Sequential double MRI (Magnetic Resonance Imaging) signal switching type contrast agent for targeted positioning of tumor as well as preparation method and application of contrast agent

Abstract

The invention provides a sequential double MRI (Magnetic Resonance Imaging) signal switching type contrast agent for targeted positioning of tumors as well as a preparation method and application thereof, and belongs to the technical field of MRI diagnosis contrast agents, Angiopep-2 peptide is taken as a targeted peptide crossing BBB, and two key biomarkers of weak acid and high GSH in brain glioma are simultaneously utilized, so that distinguishable MRI signal expression, namely T1-T2-T1 signal change, is presented, and the contrast agent can be used for diagnosis of brain glioma. According to the method, the endogenous stimulation interference can be effectively avoided, the accuracy and reliability of brain glioma diagnosis are greatly improved, accurate description of brain glioma is expected to be realized, and a powerful technical support is provided for early diagnosis and accurate treatment of brain glioma.

Description

Technical Field

[0001] The present invention belongs to the technical field of MRI diagnostic contrast agents, and relates to a sequential dual - time MRI signal - switching contrast agent for targeted tumor localization, a preparation method thereof, and an application thereof. 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 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 grow infiltratively along white matter fiber bundles, conventional T 1 and T 2 - weighted enhanced MRI is difficult to accurately distinguish the tumor parenchyma from the surrounding edema zone, resulting in difficult precise planning of the surgical resection range; Second, the heterogeneity within the tumor (such as necrosis, angiogenesis, and cell density differences) also makes traditional MRI difficult 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 mainly focus on two major directions: "active targeted transport and penetration" and "local barrier regulation". The active targeted transport and penetration strategy mainly relies on the surface modification of the nanoparticle system with targeted ligands, which can specifically recognize the receptors overexpressed on the surface of BBB endothelial cells or glioma cells, and efficiently achieve cross - BBB transport through receptor - mediated endocytosis. 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 and 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 and penetration strategy has been more widely applied in the field of brain delivery.

[0005] Currently, in the practical application of the active targeting transport penetration strategy, Angiopep-2 peptide plays an important role. As a type of Kunitz-type protease inhibitor, Angiopep-2 peptide has unique targeting recognition ability and can specifically recognize the highly expressed receptor LRP-1 on the surface of BBB endothelial cells and glioma cells. This property enables 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) switch 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 are 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, etc. And single-response probes usually lack absolute specificity for pathological tissues. Therefore, further improving the specificity of the contrast agent is an urgent problem to be solved by 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. Using Angiopep-2 peptide as a targeting peptide to cross the BBB, and at the same time utilizing two key biomarkers, weak acid and high GSH, in glioblastoma, distinguishable MRI signal performances are presented 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 to the outside, 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 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 to cross the BBB, and simultaneously utilizes two key biomarkers of gliomas, weak acidity and high GSH, to present distinguishable MRI signal manifestations on 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. The Angiopep-2 peptide modified on the surface of the first contrast agent and the second contrast agent can specifically recognize the highly expressed receptor LRP-1 on the surface of BBB endothelial cells and glioma cells, so that it can not only cross the BBB, but also further target 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 is transformed 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 is transformed 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, and it is expected to achieve accurate depiction 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 biotoxicity, 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 pentafluorophenyl 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 N 3 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 NH 2 -S-S-mPEGa-N 3 ; 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 modification 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 NH 2 -mPEGa-NH 2 ; 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 modification 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.

[0037] It should be noted that under mildly 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 ESIONPs. Then, under the action of click chemistry, ESIONPs aggregate, thereby achieving the first signal switching in the double 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 NH 2 -S-S-mPEGa-DBCO; wherein, a is 1800 - 2200.

[0040] It should be noted that in the environment of high-concentration GSH in glioma cells, the GSH-responsive group is slowly reduced and broken, causing the aggregated ESIONPs to dissociate, thereby achieving the second signal switching in the double signal switching, that is, the transformation 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 modification group in the nanoparticle for connection.

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

[0043] The maleimide group in the second polymer forms 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), and 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 type 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 type contrast agent;

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

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

[0051] In the present invention, the preparation method of the nanoparticles in step (1) and step (2) includes: mixing hydrophobic Fe 2 O 3 nanoparticles, modification groups, and a solvent, and performing a ligand exchange reaction to obtain modified Fe 2 O 3 nanoparticles.

[0052] In the present invention, the hydrophobic Fe 2 O 3 nanoparticles include fatty acid - modified Fe 2 O 3 nanoparticles (ESIONPs - OA).

[0053] In the present invention, the hydrophobic Fe 2O 3 The nanoparticles can be prepared by conventional methods of the existing technology and will not be specifically limited herein. The hydrophobic Fe 2 O 3 The preparation method of the nanoparticles specifically includes: mixing iron salt, fatty acid salt and a mixed solvent, and through vacuum treatment and thermal decomposition reaction, the hydrophobic Fe 2 O 3 nanoparticles are obtained.

[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 pentafluorophenol ester (DOPAC - PFP). The preparation method of the pentafluorophenol ester specifically includes: dissolving 3,4 - dihydroxybenzoic 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 at room temperature under nitrogen protection for 20 - 30 h. After the reaction ends, it is purified by silica gel column chromatography to obtain pentafluorophenol 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 Fe 2 O 3 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 hydrophobic Fe 2 O 3 nanoparticles in a first solvent, then adding a modifying group thereto, and performing a ligand exchange reaction in a protective atmosphere. After the reaction is completed, it is concentrated by rotary evaporation and dialyzed for several days to remove the excess modifying groups 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 NH 2 -mPEGa-NH 2 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), and 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 sequence of the first polymer / third polymer and the amino-terminated polyethylene glycol is relatively important for the contrast agent. If the amino-terminated polyethylene glycol is added 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, and 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), and 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), and 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, and for example, it can be 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C or 28 °C, etc.

[0077] Preferably, the reaction 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, and for example, they 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 successively 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 is ≥ 30 KDa. Excessive mPEG is removed through the first dialysis.

[0080] In the present invention, the pH adjustment means adjusting the pH value of the solution to 8 - 9, and 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 successively carried out.

[0082] In the present invention, the precipitation treatment is to precipitate once in ice - ether, and the collected precipitate is dissolved with 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 is ≥ 30 KDa. Excess molecules are removed through 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 - time MRI signal - switching 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 - time MRI signal - switching contrast agent described in the first aspect, and the sequential dual - time MRI signal - switching contrast agent is used for preparing tumor magnetic resonance imaging diagnostic or detection reagents.

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

[0088] The numerical ranges described in the present invention include not only the above-listed point values, but also any point values between the above numerical ranges that are 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 described ranges.

[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 T 1 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, and at this time, the first contrast signal transformation is achieved, that is, from T 1 to T 2 ; 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 crosslinked 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 T 2 to T 1 ;

[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 the two key biomarkers of glioma weak acid and high GSH to present distinguishable MRI signal performances 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 gliomas, providing strong technical support for the early diagnosis and precise treatment of gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0093] Figure 2 is the transmission electron micrograph 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 4It is the transmission electron microscopy image of the ESCA-CA contrast agent in Example 1 of the present invention.

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

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

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

[0099] Figure 8 It is the comparison chart of the longitudinal relaxation rate (r 1 ), and the transverse relaxation rate (r 2 ) of the contrast agent obtained in Example 1 of the present invention under non-responsive and different response conditions.

[0100] Figure 9 It is the comparison chart of the longitudinal relaxation rate (r 1 ), and the transverse relaxation rate (r 2 ) of the contrast agent obtained in Comparative Example 1 of the present invention under non-responsive and different response conditions.

[0101] Figure 10 It is the comparison chart of the longitudinal relaxation rate (r 1 ), and the transverse relaxation rate (r 2 ) of the contrast agent obtained in Comparative Example 2 of the present invention under non-responsive and different response conditions.

[0102] Figure 11 It is the comparison chart of the longitudinal relaxation rate (r 1 ), and the transverse relaxation rate (r 2 ) of the contrast agent obtained in Comparative Example 3 of the present invention under non-responsive and different response conditions.

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

[0104] Figure 13This is the in vivo T1- and T2-weighted MRI images of the contrast agents in the orthotopic glioma mice in Example 1 and Comparative Examples 1-3 of the present invention. Detailed implementation mode

[0105] The technical solution of the present invention will be further described below through specific implementation modes. 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 specific implementation mode of the present invention provides 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 a nanoparticle, 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 independently connected to the nanoparticle, and the first responsive shielding bifunctional layer is connected to the first targeting layer;

[0108] The second contrast agent sequentially includes a nanoparticle, 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 independently connected to the nanoparticle, 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 N 3 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 nanoparticle is iron oxide with a modified group connected to the surface; the modified group includes pentafluorophenol ester; the average particle size of the iron 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 nanoparticle in sequence to obtain the first contrast agent;

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

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

[0116] Steps (1) and (2) are not in a specific 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, NH 2 -S-S-mPEGa-N 3 、NH 2 -mPEGa-NH 2 、NH 2 -S-S-mPEGa-DBCO and Mal-mPEGn-OH are all purchased from Xi'an Ruixi Biotechnology Co., Ltd.; SH-Angiopep-2 (sequence TFFYGGSRGKRNNFKTEEYC) is purchased from Suzhou Modifu Biotechnology Co., Ltd. In the following examples and comparative examples, the hydrophobic Fe 2 O 3 nanoparticles, pentafluorophenyl esters, nanoparticles with pentafluorophenyl esters connected to the surface, PEG10000-CHO, and the second polymer (Mal-mPEG10000-CHO) are all prepared by the following preparation methods.

[0119] Among them, the hydrophobic Fe 2 O 3 nanoparticles are 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, then reflux and stir at a temperature of 70 °C for 4 h. After the reaction, cool to room temperature, wash and rotary evaporate, and then dry at a temperature of 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 described in step (1) (1.8 g, 2 mmol) in diphenyl ether (10 g, 58 mmol), then conduct a vacuum treatment at 70 °C for 2 h, and then under nitrogen protection, heat 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 Fe 2 O 3 nanoparticles (ESIONPs-OA).

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

[0123] Among them, the pentafluorophenol ester is prepared by the following method:

[0124] Dissolve 3,4-dihydroxy phenylacetic acid (3.6 g, 20 mmol) and pentafluorophenol (4.4 g, 24 mmol) in 100 mL of anhydrous 1,4-dioxane, and drop N,N'-dicyclohexylcarbodiimide (5 g, 24 mmol) dissolved in 20 mL of anhydrous 1,4-dioxane into the above solution. React at room temperature for 24 h under argon protection. After filtration and rotary evaporation, use a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 3:1 as the eluent for column purification 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 to it, and then conduct a ligand exchange reaction at 50 °C for 72 h under nitrogen protection. After the reaction, perform rotary evaporation, and then dialyze with 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, and 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] Mal-mPEG10000-OH (2 g, 0.2 mmol) and p-formylbenzoic acid (240.21 mg, 1.6 mmol) were dissolved in 40 mL of ultradry dichloromethane solution. Then, DCC (330 mg, 1.6 mmol) and DMAP (24 mg, 0.2 mmol) were added thereto and stirred to mix evenly. Subsequently, the reaction was stirred at room temperature for 48 h under argon protection. After the reaction, rotary evaporation was performed. Then, the obtained solid was dissolved in a small amount of deionized water and then filtered to remove the insoluble substances therein. The obtained aqueous solution was extracted with dichloromethane solution (3×15 mL). Anhydrous magnesium sulfate was added to the extracted organic phase and dried at room temperature for 24 h. Finally, the solution was filtered and concentrated. The concentrated solution was precipitated three times in ice-cold anhydrous ether, and the precipitate was collected and dried under vacuum to obtain the second polymer (Mal-mPEG10000-CHO).

[0130] The 1H NMR spectrum of Mal-mPEG10000-CHO is as Figure 3 shown, which corroborates the successful synthesis of Mal-mPEG10000-CHO.

[0131] Example 1

[0132] This example provides a sequential dual MRI signal switching contrast agent for targeted tumor localization 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 a nanoparticle, 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 respectively independently connected to the nanoparticle, and the first responsive shielding bifunctional layer is connected to the first targeting layer;

[0134] The second contrast agent sequentially includes a nanoparticle, 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 respectively independently connected to the nanoparticle, and the second responsive shielding bifunctional layer is connected to the second targeting layer;

[0135] The nanoparticle is Fe 2 O 3 connected with pentafluorophenol ester on the surface; the average particle size of the nanoparticle 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 NH 2-S-S-mPEG2000-N 3 ; An amide bond is formed between the amino group in the first polymer and the pentafluorophenol ester group in the nanoparticle for connection;

[0137] The preparation materials of the first response shielding bifunctional layer and the second response 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] wherein, n is 10000;

[0140] The structural general formula of the amino-terminated polyethylene glycol is NH 2 -mPEG2000-NH 2 ; An amide bond is formed between the amino group at one end of the amino-terminated polyethylene glycol and the pentafluorophenol ester group in the nanoparticle for connection, and a benzoic imine bond is formed between the amino group at the other end and the benzaldehyde group in the second polymer for connection;

[0141] The preparation materials of the second response click bifunctional layer include a third polymer containing a GSH response group and a second click group; the structural general formula of the third polymer is NH 2 -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 preparation materials of 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 thiol 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 DMSO, then dropwise add 0.55 mg NH 2 -S-S-mPEG2000-N 3 dissolved in 5 mL DMSO to it, then add 10 μL DIPEA to it, and then react for 24 h at room temperature for the first time; then dissolve 5 mg NH 2 -mPEG2000-NH 2 in 10 mL DMSO, and then dropwise add the above first reaction product to NH 2 -mPEG2000-NH 2In the solution, the second reaction was carried out at room temperature for 24 h. The second reaction product was transferred into a dialysis bag with a molecular weight cut-off of 35 kDa and dialyzed in deionized water for the first time for 1 day, and then transferred into a flask. The pH of the solution was adjusted to 8.5. Subsequently, 60 mg of Mal-mPEG10000-CHO was added to the above solution, and the third reaction was carried out at room temperature for 36 h. Then, 0.5 mg of SH-Angiopep-2 was added to the third reaction product, and the fourth reaction was carried out at room temperature for 24 h. Finally, the fourth reaction product was dropped into ice-cold anhydrous ether for precipitation treatment. The obtained precipitate was dissolved in a small amount of deionized water, and then the obtained solution was transferred into a dialysis bag with a molecular weight cut-off of 35 kDa and dialyzed in deionized water for the second time for 2 days to obtain the first contrast agent (ESCNA).

[0145] (2) Step (2) was carried out with reference to step (1), except that "0.55 mg NH 2 -S-S-mPEG2000-N 3 " was replaced with "0.75 mg NH 2 -S-S-mPEG2000-DBCO", and the rest were carried out with reference to step (1) to obtain the second contrast agent (ESCDA);

[0146] (3) The first contrast agent in step (1), the second contrast agent in step (2) and normal saline were mixed 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 for crossing the BBB and taking advantage of the two key biomarkers of weak acid and high GSH in gliomas, distinguishable MRI signal manifestations are presented at different time scales, that is, T 1 -T 2 -T 1 signal changes, which greatly improve 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 NH 2-S-S-mPEG1800-N 3 ; The structural general formula of the second polymer is Mal-mPEG9000-CHO; The structural general formula of the amino-terminated polyethylene glycol is NH 2 -mPEG1800-NH 2 ; The structural general formula of the third polymer is NH 2 -S-S-mPEG2200-DBCO; Other conditions are the same as those in Example 1.

[0152] The sequential dual - time - scale MRI signal - switching contrast agent obtained in this example uses Angiopep - 2 peptide as the targeting peptide for crossing the BBB, and simultaneously utilizes two key biomarkers, weak acid and high GSH, in gliomas, presenting distinguishable MRI signal manifestations at different time scales, namely T 1 -T 2 -T 1 signal changes, greatly improving the accuracy of glioma diagnosis.

[0153] Example 3

[0154] This example provides a sequential dual - time - scale MRI signal - switching contrast agent for targeted tumor localization and its preparation method. Except that the structural general formula of the first polymer is NH 2 -S-S-mPEG2200-N 3 ; The structural general formula of the second polymer is Mal-mPEG12000-CHO; The structural general formula of the amino-terminated polyethylene glycol is NH 2 -mPEG2200-NH 2 ; The structural general formula of the third polymer is NH 2 -S-S-mPEG1800-DBCO; Other conditions are the same as those in Example 1.

[0155] The sequential dual - time - scale MRI signal - switching contrast agent obtained in this example uses Angiopep - 2 peptide as the targeting peptide for crossing the BBB, and simultaneously utilizes two key biomarkers, weak acid and high GSH, in gliomas, presenting distinguishable MRI signal manifestations at different time scales, namely T 1 -T 2 -T 1 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 that the "first responsive click bifunctional layer" and the "second responsive click bifunctional layer" are respectively replaced by the "first click bifunctional layer" and the "second click bifunctional layer", that is, "NH2 -S-S-PEG2000-N 3 ” and “NH 2 -S-S-PEG2000-DBCO” are respectively replaced with an equal amount of “NH 2 -PEG2000-N 3 ” and “NH 2 -PEG2000-DBCO”. Other conditions are all referred to Example 1 to obtain a pH-responsive MRI contrast agent (ECA-CA).

[0158] The transmission electron microscopy image 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 that “the second polymer Mal-mPEG10000-CHO” is replaced with an equal amount of “Mal-mPEG10000-NHS”, other conditions are all referred to Example 1 to obtain a GSH-responsive MRI contrast agent (ESA-CA).

[0161] The transmission electron microscopy image 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 that “the first responsive click bifunctional layer” and “the second responsive click bifunctional layer” are respectively replaced with “the first click bifunctional layer” and “the second click bifunctional layer”, that is, “NH 2 -S-S-PEG2000-N 3 ” and “NH 2 -S-S-PEG2000-DBCO” are respectively replaced with an equal amount of “NH 2 -PEG2000-N 3 ” and “NH 2 -PEG2000-DBCO” and “the second polymer Mal-mPEG10000-CHO” is replaced with an equal amount of “Mal-mPEG10000-NHS”, other conditions are all referred to Example 1 to obtain a non-responsive MRI contrast agent (EA-CA).

[0164] The transmission electron microscopy image of the non-responsive MRI contrast agent obtained in this comparative example is as Figure 7 shown. It can be seen fromFigure 7 It can be seen 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) and reacting for 2 h, 1 mg / 1 mL and 10 μL of CY5.5-mPEG2000-SH were further added thereto, and the fourth reaction and the eighth reaction were carried out at room temperature for 24 h, other conditions were carried out with reference to Example 1.

[0168] Response performance test

[0169] The longitudinal relaxation time and transverse relaxation time of Example 1 of the present invention and Comparative Examples 1-3 were 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, and after testing on a 0.5 T MRI tester, taking the iron ion concentration (mM) as the abscissa and the reciprocals of the longitudinal relaxation time and transverse relaxation time as the ordinates for linear fitting to obtain the longitudinal relaxation rate (r 1 ) and transverse relaxation rate (r 2 ) 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 of the present invention and Comparative Examples 1-3 were tested on a 0.5 T MRI tester for the changes in the longitudinal relaxation time and transverse relaxation time in the in vitro simulated mild acid and then in the in vitro simulated GSH environment. The characterization operation method is as shown above.

[0171] Among them, r 2 / r 1 is the gold standard for evaluating the type of contrast agent. Generally, when r 2 / r 1 is less than 5, it can be considered a typical T1 contrast agent; when r 2 / r 1 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] From Figure 8It can be seen that the r of the ESCA-CA contrast agent (when not responsive) in Example 1 1 and r 2 are 3.037 ± 0.179 mM -1 s -1 and 7.107 ± 0.184 mM -1 s -1 respectively, and the r 2 / r 1 ratio is 2.352 ± 0.08. This indicates that the ESCA-CA contrast agent is initially a typical T1 contrast agent. When the contrast agent obtained in Example 1 is simulated for microacid response in vitro, r 1 and r 2 are 1.706 ± 0.030 mM -1 s -1 and 21.796 ± 0.468 mM -1 s -1 respectively, and the r 2 / r 1 ratio is 12.773 ± 0.063. After that, when it is simulated for GSH response in vitro, r 1 and r 2 become 3.907 ± 0.14 mM -1 s -1 and 9.648 ± 0.202 mM -1 s -1 respectively, and the r 2 / r 1 ratio is 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. After that, under the GSH simulation condition, 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 again.

[0173] It can be seen from Figure 9 that the r of the ECA-CA contrast agent (when not responsive) in Comparative Example 1 1 and r 2 are 2.710 ± 0.039 mM -1 s -1 and 4.973 ± 0.123 mM -1 s -1 respectively, and the r 2 / r 1 ratio is 1.834 ± 0.026. This indicates that the contrast agent is initially a typical T1 contrast agent. When the contrast agent obtained in Comparative Example 1 is simulated for microacid response in vitro, r 1 and r 2were 1.602 ± 0.045 mM respectively -1 s -1 and 22.242 ± 0.253 mM -1 s -1 , r 2 / r 1 The ratio was 13.913 ± 0.412. After simulating the GSH response in vitro, r 1 and r 2 became 1.759 ± 0.055 mM -1 s -1 and 23.422 ± 0.181 mM -1 s -1 , r 2 / r 1 The 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 simulating the GSH condition in vitro, since the contrast agent obtained in Comparative Example 1 does not contain disulfide bonds, 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, r 1 and r 2 were 2.494 ± 0.049 mM -1 s -1 and 5.598 ± 0.078 mM -1 s -1 , r 2 / r 1 The ratio was 2.246 ± 0.040. This indicates that the contrast agent was initially a typical T1 contrast agent. When the contrast agent obtained in Comparative Example 2 was simulated to respond to weak acidity in vitro, r 1 and r 2 were 2.803 ±0.067 mM -1 s -1 and 5.260 ± 0.073 mM -1 s -1 , r 2 / r 1 The ratio was 1.878 ± 0.023. After simulating the GSH response in vitro, r 1 and r 2 became 2.925 ± 0.035 mM -1 s -1 and 6.982 ± 0.379 mM -1 s -1 , r2 / r 1 The 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] From Figure 11 it can be seen that for the EA-CA contrast agent in Comparative Example 3, the r 1 and r 2 are 2.868 ± 0.015 mM -1 s -1 and 5.141 ± 0.057 mM -1 s -1 respectively, and the r 2 / r 1 ratio is 1.792 ± 0.021. This indicates that this 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, the r 1 and r 2 are 3.229 ±0.136 mM -1 s -1 and 6.098 ± 0.173 mM -1 s -1 respectively, and the r 2 / r 1 ratio is 1.893 ± 0.045. After that, when it is simulated to respond to GSH in vitro, the r 1 and r 2 become 3.193 ± 0.029 mM -1 s -1 and 5.400 ± 0.028 mM -1 s -1 respectively, and the r 2 / r 1 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] Trans-blood-brain barrier performance test

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

[0178] First, establish a Transwell model. Thaw and resuscitate Bend.3 cells and wait for them to proliferate to the exponential growth phase. Prepare a 12-well cell culture plate. Take Matrigel matrix glue and slowly melt it on ice, then evenly coat it on the bottom of the upper chamber of the Transwell insert. Incubate it under appropriate conditions for 1 - 2 h to allow it to solidify fully. Next, digest the Bend.3 cells in the exponential growth phase to prepare a cell suspension, and inoculate it into the upper chamber of the Transwell insert at a density of 5×10 4 cells per 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 continue culturing for 7 - 14 days. During the culture process, use a TEER instrument to detect the transendothelial electrical resistance value inside and outside the Transwell insert in real time. When the TEER value is greater than 100 Ω·cm 2 and remains stable, it indicates that the Bend.3 cells have formed a tight monolayer at the bottom of the upper chamber, successfully simulating the BBB barrier. After the formation of the above-mentioned tight monolayer, inoculate GL261 cells in the lower layer of the Transwell insert to complete the establishment of the in vitro blood-brain barrier (BBB) model.

[0179] After the model is established, divide the Transwell model into three groups: the experimental group, the control group, and the blank group, with two replicates in each group. After the model is successfully established, replace the upper-layer cell culture medium in the Transwell insert of the experimental group with fresh culture medium containing CY5.5-labeled ESCA-CA (Fe dose is 1 mM); replace the upper-layer cell culture medium in the Transwell insert of the control group with fresh culture medium containing CY5.5-labeled ESC-CA (Fe dose is 1 mM); replace the upper-layer cell culture medium in the Transwell insert of the blank group with fresh culture medium without the material. After culturing in a standard incubator for 4 h, discard the culture media in the upper and lower layers, 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] Thus, it can be seen that because Angiopep-2 is surface-modified on ESCA-CA, ESCA-CA 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 experiments 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 the SPF criteria (aged 4 - 6 weeks, weighing 20 g) for establishing an orthotopic glioma model. All animal experiments strictly followed the protocol approved by the Institute of Animal Protection, Chinese Academy of Sciences.

[0184] Divide 12 glioma-bearing mice into 4 groups: ESCA-CA, ECA-CA, ESA-CA, and EA-CA, with 3 mice in each group. First, anesthetize the tumor-bearing mice with 100 μL of 24% urethane solution. After anesthesia, use a 3 T magnetic resonance imaging scanner to perform T 2 -weighted imaging scans on the mice as a blank reference before injecting the material. Then, inject saline solutions containing ESCA-CA, ECA-CA, ESA-CA, and EA-CA into 3 groups of tumor-bearing mice via the tail vein (150 μL, with an Fe dose of 0.1 mmol / kg for all). Continuously obtain T 2 -weighted MRI images at different time points after injection (2 h, 4 h, 6 h, 8 h, 10 h, 12 h) using a 3 T magnetic resonance imaging scanner.

[0185] As Figure 13 shown, in the comparative studies 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, with the dynamic change of time, its imaging at the glioma site showed a trend of getting brighter - darker - brighter. At 2 h after injection, the brightness at 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 time for the benzoic acid imine bond to undergo responsive cleavage in an acidic environment, during this process, ESCA-CA exhibits typical T 1 -contrast agent characteristics; starting from 2 h after injection, the brightness at the tumor site showed a gradually decreasing trend, and by 8 h, the signal intensity dropped to the lowest, 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 imine bond gradually breaks, resulting in the exposure of the click groups inside ESCA-CA. These exposed click groups trigger the aggregation of ESIONPs, and then exhibit typical T 2Contrast agent characteristics. Starting from 8 h, the brightness at the tumor site began to gradually recover. By the end of the detection, its signal intensity increased to 113.7 ± 0.8%. This is because the nanoclusters formed by aggregation, with their relatively large particle sizes, further internalize and remain in the cytoplasm. The reduction reaction occurs between the high-concentration GSH in the cytoplasm and the disulfide bonds of the nanoclusters, prompting the dissociation of the nanoparticles, and the system finally exhibits a typical T 1 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 precisely determines the contour of glioma for us, enabling its accurate diagnosis.

[0186] In the study of the ECA-CA group that only responds to pH (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 to form a cluster structure in an acidic environment, 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 glioma. On the contrary, in the ESA-CA group that only responds to GSH (Comparative Example 2), at 2 h after injection, the brightness at the tumor site also became brighter due to the enrichment of the 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 a T 1 Contrast agent characteristics. And the non-responsive EA-CA group (Comparative Example 3) also always serves as a T 1 Contrast agent presence. 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 benzimidate bonds and disulfide bonds in realizing the precise multiple response function of the material to the complex tumor microenvironment.

[0187] The above is only the specific implementation manner 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 those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and disclosure scope of the present invention.

Claims

1. A sequential double MRI signal switching contrast agent for targeting tumors, characterized in that: The sequential double MRI signal switching contrast agent is a mixture of a first contrast agent and a second contrast agent; The first contrast agent includes nanoparticles, a first response click dual-functional layer, a first response shielding dual-functional layer and a first targeting layer in order from the inside to the outside, the first response click dual-functional layer and the first response shielding dual-functional layer are independently connected to the nanoparticles, and the first response shielding dual-functional layer is connected to the first targeting layer; The second contrast agent includes nanoparticles, a second responsive click dual-functional layer, a second responsive shielding dual-functional layer, and a second targeting layer in order from the inside to the outside, the second responsive click dual-functional layer and the second responsive shielding dual-functional layer are independently connected to the nanoparticles, and the second responsive shielding dual-functional layer is connected to the second targeting layer; The first responsive click bifunctional layer contains a GSH responsive group and a first click group; The second click-responsive bifunctional layer contains a GSH-responsive group and a second click group; The first responsive shielding double-functional layer and the second responsive shielding double-functional layer both contain pH responsive groups and shielding groups.

2. The sequential double MRI signal switching contrast agent according to claim 1, characterized in that: The nanoparticles are ferric oxide with modified groups connected to the surface; The modifying group includes pentafluorophenol ester; The average particle size of the ferric oxide is less than 4 nm.

3. The sequential double MRI signal switching contrast agent according to claim 1, characterized in that: The GSH response group is a -SS- functional group; The first click group is an N3 functional group; The second click group is a DBCO functional group; The pH response group is a benzoic acid imide functional group; The shielding group includes an mPEGn functional group, wherein n is 9,000-12,000.

4. The sequential double MRI signal switching contrast agent according to claim 1, characterized in that: The raw material for preparing the first responsive click dual-functional layer includes a first polymer containing a GSH responsive group and a first click group; the first polymer has an amino group at the end; The general structural formula of the first polymer is NH2-SS-mPEGa-N3; wherein a is 1800-2200; The amino group in the first polymer is connected to the modification group in the nanoparticle by forming an amide bond; The raw materials for preparing the first responsive shielding dual-functional layer and the second responsive shielding dual-functional layer both include amino-terminated polyethylene glycol and a second polymer of the general structural formula shown in Formula I); Formula I) Wherein, n is 9000-12000; The general structural formula of the amino-terminated polyethylene glycol is NH2-mPEGa-NH2; wherein a is 1800-2200; The amino group at one end of the amino-terminated polyethylene glycol is connected to the modified group in the nanoparticle by forming an amide bond, and the amino group at the other end is connected to the benzaldehyde group in the second polymer by forming a benzoic acid imide bond; The raw material for preparing the second responsive click dual-functional layer includes a third polymer containing a GSH responsive group and a second click group; the third polymer has an amino group at the end; The general structural formula of the third polymer is NH2-SS-mPEGa-DBCO; wherein a is 1800-2200; The amino group in the third polymer is connected to the modification group in the nanoparticle by forming an amide bond; The raw materials for preparing the first targeting layer and the second targeting layer both include targeting peptides, and the targeting peptides include SH-Angiopep-2; The maleimide group in the second polymer is connected to the thiol group in the targeting peptide by forming a thioether bond.

5. The sequential double MRI signal switching contrast agent according to claim 1, characterized in that: The molar ratio of the first contrast agent to the second contrast agent is 1:(1-1.2).

6. A method for preparing a sequential double MRI signal switching contrast agent as claimed in any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) performing a first response click double functional layer connection reaction, a first response shielding double functional layer connection reaction, and a first targeting layer connection reaction on the nanoparticles in sequence to obtain a first contrast agent; (2) performing a second response click double functional layer connection reaction, a second response shielding double functional layer connection reaction, and a second targeting layer connection reaction on the nanoparticles in sequence to obtain a second contrast agent; (3) mixing the first contrast agent of step (1) and the second contrast agent of step (2) to obtain the sequential dual MRI signal switching contrast agent; Steps (1) and (2) are performed in no particular order.

7. The preparation method according to claim 6, characterized in that: Step (1) The method of the first responsive click dual-functional layer connection reaction comprises: mixing nanoparticles, a first polymer containing a GSH responsive group and a first click group, and a first solvent, performing a first reaction, and obtaining a first intermediate; Step (1) The method of the first responsive shielding dual-functional layer connection reaction comprises: mixing a first intermediate, amino-terminated polyethylene glycol and a first solvent, performing a second reaction, then adding a second polymer thereto, performing a third reaction, and obtaining a second intermediate; The method of step (1) the first targeting layer connection reaction comprises: mixing the second intermediate and the targeting peptide, performing a fourth reaction, and obtaining a first contrast agent; Step (2) the method for the second responsive click dual-functional layer connection reaction comprises: 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; Step (2) The method for the second responsive shielding dual-functional layer connection reaction comprises: mixing a third intermediate, amino-terminated polyethylene glycol and a second solvent, performing a sixth reaction, and then adding a second polymer thereto, performing a seventh reaction, and obtaining a fourth intermediate; The method of step (2) the second targeting layer connection reaction comprises: mixing the fourth intermediate and the targeting peptide, performing an eighth reaction, and obtaining a second contrast agent.

8. The preparation method according to claim 7, characterized in that: The molar ratio of the first polymer to the amino-terminated polyethylene glycol is 1:(6-15); The molar ratio of the second polymer to the amino-terminated polyethylene glycol is ≥2:1; The molar ratio of the third polymer to the amino-terminated polyethylene glycol is 1:(6-15); The molar ratio of the targeting peptide to the second polymer is 1:(20-40); The first solvent and the second solvent both 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 time 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 ≥ 20h; After the second reaction and the sixth reaction, the first dialysis and the pH adjustment to 8-9 were performed in sequence; The fourth reaction and the eighth reaction were followed by precipitation treatment and a second dialysis.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a drug, a pharmaceutically acceptable pharmaceutical carrier and the sequential double MRI signal switching contrast agent according to any one of claims 1 to 5.

10. A use of the sequential double MRI signal switching contrast agent according to any one of claims 1 to 5, characterized in that: The sequential double MRI signal switching contrast agent is used in preparing tumor magnetic resonance imaging diagnosis or detection reagents.

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