - CHO provided in the specific embodiment of the present invention.

[0095] Figure 4 is the transmission electron microscopy image of the ESCA - CA contrast agent in Example 1 of the present invention.

[0096] Figure 5 is the transmission electron microscopy image of the ECA - CA contrast agent in Comparative Example 1 of the present invention.

[0097] Figure 6 is the transmission electron microscopy image of the ESA - CA contrast agent in Comparative Example 2 of the present invention.

[0098] Figure 7 is the transmission electron microscopy image of the EA - CA contrast agent in Comparative Example 3 of the present invention.

[0099] Figure 8 The contrast diagram of the longitudinal relaxation rate (r1) and the transverse relaxation rate (r2) of the contrast agent obtained in Example 1 of the present invention under non-responsive and different response conditions.

[0100] Figure 9 The contrast diagram of the longitudinal relaxation rate (r1) and the transverse relaxation rate (r2) of the contrast agent obtained in Comparative Example 1 of the present invention under non-responsive and different response conditions.

[0101] Figure 10 The contrast diagram of the longitudinal relaxation rate (r1) and the transverse relaxation rate (r2) of the contrast agent obtained in Comparative Example 2 of the present invention under non-responsive and different response conditions.

[0102] Figure 11 The contrast diagram of the longitudinal relaxation rate (r1) and the transverse relaxation rate (r2) of the contrast agent obtained in Comparative Example 3 of the present invention under non-responsive and different response conditions.

[0103] Figure 12 The verification diagram of the contrast agent obtained in Example 1 of the present invention for crossing the BBB and targeting glioma on the Transwell model; (a) is the proportion diagram of Dil-positive cells detected by flow cytometry after co-incubation of the blank group, the control group, and the experimental group with bend.3 cells; (b) is the confocal microscopy fluorescence diagram of bend.3 cells taking up the particles of the blank group, the control group, and the experimental group, with DAPI staining the cell nucleus; (c) is the proportion diagram of Dil-positive cells detected by flow cytometry after co-incubation of the blank group, the control group, and the experimental group with GL261 cells; (d) is the confocal microscopy fluorescence diagram of GL261 cells taking up the particles of the blank group, the control group, and the experimental group, with DAPI staining the cell nucleus.

[0104] Figure 13 The in vivo T1 and T2 weighted MRI imaging diagrams of the contrast agents in Examples 1 and Comparative Examples 1-3 of the present invention in orthotopic glioma mice. Detailed implementation manners

[0105] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0106] The detailed implementation manners of the present invention provide a sequential dual MRI signal switching type contrast agent for targeted tumor localization and its preparation method. The sequential dual MRI signal switching type contrast agent is a mixture of a first contrast agent and a second contrast agent;

[0107] The first contrast agent sequentially includes nanoparticles, a first responsive click bifunctional layer, a first responsive shielding bifunctional layer, and a first targeting layer from the inside to the outside. The first responsive click bifunctional layer and the first responsive shielding bifunctional layer are each independently connected to the nanoparticles, and the first responsive shielding bifunctional layer is connected to the first targeting layer;

[0108] The second contrast agent sequentially includes nanoparticles, a second responsive click bifunctional layer, a second responsive shielding bifunctional layer, and a second targeting layer from the inside to the outside. The second responsive click bifunctional layer and the second responsive shielding bifunctional layer are each independently connected to the nanoparticles, and the second responsive shielding bifunctional layer is connected to the second targeting layer;

[0109] The first responsive click bifunctional layer contains a GSH-responsive group and a first click group; the second responsive click bifunctional layer contains a GSH-responsive group and a second click group; the GSH-responsive group is a -S-S- functional group; the first click group is an N3 functional group; the second click group is a DBCO functional group;

[0110] Both the first responsive shielding bifunctional layer and the second responsive shielding bifunctional layer contain a pH-responsive group and a shielding group; the pH-responsive group is a benzoic acid imide functional group; the shielding group includes an mPEGn functional group, where n is 9000 - 12000;

[0111] The nanoparticles are iron(III) oxide with a modification group connected to the surface; the modification group includes pentafluorophenyl ester; the average particle size of the iron(III) oxide is <4 nm.

[0112] The preparation method includes the following steps:

[0113] (1) Perform a first responsive click bifunctional layer connection reaction, a first responsive shielding bifunctional layer connection reaction, and a first targeting layer connection reaction on the nanoparticles in sequence to obtain the first contrast agent;

[0114] (2) Perform a second responsive click bifunctional layer connection reaction, a second responsive shielding bifunctional layer connection reaction, and a second targeting layer connection reaction on the nanoparticles in sequence to obtain the second contrast agent;

[0115] (3) Mix the first contrast agent obtained in step (1) and the second contrast agent obtained in step (2) to obtain the sequential dual - MRI signal switching type contrast agent;

[0116] Steps (1) and (2) are not in a sequential order.

[0117] Unless otherwise specified, the raw materials and reagents used in the following examples and comparative examples are all commercially available products (customized products), or can be prepared by known methods. For the following examples and comparative examples, if the specific technical or condition parameters are not indicated, they can all be carried out according to the techniques or conditions described in the literature in this field, or according to the product instructions.

[0118] In the following examples and comparative examples, NH2-S-S-mPEGa-N3, NH2-mPEGa-NH2, NH2-S-S-mPEGa-DBCO and Mal-mPEGn-OH were all purchased from Xi'an Ruixi Biotechnology Co., Ltd.; SH-Angiopep-2 (sequence TFFYGGSRGKRNNFKTEEYC) was purchased from Suzhou Modif Biotechnology Co., Ltd. In the following examples and comparative examples, hydrophobic Fe2O3 nanoparticles, pentafluorophenyl esters, nanoparticles with pentafluorophenyl esters connected to the surface, PEG10000-CHO and the second polymer (Mal-mPEG10000-CHO) were all prepared by the following preparation methods.

[0119] Among them, the hydrophobic Fe2O3 nanoparticles were prepared by the following method:

[0120] (a) Dissolve sodium oleate (18.3 g, 60 mmol) and ferric chloride hexahydrate (5.4 g, 20 mmol) in a mixed solvent of 30 mL of water, 40 mL of absolute ethanol and 70 mL of n-hexane, and then reflux and stir at 70 °C for 4 h. After the reaction, cool to room temperature, wash and rotary evaporate, and then dry at 70 °C for 24 h to obtain an iron oleate complex precursor.

[0121] (b) Dissolve oleic acid (0.57 g, 2 mmol), oleyl alcohol (1.61 g, 6 mmol) and the iron oleate complex precursor (1.8 g, 2 mmol) described in step (1) in diphenyl ether (10 g, 58 mmol), and then perform vacuum treatment at 70 °C for 2 h. Then, under nitrogen protection, heat up to 250 °C at a heating rate of 10 °C / min for thermal decomposition reaction and keep warm for 30 min. After the reaction, cool to room temperature and wash several times with acetone to obtain oleic acid-modified Fe2O3 nanoparticles (ESIONPs-OA).

[0122] The transmission electron microscope image of ESIONPs-OA is as Figure 1 shown, and it can be seen from Figure 1 that the prepared ESIONPs-OA have uniform particle size and good dispersibility.

[0123] Among them, the pentafluorophenyl ester was prepared by the following method:

[0124] Dissolve 3,4-dihydroxyphenylacetic acid (3.6 g, 20 mmol) and pentafluorophenol (4.4 g, 24 mmol) in 100 mL of anhydrous 1,4-dioxane, and add dropwise N,N'-dicyclohexylcarbodiimide (5 g, 24 mmol) dissolved in 20 mL of anhydrous 1,4-dioxane to the above solution. React at room temperature for 24 h under argon protection. After filtration and rotary evaporation, column purification is carried out using a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 3:1 as the eluent to obtain pentafluorophenol ester (DOPAC-PFP).

[0125] Among them, the nanoparticles with pentafluorophenol ester connected to the surface are prepared by the following method:

[0126] Dissolve 60 mg of ESIONPs-OA in 50 mL of THF, then add 600 mg of DOPAC-PFP dissolved in 20 mL of THF thereto. Subsequently, carry out ligand exchange reaction at 50 °C under nitrogen protection for 72 h. After the reaction is completed, carry out rotary evaporation, and then dialyze using a dialysis bag with a molecular weight of 2 KDa for at least 3 days to obtain nanoparticles with pentafluorophenol ester connected to the surface (ESIONPs-PFP).

[0127] The transmission electron microscopy image of ESIONPs-PFP is as Figure 2 shown. It can be seen from Figure 2 that the prepared ESIONPs-PFP has uniform particle size and good dispersibility.

[0128] Among them, the second polymer is prepared by the following method:

[0129] Dissolve Mal-mPEG10000-OH (2 g, 0.2 mmol) and p-formylbenzoic acid (240.21 mg, 1.6 mmol) in 40 mL of ultradry dichloromethane solution, then add DCC (330 mg, 1.6 mmol) and DMAP (24 mg, 0.2 mmol) thereto and stir to mix evenly. Subsequently, stir and react at room temperature for 48 h under argon protection. After the reaction is completed, carry out rotary evaporation. Then dissolve the obtained solid in a small amount of deionized water, and then filter to remove the insoluble substances therein. Extract the obtained aqueous solution with dichloromethane solution (3×15 mL), add anhydrous magnesium sulfate to the extracted organic phase and dry at room temperature for 24 h. Finally, filter the solution and carry out concentration treatment. The concentrated solution is precipitated three times in ice-cold anhydrous ether, collect the precipitate, and vacuum dry to obtain the second polymer (Mal-mPEG10000-CHO).

[0130] The nuclear magnetic resonance hydrogen spectrum of Mal-mPEG10000-CHO is as Figure 3As shown, it corroborates the successful synthesis of Mal-mPEG10000-CHO.

[0131] Example 1

[0132] This example provides a sequential dual MRI signal switching contrast agent for targeting tumors and its preparation method. The sequential dual MRI signal switching contrast agent is a mixture of a first contrast agent and a second contrast agent with a molar ratio of 1:1.

[0133] The first contrast agent sequentially includes nanoparticles, a first responsive click bifunctional layer, a first responsive shielding bifunctional layer, and a first targeting layer from the inside out. The first responsive click bifunctional layer and the first responsive shielding bifunctional layer are independently connected to the nanoparticles, and the first responsive shielding bifunctional layer is connected to the first targeting layer.

[0134] The second contrast agent sequentially includes nanoparticles, a second responsive click bifunctional layer, a second responsive shielding bifunctional layer, and a second targeting layer from the inside out. The second responsive click bifunctional layer and the second responsive shielding bifunctional layer are independently connected to the nanoparticles, and the second responsive shielding bifunctional layer is connected to the second targeting layer.

[0135] The nanoparticles are Fe2O3 with pentafluorophenol ester connected to the surface; the average particle size of the nanoparticles is 3 nm.

[0136] The preparation raw materials of the first responsive click bifunctional layer include a first polymer containing a GSH-responsive group and a first click group; the structural general formula of the first polymer is NH2-S-S-mPEG2000-N3; the amino group in the first polymer forms an amide bond with the pentafluorophenol ester group in the nanoparticles for connection.

[0137] The preparation raw materials of the first responsive shielding bifunctional layer and the second responsive shielding bifunctional layer both include amino-terminated polyethylene glycol and a second polymer (Mal-mPEG10000-CHO) with the structural general formula shown in formula (I).

[0138] Formula (I)

[0139] Among them, n is 10000.

[0140] The structural general formula of the amino-terminated polyethylene glycol is NH2-mPEG2000-NH2; one amino group at one end of the amino-terminated polyethylene glycol forms an amide bond with the pentafluorophenol ester group in the nanoparticles for connection, and the other amino group at the other end forms a benzoic acid imine bond with the benzaldehyde group in the second polymer for connection.

[0141] The raw materials for preparing the second responsive click bilayer include a third polymer containing a GSH-responsive group and a second click group; the structural general formula of the third polymer is NH2-S-S-mPEG2000-DBCO; an amide bond is formed between the amino group in the third polymer and the pentafluorophenol ester group in the nanoparticle for connection;

[0142] The raw materials for preparing the first targeting layer and the second targeting layer both include a targeting peptide, and the targeting peptide is SH-Angiopep-2; a thioether bond is formed between the maleimide group in the second polymer and the sulfhydryl group in the targeting peptide for connection.

[0143] The preparation method includes the following steps:

[0144] (1) Disperse 0.0224 mM ESIONPs-PFP in 15 mL of DMSO, then dropwise add 0.55 mg of NH2-S-S-mPEG2000-N3 dissolved in 5 mL of DMSO thereto, then add 10 μL of DIPEA thereto, and then react for the first time at room temperature for 24 h; then dissolve 5 mg of NH2-mPEG2000-NH2 in 10 mL of DMSO, and then dropwise add the above first reaction product to the NH2-mPEG2000-NH2 solution, react for the second time at room temperature for 24 h, transfer the second reaction product to a dialysis bag with a molecular weight cut-off of 35 kDa, dialyze for the first time in deionized water for 1 day, then transfer it to a flask, adjust the pH of the solution to 8.5, then add 60 mg of Mal-mPEG10000-CHO to the above solution, and react for the third time at room temperature for 36 h; then add 0.5 mg of SH-Angiopep-2 to the third reaction product, and react for the fourth time at room temperature for 24 h; finally, drop the fourth reaction product into ice-cooled anhydrous ether for precipitation treatment, dissolve the obtained precipitate in a small amount of deionized water, and then transfer the obtained solution to a dialysis bag with a molecular weight cut-off of 35 kDa, and dialyze for the second time in deionized water for 2 days to obtain the first contrast agent (ESCNA).

[0145] (2) Step (2) is carried out with reference to step (1), except that "0.55 mg of NH2-S-S-mPEG2000-N3" is replaced with "0.75 mg of NH2-S-S-mPEG2000-DBCO", and the others are all carried out with reference to step (1) to obtain the second contrast agent (ESCDA);

[0146] (3) Mix the first contrast agent in step (1), the second contrast agent in step (2) and physiological saline to obtain the sequential dual MRI signal switching contrast agent (ESCA-CA);

[0147] Among them, steps (1) and (2) have no sequential order.

[0148] The transmission electron micrograph of the sequential dual - MRI signal - switching contrast agent obtained in this example is as Figure 4 shown. It can be seen from Figure 4 that the morphology of the ESCA - CA contrast agent is relatively dispersed.

[0149] For the sequential dual - MRI signal - switching contrast agent obtained in this example, using Angiopep - 2 peptide as the targeting peptide to cross the BBB, and simultaneously utilizing the two key biomarkers of weak acid and high GSH in gliomas, it presents distinguishable MRI signal manifestations at different time scales, namely T1 - T2 - T1 signal changes, greatly improving the accuracy of glioma diagnosis.

[0150] Example 2

[0151] This example provides a sequential dual - MRI signal - switching contrast agent for targeted tumor localization and its preparation method. Except that the structural general formula of the first polymer is NH2 - S - S - mPEG1800 - N3; the structural general formula of the second polymer is Mal - mPEG9000 - CHO; the structural general formula of the amino - terminated polyethylene glycol is NH2 - mPEG1800 - NH2; the structural general formula of the third polymer is NH2 - S - S - mPEG2200 - DBCO; other conditions are the same as those in Example 1.

[0152] For the sequential dual - MRI signal - switching contrast agent obtained in this example, using Angiopep - 2 peptide as the targeting peptide to cross the BBB, and simultaneously utilizing the two key biomarkers of weak acid and high GSH in gliomas, it presents distinguishable MRI signal manifestations at different time scales, namely T1 - T2 - T1 signal changes, greatly improving the accuracy of glioma diagnosis.

[0153] Example 3

[0154] This example provides a sequential dual - MRI signal - switching contrast agent for targeted tumor localization and its preparation method. Except that the structural general formula of the first polymer is NH2 - S - S - mPEG2200 - N3; the structural general formula of the second polymer is Mal - mPEG12000 - CHO; the structural general formula of the amino - terminated polyethylene glycol is NH2 - mPEG2200 - NH2; the structural general formula of the third polymer is NH2 - S - S - mPEG1800 - DBCO; other conditions are the same as those in Example 1.

[0155] The sequential dual - time - point MRI signal - switching contrast agent obtained in this example uses Angiopep - 2 peptide as the targeting peptide for crossing the BBB. At the same time, by taking advantage of the two key biomarkers, weak acid and high GSH, in gliomas, it shows distinguishable MRI signal manifestations on different time scales, namely, T1 - T2 - T1 signal changes, greatly improving the accuracy of glioma diagnosis.

[0156] Comparative Example 1

[0157] This comparative example provides a pH - responsive MRI contrast agent. Except for replacing the "first - responsive click bifunctional layer" and "second - responsive click bifunctional layer" with "first click bifunctional layer" and "second click bifunctional layer" respectively, that is, replacing "NH2 - S - S - PEG2000 - N3" and "NH2 - S - S - PEG2000 - DBCO" with equal amounts of "NH2 - PEG2000 - N3" and "NH2 - PEG2000 - DBCO", other conditions are carried out with reference to Example 1 to obtain a pH - responsive MRI contrast agent (ECA - CA).

[0158] The transmission electron micrograph of the pH - responsive MRI contrast agent obtained in this comparative example is as Figure 5 shown. It can be seen from Figure 5 that the morphology of the ECA - CA contrast agent is relatively dispersed.

[0159] Comparative Example 2

[0160] This comparative example provides a GSH - responsive MRI contrast agent. Except for replacing the "second polymer Mal - mPEG10000 - CHO" with an equal amount of "Mal - mPEG10000 - NHS", other conditions are carried out with reference to Example 1 to obtain a GSH - responsive MRI contrast agent (ESA - CA).

[0161] The transmission electron micrograph of the GSH - responsive MRI contrast agent obtained in this comparative example is as Figure 6 shown. It can be seen from Figure 6 that the morphology of the ESA - CA contrast agent is relatively dispersed.

[0162] Comparative Example 3

[0163] This comparative example provides a non-responsive MRI contrast agent. Except for replacing the "first responsive click bifunctional layer" and the "second responsive click bifunctional layer" with the "first click bifunctional layer" and the "second click bifunctional layer" respectively, that is, replacing "NH2-S-S-PEG2000-N3" and "NH2-S-S-PEG2000-DBCO" with equal amounts of "NH2-PEG2000-N3" and "NH2-PEG2000-DBCO" respectively, and replacing the "second polymer Mal-mPEG10000-CHO" with an equal amount of "Mal-mPEG10000-NHS", other conditions are carried out with reference to Example 1 to obtain a non-responsive MRI contrast agent (EA-CA).

[0164] The transmission electron micrograph of the non-responsive MRI contrast agent obtained in this comparative example is as Figure 7 shown. It can be seen from Figure 7 that the morphology of the EA-CA contrast agent is relatively dispersed.

[0165] Comparative Examples 4 - 5

[0166] The above comparative examples provide CY5.5-labeled MRI contrast agents, namely CY5.5-labeled ESCA-CA and CY5.5-labeled ECA-CA respectively.

[0167] Except that after adding 0.5 mg of SH-Angiopep-2 in steps (1) and (2) for 2 h, then adding 1 mg / 1 mL and 10 μL of CY5.5-mPEG2000-SH and reacting for the fourth and eighth times at room temperature for 24 h, other conditions are carried out with reference to Example 1.

[0168] Response performance test

[0169] The longitudinal relaxation time and transverse relaxation time of Example 1 and Comparative Examples 1 - 3 of the present invention are tested on a 0.5 T MRI tester. The characterization operation method includes: respectively preparing the above samples with iron concentrations of 0.289 mM, 0.578 mM, 1.157 mM, 2.315 mM, and 4.630 mM, testing them on a 0.5 T MRI tester, and then performing linear fitting with the iron ion concentration (mM) as the abscissa and the reciprocals of the longitudinal relaxation time and transverse relaxation time as the ordinates to obtain the longitudinal relaxation rate (r1) and transverse relaxation rate (r2) of the above samples;

[0170] An in vitro mild acid simulation experiment was carried out by adjusting the pH of the whole system to 6.5 and incubating for 6 h, and then the reaction system was co-incubated in a 10 mM DTT solution at 37 °C for 6 h for an in vitro GSH simulation experiment. The contrast agents obtained in Example 1 and Comparative Examples 1-3 of the present invention were tested on a 0.5 T MRI tester. First, the in vitro simulation of mild acid was carried out, and then the changes in the longitudinal relaxation time and transverse relaxation time were measured twice under the in vitro simulated GSH environment. The characterization operation method is as shown above.

[0171] Among them, r2 / r1 is the gold standard for evaluating the type of contrast agent. Generally, when r2 / r1 is less than 5, it can be considered a typical T1 contrast agent; when r2 / r1 is greater than 8, it can be considered a typical T2 contrast agent. The fitting results of the above samples are as Figures 8 - 11 shown.

[0172] It can be Figure 8 seen from that the r1 and r2 of the ESCA-CA contrast agent (when not responsive) in Example 1 were 3.037 ± 0.179 mM -1 s -1 and 7.107 ± 0.184 mM -1 s -1 , and the r2 / r1 ratio was 2.352 ± 0.08. This indicates that the ESCA-CA contrast agent was initially a typical T1 contrast agent. When the contrast agent obtained in Example 1 was responsive to in vitro simulated mild acid, the r1 and r2 were 1.706 ± 0.030 mM -1 s -1 and 21.796 ± 0.468 mM -1 s -1 , and the r2 / r1 ratio was 12.773 ± 0.063. After that, when it was responsive to in vitro simulated GSH, the r1 and r2 became 3.907 ± 0.14 mM -1 s -1 and 9.648 ± 0.202 mM -1 s -1 , and the r2 / r1 ratio was 2.474 ± 0.051. This indicates that: under acidic conditions, the pH-responsive group of ESCA-CA dissociates, causing the shielding group to break, the nanoparticles to aggregate, and the contrast signal to change from the initial T1 to T2. Then, under the GSH simulation conditions, the disulfide bond is slowly reduced and broken, and the nanoparticles return to the dispersed state, and the contrast signal changes from T2 to T1.

[0173] It can be Figure 9 seen from that the r1 and r2 of the ECA-CA contrast agent (when not responsive) in Comparative Example 1 were 2.710 ± 0.039 mM -1 s -1and 4.973 ± 0.123 mM -1 s -1 The r2 / r1 ratio was 1.834 ± 0.026. This indicates that the contrast agent was initially a typical T1 contrast agent. After the contrast agent obtained in Comparative Example 1 was simulated for micro-acid response in vitro, r1 and r2 were 1.602 ± 0.045 mM -1 s -1 and 22.242 ± 0.253 mM -1 s -1 The r2 / r1 ratio was 13.913 ± 0.412. After the contrast agent was simulated for GSH response in vitro, r1 and r2 became 1.759 ± 0.055 mM -1 s -1 and 23.422 ± 0.181 mM -1 s -1 The r2 / r1 ratio was 13.361 ± 0.495. This indicates that: under acidic conditions, the pH-responsive group of ECA-CA dissociates, causing the shielding group to break, nanoparticle aggregation, and the contrast signal to change from the initial T1 to T2. After that, under the GSH simulation condition, since the contrast agent obtained in Comparative Example 1 does not contain a disulfide bond, it cannot respond to the GSH environment and remains a T2 contrast signal.

[0174] It can be seen from Figure 10 that for the ESA-CA contrast agent in Comparative Example 2, r1 and r2 were 2.494 ± 0.049 mM -1 s -1 and 5.598 ± 0.078 mM -1 s -1 The r2 / r1 ratio was 2.246 ± 0.040. This indicates that the contrast agent was initially a typical T1 contrast agent. After the contrast agent obtained in Comparative Example 2 was simulated for micro-acid response in vitro, r1 and r2 were 2.803 ± 0.067 mM -1 s -1 and 5.260 ± 0.073 mM -1 s -1 The r2 / r1 ratio was 1.878 ± 0.023. After the contrast agent was simulated for GSH response in vitro, r1 and r2 became 2.925 ± 0.035 mM -1 s -1 and 6.982 ± 0.379 mM -1 s -1, the r2 / r1 ratio is 2.387 ± 0.137. This indicates that: since ESA-CA does not contain a pH-responsive group and cannot shed the shielding group, it cannot exhibit any response in both simulated environments and always maintains the T1 contrast signal.

[0175] It can be seen from Figure 11 that in Comparative Example 3, the r1 and r2 of the EA-CA contrast agent are 2.868 ± 0.015 mM -1 s -1 and 5.141 ± 0.057 mM -1 s -1 , and the r2 / r1 ratio is 1.792 ± 0.021. This shows that the contrast agent is initially a typical T1 contrast agent. When the contrast agent obtained in Comparative Example 3 is simulated to respond to mild acidity in vitro, r1 and r2 are 3.229 ± 0.136 mM -1 s -1 and 6.098 ± 0.173 mM -1 s -1 , and the r2 / r1 ratio is 1.893 ± 0.045. After that, when it is simulated to respond to GSH in vitro, r1 and r2 become 3.193 ± 0.029 mM -1 s -1 and 5.400 ± 0.028 mM -1 s -1 , and the r2 / r1 ratio is 1.691 ± 0.007. This indicates that: since EA-CA does not contain a pH-responsive group and a GSH-responsive group and cannot shed the shielding group, it cannot exhibit any response in both simulated environments and always maintains the T1 contrast signal.

[0176] Blood-brain barrier permeability test

[0177] To investigate the blood-brain barrier permeability of the MRI contrast agent obtained in Example 1 of the present invention, the operation method includes:

[0178] First, establish a Transwell model, thaw and resuscitate Bend.3 cells until they reach the exponential growth phase. Prepare a 12-well cell culture plate, take Matrigel matrix glue and slowly melt it on ice, then evenly spread it on the bottom of the upper chamber of the Transwell insert, and incubate it under appropriate conditions for 1-2 h to allow it to solidify fully. Then, digest the Bend.3 cells in the exponential growth phase to prepare a cell suspension, and according to 5×10 4Inoculate the cells at a density of per chamber into the upper chamber of the Transwell chamber. At the same time, add the culture medium with the same medium as the upper chamber to the lower chamber to maintain the same osmotic pressure inside and outside the chamber. Thereafter, change the medium every 2-3 days and culture for 7-14 days. During the culture process, use a TEER instrument to detect the transendothelial resistance value inside and outside the Transwell chamber in real time. When the TEER value is greater than 100 Ω·cm 2 and remains stable, it indicates that Bend.3 cells have formed a tight monolayer at the bottom of the upper chamber, successfully simulating the BBB barrier. After the above-mentioned tight monolayer is formed, inoculate GL261 cells in the lower layer of the Transwell chamber to complete the establishment of an in vitro blood-brain barrier (BBB) model.

[0179] After the model is established, divide the Transwell model into three groups: experimental group, control group, and blank group, with two replicate wells in each group. After the model is successfully established, replace the upper layer cell culture medium in the experimental group Transwell chamber with fresh culture medium containing CY5.5-labeled ESCA-CA (Fe dose is 1 mM); replace the upper layer cell culture medium in the control group Transwell chamber with fresh culture medium containing CY5.5-labeled ESC-CA (Fe dose is 1 mM); replace the upper layer cell culture medium in the blank group Transwell chamber with fresh culture medium without the material. After culturing in a standard incubator for 4 h, discard the upper and lower layer culture solutions, rinse several times with PBS, and perform confocal and flow cytometry analysis on the cells in the upper and lower wells (as Figure 12 shown).

[0180] It can be seen that since ESCA-CA is surface-modified with Angiopep-2, it can not only penetrate endothelial cells but also further target glioma cells GL261.

[0181] MRI imaging experiment test

[0182] Explore the in vivo MRI imaging experiment of the MRI contrast agents obtained in Example 1 and Comparative Examples 1-3 of the present invention. The operation method includes:

[0183] Purchase a batch of C57 mice meeting SPF conditions (4-6 weeks old, 20 g in weight) for establishing an orthotopic glioma model. All animal experiments strictly follow the protocol approved by the Institute of Animal Protection, Chinese Academy of Sciences.

[0184] Twelve mice with brain glioma were divided into four groups: ESCA-CA, ECA-CA, ESA-CA and EA-CA, with three mice in each group. First, the tumor-bearing mice were anesthetized with 100 μL of 24% urethane solution, and the anesthetized mice were scanned with T2-weighted imaging using a 3 T magnetic resonance imaging scanner as a blank reference before injection of the material. Then, saline solutions containing ESCA-CA, ECA-CA, ESA-CA and EA-CA were injected into the three groups of tumor-bearing mice through the tail vein (150 μL, Fe dose was 0.1 mmol / kg), and T2-weighted MRI images at different time points after injection (2h, 4h, 6h, 8h, 10h, 12h) were continuously acquired using a 3 T magnetic resonance imaging scanner.

[0185] like Figure 13 As shown, in the comparative study of different groups, the experimental group and the control group showed different signal change characteristics. In the study of the experimental group ESCA-CA (Example 1), compared with the blank group before injection, its imaging at the glioma site showed a trend of brightening-darkening-brightening with the dynamic change of time. 2 hours after injection, the brightness of the tumor site began to increase significantly, and the signal intensity ratio reached 107.8 ± 0.9%. This phenomenon is mainly attributed to the efficient transport mechanism mediated by Angiopep-2, which enables the contrast agent to quickly cross the BBB and be accurately delivered to the glioma site. At the same time, since it takes a certain amount of time for the responsive cleavage of the benzoic acid imide bond in an acidic environment, ESCA-CA exhibits typical T1 contrast agent characteristics during this process; since the 2nd hour after injection, the brightness of the tumor site showed a trend of gradual weakening, and by the 8th hour, the signal intensity dropped to a minimum of only 90 ± 0.6%. Based on this phenomenon, we speculate that at this time ESCA-CA has been fully exposed to the acidic microenvironment of glioma, and the benzoic acid imide bond gradually breaks, causing the click groups inside ESCA-CA to be exposed. These exposed click groups trigger the aggregation of ESIONPs, which then show typical T2 contrast agent characteristics. From 8 h, the brightness of the tumor site began to gradually recover. Until the end of the detection, its signal intensity rose to 113.7 ± 0.8%. This is because the aggregated nanoclusters are further internalized and retained in the cytoplasm by virtue of their larger particle size. The high concentration of GSH in the cytoplasm undergoes a reduction reaction with the disulfide bonds between the nanoclusters, causing the nanoparticles to dissociate, and the system finally presents typical T1 contrast agent characteristics. This dynamic change process not only intuitively demonstrates the unique response mechanism of ESCA-CA in a complex physiological environment, but also accurately determines the outline of glioma for us, enabling accurate diagnosis of it.

[0186] In the study of the pH-responsive only ECA-CA group (Comparative Example 1), at 2 h after injection, the brightness at the tumor site was significantly enhanced due to the just-enriched nanomaterials. However, over time, the brightness at the tumor site gradually decreased and finally dropped to 84.3 ± 0.8%. This is because ECA-CA responds and crosslinks in an acidic environment to form a clustered structure, but due to the lack of disulfide bonds, the dissociation process cannot be achieved. This non-dissociable state causes the tumor site to continuously show a dark signal, limiting the clear resolution of gliomas. On the contrary, in the GSH-responsive only ESA-CA group (Comparative Example 2), at 2 h after injection, the tumor site also became brighter due to the enrichment of nanomaterials. But as time progresses, the nanoparticles are gradually metabolized and the brightness gradually decreases, and finally it still remains at 114.1 ± 0.16%, always showing the characteristics of a T1 contrast agent. And the non-responsive EA-CA group (Comparative Example 3) also always exists as a T1 contrast agent. The experimental results of these three control groups are in sharp contrast to the rich and effective multi-stage response presented by ESCA-CA, highlighting the key role of the synergistic effect of benzoic imine bonds and disulfide bonds in realizing the precise multiple response function of the material to the complex tumor microenvironment.

[0187] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A sequential dual - time MRI signal - switching contrast agent for targeted tumor localization, characterized in that, The sequential dual - MRI signal - switching contrast agent is a mixture of a first contrast agent and a second contrast agent; The first contrast agent includes, from the inside to the outside, nanoparticles, a first response click bifunctional layer, a first response shielding bifunctional layer, and a first targeting layer in sequence; The second contrast agent includes, from the inside to the outside, nanoparticles, a second response click bifunctional layer, a second response shielding bifunctional layer, and a second targeting layer in sequence; The nanoparticles are iron(III) oxide with a modification group connected to the surface; the modification group includes pentafluorophenyl ester; the average particle size of the iron(III) oxide is < 4 nm; The preparation raw materials of the first response click bifunctional layer include a first polymer containing a GSH - responsive group and a first click group; the structural general formula of the first polymer is NH2 - S - S - mPEGa - N3; where a is 1800 - 2200; the amino group in the first polymer forms an amide bond with the modification group in the nanoparticles for connection; The preparation raw materials of the second response click bifunctional layer include a third polymer containing a GSH - responsive group and a second click group; the structural general formula of the third polymer is NH2 - S - S - mPEGa - DBCO; where a is 1800 - 2200; the amino group in the third polymer forms an amide bond with the modification group in the nanoparticles for connection; Both the first response shielding bifunctional layer and the second response shielding bifunctional layer contain a pH - responsive group and a shielding group; the preparation raw materials of both the first response shielding bifunctional layer and the second response shielding bifunctional layer include amino - terminated polyethylene glycol and a second polymer with the structural general formula shown in formula (I); Formula I) where n is 9000 - 12000; The structural general formula of the amino - terminated polyethylene glycol is NH2 - mPEGa - NH2; where a is 1800 - 2200; one amino group at one end of the amino - terminated polyethylene glycol forms an amide bond with the modification group in the nanoparticles for connection, and the other amino group at the other end forms a benzoic acid imine bond with the benzaldehyde group in the second polymer for connection; The preparation raw materials of both the first targeting layer and the second targeting layer include a targeting peptide, and the targeting peptide includes SH - Angiopep - 2; the maleimide group in the second polymer forms a thioether bond with the sulfhydryl group in the targeting peptide for connection.

2. The sequential dual - MRI signal - switching contrast agent according to claim 1, characterized in that, The GSH - responsive group is a - S - S - functional group; The first click group is an N3 functional group; The second click group is a DBCO functional group; The pH - responsive group is a benzoic acid imine functional group; The shielding group includes an mPEGn functional group, where n is 9000 - 12000.

3. The sequential dual - time MRI signal - switching contrast agent according to claim 1, characterized in that, The first polymer has an amino group at the end; The third polymer has an amino group at the end; 4. The sequential dual - time MRI signal - switching contrast agent according to claim 1, wherein, The molar ratio of the first contrast agent to the second contrast agent is 1:(1 - 1.2).

5. A method for preparing a sequential dual - time MRI signal - switching contrast agent according to any one of claims 1 - 4, characterized in that, The preparation method includes the following steps: (1) Conduct a first response click bifunctional layer connection reaction, a first response shielding bifunctional layer connection reaction, and a first targeting layer connection reaction on the nanoparticles in sequence to obtain the first contrast agent; (2) The nanoparticles are successively subjected to a second responsive click bifunctional layer linking reaction, a second responsive shielding bifunctional layer linking reaction, and a second targeting layer linking reaction to obtain a second contrast agent; (3) The first contrast agent obtained in step (1) and the second contrast agent obtained in step (2) are mixed to obtain the sequential dual-MRI signal switching contrast agent; Steps (1) and (2) have no sequential order.

6. The preparation method according to claim 5, characterized in that, The method for the first responsive click bifunctional layer linking reaction in step (1) includes: mixing nanoparticles, a first polymer containing a GSH-responsive group and a first click group, and a first solvent, and performing a first reaction to obtain a first intermediate; The method for the first responsive shielding bifunctional layer linking reaction in step (1) includes: mixing the first intermediate, amino-terminated polyethylene glycol, and a first solvent, performing a second reaction, and then adding a second polymer thereto to perform a third reaction to obtain a second intermediate; The method for the first targeting layer linking reaction in step (1) includes: mixing the second intermediate and a targeting peptide, and performing a fourth reaction to obtain a first contrast agent; The method for the second responsive click bifunctional layer linking reaction in step (2) includes: mixing nanoparticles, a third polymer containing a GSH-responsive group and a second click group, and a second solvent, and performing a fifth reaction to obtain a third intermediate; The method for the second responsive shielding bifunctional layer linking reaction in step (2) includes: mixing the third intermediate, amino-terminated polyethylene glycol, and a second solvent, performing a sixth reaction, and then adding a second polymer thereto to perform a seventh reaction to obtain a fourth intermediate; The method for the second targeting layer linking reaction in step (2) includes: mixing the fourth intermediate and a targeting peptide, and performing an eighth reaction to obtain a second contrast agent.

7. The preparation method according to claim 6, characterized in that, The molar ratio of the first polymer to amino-terminated polyethylene glycol is 1:(6 - 15); The molar ratio of the second polymer to amino-terminated polyethylene glycol ≥ 2:1; The molar ratio of the third polymer to amino-terminated polyethylene glycol is 1:(6 - 15); The molar ratio of the targeting peptide to the second polymer is 1:(20 - 40); Both the first solvent and the second solvent include dimethyl sulfoxide; The temperatures of the first reaction, the second reaction, the third reaction, the fourth reaction, the fifth reaction, the sixth reaction, the seventh reaction, and the eighth reaction are all 20 - 30 °C; The times of the first reaction, the second reaction, the third reaction, the fourth reaction, the fifth reaction, the sixth reaction, the seventh reaction, and the eighth reaction are all ≥ 20 h; After the second reaction and the sixth reaction, first dialysis and pH adjustment to 8 - 9 are successively carried out; After the fourth reaction and the eighth reaction, precipitation treatment and second dialysis are successively carried out.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes a drug, a pharmaceutically acceptable pharmaceutical carrier, and the sequential dual-MRI signal switching contrast agent according to any one of claims 1 - 4.

9. Use of a sequential dual - time MRI signal - switching contrast agent according to any one of claims 1 - 4, characterized in that, The sequential dual-MRI signal switching contrast agent is used in the preparation of a tumor magnetic resonance imaging diagnostic or detection reagent.

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