A pH-responsive magnetic resonance imaging contrast agent, its preparation method and application

Abstract

This invention discloses a pH-responsive magnetic resonance imaging contrast agent, its preparation method, and its application. The magnetic resonance imaging contrast agent has the following structural formula: nanoparticles. The pH-responsive magnetic resonance imaging contrast agent provided by this invention can specifically respond to the weakly acidic microenvironment of tumors, leading to changes in the hydrophilic and hydrophobic properties of the surface polymer of the contrast agent particles. Through hydrophobic interactions, the ferric oxide nanoparticles aggregate, thereby achieving the switching from T1 to T2 contrast agents and activating the T2 contrast enhancement effect. It has advantages such as high sensitivity and selectivity, low toxicity, and good biocompatibility in tumor diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology and relates to a magnetic resonance imaging contrast agent, specifically a pH-responsive T1-T2 switching magnetic resonance imaging contrast agent (ESIONPs-PEG-PSDM) and its preparation method and application, such as its application in the preparation of products with tumor detection function. Background Technology

[0002] Early diagnosis of cancer is crucial for its treatment. Among various imaging techniques, magnetic resonance imaging (MRI) is widely used in the clinical diagnosis of tumors due to its advantages such as high spatial resolution, no radiation damage, and multi-planar scanning. However, MRI has a limitation of relatively low sensitivity, sometimes making it difficult to distinguish between diseased and normal tissues. Therefore, contrast agents are often introduced to address this issue. Gadolinium-based small molecule contrast agents are commonly used clinically, as they can improve the contrast between tumors and normal tissues. However, they still carry the risk of causing renal systemic fibrosis. Furthermore, these small molecule contrast agents have drawbacks such as low relaxation rates and lack of specificity for tumor tissues. Therefore, with a deeper understanding of tumor tissues, responsive contrast agents have gradually attracted attention. These contrast agents alter signals in response to endogenous stimuli at the tumor site, enhancing the specificity of tumor tissue and the MRI signal, thereby improving diagnostic accuracy. For example, researchers have synthesized biblock copolymers using polyethylene glycol and polyhistidine, and then attached small-molecule gadolinium chelates to the ends of the copolymers, which self-assembled to form pH-responsive polymer micelles. These micelles can disperse in the weakly acidic tumor microenvironment through protonation of the imidazole groups in the polyhistidine, selectively enhancing T1-weighted imaging signals. While these stimulus-responsive contrast agents can enhance MRI imaging at tumor sites, they still produce signals in normal tissues that interfere with diagnosis. Therefore, T1-T2 switching contrast agents are being developed to reduce background interference and specifically enhance imaging effects by responding to endogenous stimuli and switching the imaging signal at the tumor site.

[0003] Currently, there are few reports on this type of T1-T2 switching contrast agent. The inventors of this case have previously explored the effective aggregation of ferric oxide nanoparticles in a weakly acidic tumor environment by modifying the surface of the nanoparticles with two charge-switching responsive polymers. Although this system can achieve T1-T2 contrast effects, it requires the simultaneous preparation and mixing of two types of nanoparticles, which increases the complexity of material preparation and the uncertainty of in vivo effects. Summary of the Invention

[0004] The main objective of this invention is to provide a pH-responsive magnetic resonance imaging contrast agent, its preparation method, and its application, thereby overcoming the shortcomings of the prior art.

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0006] This invention provides a pH-responsive magnetic resonance imaging contrast agent with the following structural formula:

[0007]

[0008] Wherein, X is an assembly formed by adamantane and cyclodextrin;

[0009] Y is m is 30–50, n is 5–20, and M is ferric oxide nanoparticles.

[0010] This invention also provides a method for preparing a pH-responsive magnetic resonance imaging contrast agent, comprising:

[0011] A nucleophilic acyl substitution reaction was carried out on a first homogeneous mixture containing sulfadimethoxypyrimidine and methacryloyl chloride to prepare sulfadimethoxypyrimidine containing methacryloyl chloride.

[0012] A second homogeneous mixed reaction system comprising sulfadimethoxypyrimidine containing methpropylene, 4-cyano-4-(thiobenzoyl)valerate and azobisisobutyronitrile is subjected to a reversible addition-fracture chain transfer polymerization reaction under a protective atmosphere to obtain carboxyl-containing polysulfadimethoxypyrimidine.

[0013] A condensation reaction is carried out in a third homogeneous mixture containing the carboxyl-containing polysulfonamide dimethoxypyrimidine, a fully amino betacyclodextrin, and a 1-hydroxybenzotriazole to obtain polysulfonamide dimethoxypyrimidine containing cyclodextrin groups.

[0014] A carboxyl-activated ester-containing adamantane molecule was prepared by subjecting a fourth homogeneous mixed reaction system containing 1-adamantanecarboxylic acid and pentafluorophenol to a carboxyl activation reaction under a protective atmosphere.

[0015] A fifth homogeneous mixing reaction system containing adamantane molecules with carboxyl-activated esters and polyethylene glycol with amino and ditert-butyl dicarbonate-protected amino groups undergoes a condensation reaction under an alkaline environment to obtain polyethylene glycol with adamantane and amino groups at the end.

[0016] A sixth uniform mixing reaction system containing polyethylene glycol with terminal groups of adamantyl and amino is reacted with ferric oxide nanoparticles with surface-modified carboxyl activated esters to obtain ferric oxide nanoparticles with surface partially modified with adamantyl groups by polyethylene glycol.

[0017] The host-guest self-assembly of the seventh homogeneous mixing reaction system of ferric oxide nanoparticles with polyethylene glycol partially modified adamantyl group and polysulfamine dimethoxypyrimidine containing cyclodextrin group was carried out to obtain ferric oxide nanoparticles with polysulfamine dimethoxypyrimidine on the surface, which is the pH-responsive magnetic resonance imaging contrast agent.

[0018] This invention also provides the use of the pH-stimulation-responsive magnetic resonance imaging contrast agent in the preparation of products with tumor detection functions.

[0019] Compared with the prior art, the beneficial effects of the present invention are at least as follows:

[0020] 1) The pH-responsive magnetic resonance imaging contrast agent provided by this invention uses ferric oxide nanoparticles as a carrier. It responds to and aggregates under weakly acidic conditions, accumulating in the weakly acidic tumor microenvironment and switching between T1 and T2 imaging signals. The ratio of transverse relaxation rate to longitudinal relaxation rate after the switch is 26.8, which is 2.5 times higher than the 10.87 ratio in the prior art. This indicates that the pH-responsive magnetic resonance imaging contrast agent can effectively improve sensitivity and reduce background signal interference, exhibiting specificity and selectivity for tumor imaging. Simultaneously, the rapid aggregation of this pH-responsive magnetic resonance imaging contrast agent in the tumor microenvironment leads to increased particle size, effectively enhancing high permeability and retention effect (EPR) and improving the enrichment of the contrast agent at the tumor site.

[0021] 2) The pH-responsive magnetic resonance imaging contrast agent provided by this invention is composed of ferric oxide nanoparticles with surface-modified stimuli-responsive polymers. The surface-modified stimuli-responsive polymers undergo a hydrophilic-hydrophobic transition response in a weakly acidic environment, resulting in a change from hydrophilic to hydrophobic. Compared to the existing technology with a pH response point of 6.5, this hydrophobic interaction results in a pH response point of 6.6. The higher weakly acidic pH response point better matches the tumor microenvironment and effectively enhances the aggregation effect of nanoparticles at the tumor site. Through hydrophobic interactions, this pH-responsive magnetic resonance imaging contrast agent aggregates. Therefore, this switch from a T1-class contrast agent to a T2-class contrast agent rapidly activates the T2 contrast enhancement effect at the tumor site, improving the imaging contrast of the tumor site. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is the 1H NMR spectrum of sulfadimethoxypyrimidine, the product containing methylpropene, in step (1) of Example 1 of the present invention;

[0024] Figure 2 This is the 1H NMR spectrum of polysulfonamide dimethoxypyrimidine, the product containing a carboxyl group, in step (2) of Example 1 of the present invention;

[0025] Figure 3 This is the 1H NMR spectrum of the adamantane molecule containing a carboxyl-activated ester in step (4) of Example 1 of the present invention;

[0026] Figure 4 This is the 1H NMR spectrum of polyethylene glycol containing terminal groups of adamantyl and amino in step (5) of Example 1 of the present invention;

[0027] Figure 5 This is the infrared spectrum of the iron oxide nanoparticles with polyethylene glycol partially modified adamantyl alkyl groups on the surface of the product in step (6) of Example 1 of the present invention.

[0028] Figure 6 These are the infrared spectra of polysulfonamide dimethoxypyrimidine containing cyclodextrin groups and ferric oxide nanoparticles containing polysulfonamide dimethoxypyrimidine on the surface of the product in steps (3) and (7) of Example 1 of the present invention.

[0029] Figures 7a-7b This is a comparison of the longitudinal relaxation rate (r1) and relaxation rate of the MRI contrast agent under physiological neutral (pH 7.4) and weakly acidic (pH 6.5) conditions in Example 1 of the present invention, as well as the corresponding solution imaging diagram.

[0030] Figures 8a-8b This is a comparison of the transverse relaxation rate (r2) and relaxation rate of the MRI contrast agent under physiological neutral (pH 7.4) and weakly acidic (pH 6.5) conditions in Example 1 of the present invention, as well as the corresponding solution imaging diagram.

[0031] Figure 9 This is a cytotoxicity test diagram of the MRI contrast agent in human umbilical vein endothelial cells (HUVEC) and mouse breast cancer cells (4T1) in Example 1 of the present invention;

[0032] Figure 10 This is a tissue toxicology test diagram of the MRI contrast agent in BALB-c mice in Example 1 of the present invention;

[0033] Figure 11 This is an in vivo imaging image of the MRI contrast agent in BALB-c mice with transplanted 4T1 cell tumors, as shown in Example 1 of this invention. Detailed Implementation

[0034] In view of the deficiencies of the prior art, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention, namely, by developing single modified ferric oxide nanoparticles that can be directly applied to T1-T2 switching imaging, and achieving aggregation at the tumor site based on hydrophobic interactions, thereby enhancing the imaging effect.

[0035] The following will provide a further explanation of the technical solution, its implementation process, and its principles.

[0036] One aspect of the present invention provides a magnetic resonance imaging contrast agent having the following structural formula:

[0037]

[0038] Wherein, X is an assembly formed by adamantane and cyclodextrin;

[0039] Y is m is 30–50, n is 5–20, and M is ferric oxide nanoparticles.

[0040] The magnetic resonance imaging contrast agent of the present invention is constructed by using sulfonamide drugs as monomers to form a pH-responsive hydrophilic-hydrophobic transition polymer, which is then modified onto the surface of monodisperse ferric oxide nanoparticles, thereby achieving the aggregation of nanoparticles caused by the change in the solubility of the surface polymer in the aqueous phase induced by pH.

[0041] In some more specific embodiments, the ferric oxide nanoparticles have a particle size of less than 5 nm, and the ferric oxide nanoparticles are ultra-small ferric oxide nanoparticles.

[0042] The pH-responsive magnetic resonance imaging contrast agent (ESIONPs-PEG-PSDM) provided in this invention comprises ferric oxide nanoparticles that aggregate in the weakly acidic tumor microenvironment, leading to a switching of contrast effects. These ferric oxide nanoparticles, due to their ultra-small magnetic moment and surface paramagnetic iron ions, can act as a T1-type contrast agent. Monodisperse ferric oxide nanoparticles in water are obtained by modifying the nanoparticle surface with polyethylene glycol. Subsequently, a stimulus-responsive polymer that changes from hydrophilic to hydrophobic under weak acid conditions is modified onto cyclodextrin, and then modified onto the nanoparticle surface through host-guest self-assembly with polyethylene glycol with adamantane end groups. Under the weakly acidic conditions of the tumor microenvironment, these nanoparticles aggregate hydrophobically, thereby selectively generating T2 contrast signal enhancement at the tumor site through the conversion from T1 to T2 contrast agent. Simultaneously, the increased size after particle aggregation effectively enhances the EPR effect (high permeability and retention effect), increasing the enrichment and residence time of the contrast agent in tumor tissue.

[0043] Another aspect of the present invention provides a method for preparing a pH-responsive magnetic resonance imaging contrast agent, comprising:

[0044] A nucleophilic acyl substitution reaction was carried out on a first homogeneous mixture containing sulfadimethoxypyrimidine and methacryloyl chloride to prepare sulfadimethoxypyrimidine containing methacryloyl chloride.

[0045] A second homogeneous mixed reaction system comprising sulfadimethoxypyrimidine containing methpropylene, 4-cyano-4-(thiobenzoyl)valerate and azobisisobutyronitrile is subjected to a reversible addition-fracture chain transfer polymerization reaction under a protective atmosphere to obtain carboxyl-containing polysulfadimethoxypyrimidine.

[0046] A condensation reaction is carried out in a third homogeneous mixture containing the carboxyl-containing polysulfonamide dimethoxypyrimidine, a fully amino betacyclodextrin, and a 1-hydroxybenzotriazole to obtain polysulfonamide dimethoxypyrimidine containing cyclodextrin groups.

[0047] A carboxyl-activated ester-containing adamantane molecule was prepared by subjecting a fourth homogeneous mixed reaction system containing 1-adamantanecarboxylic acid and pentafluorophenol to a carboxyl activation reaction under a protective atmosphere.

[0048] A fifth homogeneous mixing reaction system containing adamantane molecules with carboxyl-activated esters and polyethylene glycol with amino and ditert-butyl dicarbonate-protected amino groups undergoes a condensation reaction under an alkaline environment to obtain polyethylene glycol with adamantane and amino groups at the end.

[0049] A sixth uniform mixing reaction system containing polyethylene glycol with terminal groups of adamantyl and amino is reacted with ferric oxide nanoparticles with surface-modified carboxyl activated esters to obtain ferric oxide nanoparticles with surface partially modified with adamantyl groups by polyethylene glycol.

[0050] The host-guest self-assembly of the seventh homogeneous mixing reaction system of ferric oxide nanoparticles with polyethylene glycol partially modified adamantyl group and polysulfamine dimethoxypyrimidine containing cyclodextrin group was carried out to obtain ferric oxide nanoparticles with polysulfamine dimethoxypyrimidine on the surface, which is the pH-responsive magnetic resonance imaging contrast agent.

[0051] In some more specific embodiments, the preparation method specifically includes: dissolving sulfadimethoxypyrimidine in an aqueous sodium hydroxide solution and acetone to form a solution, then adding methacryloyl chloride dropwise to the solution under ice bath conditions to form a first homogeneous mixed reaction system, and carrying out a nucleophilic acyl substitution reaction for 2-6 hours to obtain the methacrylic acid-containing sulfadimethoxypyrimidine, wherein the volume of the sodium hydroxide solution is greater than the volume of the acetone.

[0052] Furthermore, the volume ratio of the sodium hydroxide aqueous solution to acetone is 1 to 3:1.

[0053] Furthermore, the molar ratio of sulfadimethoxypyrimidine to methacryloyl chloride is 3 to 1:1.

[0054] Furthermore, the alkaline aqueous solution is an aqueous solution of sodium hydroxide, but is not limited thereto.

[0055] Furthermore, the molar concentration of the sodium hydroxide aqueous solution is 0.2–0.5 mol / L.

[0056] Furthermore, the protective atmosphere includes a nitrogen atmosphere or an inert gas atmosphere.

[0057] In some more specific embodiments, the preparation method specifically includes: dissolving the methpropylene-containing sulfamethoxypyrimidine in dimethyl sulfoxide under a protective atmosphere, then adding 4-cyano-4-(thiobenzoyl)valerate and azobisisobutyronitrile solution to form a second homogeneous mixed reaction system, followed by freezing and deoxygenation treatment, and then carrying out a reversible addition-fragmentation chain transfer polymerization reaction at 70-100°C for 24-48 hours to obtain the carboxyl-containing polysulfamethoxypyrimidine.

[0058] Furthermore, the molar ratio of the methpropylene-containing sulfadimethoxypyrimidine to 4-cyano-4-(thiobenzoyl)valerate is 1:25-50.

[0059] Furthermore, the protective atmosphere includes a nitrogen atmosphere or an inert gas atmosphere.

[0060] In some more specific embodiments, the preparation method specifically includes: dissolving the carboxyl-containing polysulfonamide dimethoxypyrimidine, all-amino beta-cyclodextrin and 1-hydroxybenzotriazole in dimethylformamide to form a third uniform mixed reaction system, and carrying out a condensation reaction at 20-25°C for 24-48 hours to obtain the polysulfonamide dimethoxypyrimidine containing cyclodextrin groups.

[0061] Furthermore, the preparation method further includes: after the condensation reaction of the third uniformly mixed reaction system is completed, the obtained mixture is subjected to dialysis and freeze-drying treatment; wherein, the dialysis bag used for the dialysis treatment has a molecular weight cutoff of 5000-8000 Da, preferably 5000-7000 Da.

[0062] Furthermore, the molar ratio of the carboxyl-containing polysulfamine dimethoxypyrimidine to the fully amino betacyclodextrin is 5 to 10:1.

[0063] Furthermore, the molar ratio of the carboxyl-containing polysulfamine dimethoxypyrimidine to 1-hydroxybenzotriazole is 5 to 10:1.

[0064] In some more specific embodiments, the preparation method specifically includes: dissolving 1-adamantane carboxylic acid and pentafluorophenol in 1,4-dioxane under a protective atmosphere, then adding dicyclohexylcarbodiimide to form a fourth homogeneous mixed reaction system, and performing a carboxyl activation reaction at 20-25°C for 24-48 hours to obtain the adamantane molecule containing the carboxyl activated ester.

[0065] Furthermore, the molar ratio of pentafluorophenol to 1-adamantanecarboxylic acid is 2 to 10:1.

[0066] Furthermore, the molar ratio of pentafluorophenol to dicyclohexylcarbodiimide is 1 to 3:1.

[0067] Furthermore, the protective atmosphere includes a nitrogen atmosphere or an inert gas atmosphere.

[0068] In some more specific embodiments, the preparation method specifically includes: dissolving the adamantane molecule containing carboxyl activated ester and the polyethylene glycol with amino and ditert-butyl dicarbonate-protected end groups in dichloromethane, then adding N,N-diisopropylethylamine to form a fifth homogeneous mixed reaction system, and carrying out a condensation reaction at 20-25°C for 24-48 hours in an alkaline environment (pH value 8-10) to obtain the polyethylene glycol with adamantyl and amino end groups.

[0069] Furthermore, the molar ratio of the N,N-diisopropylethylamine to the adamantane molecule containing a carboxyl activated ester is 10 to 20:1.

[0070] Further, the molar ratio of the adamantane molecule containing carboxyl-activated ester to the polyethylene glycol with amino and di-tert-butyl dicarbonate-protected amino groups is 5-10:1. In some more specific embodiments, the preparation method specifically includes: mixing the surface-modified carboxyl-activated ester ferric oxide nanoparticles with the polyethylene glycol with adamantyl and amino end groups in chloroform, then adding N,N-diisopropylethylamine to form a sixth homogeneous mixing reaction system, and reacting at 20-25°C for 12-24 hours to obtain the ferric oxide nanoparticles with adamantyl partially modified by polyethylene glycol; wherein, the mass ratio of the surface-modified carboxyl-activated ester ferric oxide nanoparticles to the polyethylene glycol with adamantyl and amino end groups is 1:5-10.

[0071] Furthermore, the surface-modified carboxyl-activated ester ferric oxide nanoparticles include surface-modified pentafluorophenol ester ferric oxide nanoparticles.

[0072] Furthermore, the molar ratio of N,N-diisopropylethylamine to polyethylene glycol with terminal groups of adamantyl and amino is 100-200:1.

[0073] In some more specific embodiments, the preparation method specifically includes: dissolving the ferric oxide nanoparticles with polyethylene glycol partially modified adamantyl groups in an aqueous sodium hydroxide solution, then adding the polysulfonamide dimethoxypyrimidine containing cyclodextrin groups to form a seventh uniform mixed reaction system, and conducting a host-guest self-assembly reaction at 20-25°C for 24-48 hours to obtain the ferric oxide nanoparticles with polysulfonamide dimethoxypyrimidine on the surface.

[0074] Furthermore, the molar ratio of the polyethylene glycol-modified adamantyl alkyl iron oxide nanoparticles to the polysulfamine dimethoxypyrimidine containing cyclodextrin groups is 1:10-20.

[0075] Furthermore, the preparation method further includes: after the host-guest self-assembly reaction of the seventh uniformly mixed reaction system is completed, the obtained mixture is subjected to dialysis and freeze-drying treatment; wherein, the molecular weight cutoff of the dialysis bag used in the dialysis treatment is 30,000 to 50,000 Da, preferably 35,000 to 50,000 Da.

[0076] Furthermore, the alkaline aqueous solution is an aqueous solution of sodium hydroxide, but is not limited thereto.

[0077] Furthermore, the molar concentration of the sodium hydroxide aqueous solution is 1–10 μmol / L.

[0078] In some more specific embodiments, the preparation method may include:

[0079] (1) Preparation of sulfamethoxypyrimidine containing methpropylene: Sulfamethoxypyrimidine is dissolved in 0.2–0.5 mol / L sodium hydroxide aqueous solution and acetone to form a solution. Then, methacryloyl chloride is added dropwise to the solution under ice bath to form a first homogeneous mixed reaction system, and a nucleophilic acyl substitution reaction occurs for 2–6 h to obtain sulfamethoxypyrimidine containing methpropylene, wherein the volume of sodium hydroxide aqueous solution is greater than the volume of acetone. The volume ratio of sodium hydroxide aqueous solution to acetone is 1–3:1.

[0080] (2) Preparation of carboxyl-containing polysulfamethoxypyrimidine: Under a protective atmosphere, sulfamethoxypyrimidine containing methpropylene was dissolved in dimethyl sulfoxide, and then 4-cyano-4-(thiobenzoyl)valerate and azobisisobutyronitrile solution were added to form a second homogeneous mixed reaction system. After freezing and deoxygenation treatment, a reversible addition-fragmentation chain transfer polymerization reaction was carried out at 70-100℃ for 24-48h to obtain carboxyl-containing polysulfamethoxypyrimidine.

[0081] (3) Preparation of polysulfonamide dimethoxypyrimidine containing cyclodextrin groups: Carboxyl-containing polysulfonamide dimethoxypyrimidine, peraminobetacyclodextrin, and 1-hydroxybenzotriazole were dissolved in dimethylformamide to form a third homogeneous mixing reaction system, and a condensation reaction was carried out at 20–25 °C for 24–48 h to obtain polysulfonamide dimethoxypyrimidine containing cyclodextrin groups. The molar ratio of carboxyl-containing polysulfonamide dimethoxypyrimidine to peraminobetacyclodextrin was 5–10:1, and the molar ratio of carboxyl-containing polysulfonamide dimethoxypyrimidine to 1-hydroxybenzotriazole was 5–10:1.

[0082] (4) Preparation of adamantane molecules containing carboxyl-activated esters: Under a protective atmosphere, 1-adamantanecarboxylic acid and pentafluorophenol are dissolved in 1,4-dioxane, and then dicyclohexylcarbodiimide is added to form a fourth homogeneous mixed reaction system. The carboxyl activation reaction is carried out at 20-25°C for 24-48 h to obtain the adamantane molecules containing carboxyl-activated esters. The molar ratio of pentafluorophenol to 1-adamantanecarboxylic acid is 2-10:1.

[0083] (5) Preparation of polyethylene glycol with adamantyl and amino terminal groups: Adamantane molecules containing carboxyl-activated esters and polyethylene glycol with amino and di-tert-butyl dicarbonate terminal groups were dissolved in dichloromethane. N,N-diisopropylethylamine was then added to form a fifth homogeneous mixing reaction system, and a condensation reaction was carried out at 20-25°C for 24-48 h to obtain polyethylene glycol with adamantyl and amino terminal groups. The molar ratio of N,N-diisopropylethylamine to adamantane molecules containing carboxyl-activated esters was 10-20:1.

[0084] (6) Preparation of ferric oxide nanoparticles with surface partially modified with adamantyl groups by polyethylene glycol: Ferric oxide nanoparticles with surface modified with carboxyl activated esters at a mass ratio of 1:5 to 10 were mixed with polyethylene glycol with adamantyl and amino end groups in chloroform. N,N-diisopropylethylamine was then added to form a sixth homogeneous mixing reaction system, and the mixture was reacted at 20–25°C for 12–24 h to obtain ferric oxide nanoparticles with surface partially modified with adamantyl groups by polyethylene glycol. The molar ratio of N,N-diisopropylethylamine to polyethylene glycol with adamantyl and amino end groups was 100–200:1.

[0085] (7) Preparation of ferric oxide nanoparticles with polysulfamine dimethoxypyrimidine on the surface: Ferric oxide nanoparticles with a mass ratio of 1:5 to 10 and adamantyl alkyl groups on the surface modified by polyethylene glycol are mixed with polysulfamine dimethoxypyrimidine containing cyclodextrin groups in an aqueous sodium hydroxide solution and reacted at 20 to 25°C for 24 to 48 hours to obtain the ferric oxide nanoparticles with polysulfamine dimethoxypyrimidine on the surface.

[0086] In the preparation step of the methylpropene-containing sulfadimethoxypyrimidine, the methylpropene-containing sulfadimethoxypyrimidine is obtained by nucleophilic acyl substitution, and the insoluble matter is removed by filtration, the mixture is repeatedly washed with water, recrystallized with methanol, and purified by vacuum drying.

[0087] In the preparation step of the carboxyl-containing polysulfamine dimethoxypyrimidine, the carboxyl-containing polysulfamine dimethoxypyrimidine is obtained by a reversible addition-fragmentation chain transfer polymerization reaction, and an excess of diethyl ether is added dropwise to obtain a white solid. The solid is collected by filtration, centrifuged, dissolved in dichloromethane, and then precipitated with a large amount of diethyl ether. The solid is collected by filtration, dissolved in dichloromethane, and precipitated again with diethyl ether. This process is repeated 3 or more times. Then, the diethyl ether is removed by rotary evaporation, and the solid is purified by vacuum drying.

[0088] In the preparation step of the polysulfonamide dimethoxypyrimidine containing cyclodextrin groups, the polysulfonamide dimethoxypyrimidine containing cyclodextrin groups can be precipitated in a large amount of diethyl ether, the precipitate can be collected by centrifugation and dissolved in deionized water, the pH value can be adjusted to 8-9 with sodium hydroxide, and dialyzed for three days in a 1-10 μmol-L sodium hydroxide aqueous solution using a dialysis bag with a molecular weight cutoff of 5000-7000 Da, followed by freeze drying.

[0089] In the preparation step of the adamantane molecule containing carboxyl activated ester, the adamantane molecule containing carboxyl activated ester can be purified by silica gel chromatography column using dichloromethane as eluent, followed by rotary evaporation to remove dichloromethane, and then vacuum drying for purification.

[0090] In the preparation step of polyethylene glycol with adamantyl and amino end groups, the polyethylene glycol with adamantyl and amino end groups can be deprotected by a large amount of trifluoroacetic acid to remove the amino protecting group tert-butoxycarbonyl protecting group, then added dropwise to a large amount of diethyl ether, dissolved in dichloromethane and precipitated again with diethyl ether, repeated 3 or more times, then the diethyl ether is removed by rotary evaporation and purified by vacuum drying.

[0091] In the preparation step of the ferric oxide nanoparticles with polyethylene glycol partially modified adamantyl group on the surface, the ferric oxide nanoparticles with polyethylene glycol partially modified adamantyl group can be concentrated and then dropped into a large amount of diethyl ether, collected by centrifugation, dissolved in deionized water, dialyzed for three days using a dialysis bag with a molecular weight cutoff of 35,000 to 50,000 Da, and then freeze-dried.

[0092] In the preparation step of the ferric oxide nanoparticles containing polysulfamine dimethoxypyrimidine on the surface, the ferric oxide nanoparticles containing polysulfamine dimethoxypyrimidine on the surface are obtained by host-guest self-assembly, and are directly dialyzed for three days using a dialysis bag with a molecular weight cutoff of 35,000 to 50,000 Da, and then freeze-dried.

[0093] In the above embodiments of the present invention, dichloromethane, trichloromethane and dimethylformamide can also be replaced with other suitable organic solvents.

[0094] Another aspect of the present invention provides a pH-stimulation-responsive magnetic resonance imaging contrast agent prepared by the aforementioned method.

[0095] Another aspect of the present invention provides the use of the aforementioned pH-stimulation-responsive magnetic resonance imaging contrast agent in the preparation of products with tumor detection functions.

[0096] The pH-responsive magnetic resonance imaging contrast agent can rapidly respond at the tumor site and achieve efficient and stable aggregation, thereby specifically enhancing the imaging effect at the tumor site. Furthermore, the nanoparticle size increase effect prevents the backflow of ferric oxide nanoparticles from the tumor site into the blood vessels, providing a sufficient imaging time window while ensuring the contrast of the imaging.

[0097] In summary, the pH-responsive magnetic resonance imaging contrast agent provided in the embodiments of the present invention exhibits high specificity and selectivity for tumor sites, good biocompatibility, and low toxicity. Furthermore, it is rapidly metabolized in vivo, resulting in excellent imaging contrast performance and avoiding interference from background signals, thus providing high sensitivity and tumor-targeting specificity for tumor magnetic resonance imaging. Further, the increased size of the pH-responsive magnetic resonance imaging contrast agent after aggregation in the above embodiments of the present invention significantly enhances its retention effect at tumor tissue.

[0098] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention, and the experimental conditions and set parameters therein should not be considered as limitations on the basic technical solution of the invention. Furthermore, the technical features involved in the various embodiments of the invention described below can be combined with each other as long as they do not conflict with each other.

[0099] Example 1

[0100] The preparation method of a pH-responsive magnetic resonance imaging contrast agent (ESIONPs-PEG-PSDM) in this embodiment includes the following steps:

[0101] (1) Preparation of sulfamethoxypyrimidine containing methylpropene: Sulfamethoxypyrimidine was dissolved in 0.2 mol / L sodium hydroxide aqueous solution and acetone to form a solution. Then, methacryloyl chloride was added dropwise to the solution under ice bath to form a first homogeneous reaction system, and the reaction was carried out for 2 hours to obtain sulfamethoxypyrimidine containing methylpropene. The volume of sodium hydroxide aqueous solution was greater than the volume of acetone. The volume ratio of sodium hydroxide aqueous solution to acetone was 1:1. The molar ratio of sulfamethoxypyrimidine to methacryloyl chloride was 3:1. After the reaction was completed, the obtained solution was filtered to remove insoluble matter, washed repeatedly with water, and then recrystallized from methanol to obtain a white solid. After vacuum drying, it was sulfamethoxypyrimidine containing methylpropene (methacryloyl-SDM), and its 1H NMR spectrum is shown below. Figure 1 As shown;

[0102] (2) Preparation of carboxyl-containing polysulfonamide dimethoxypyrimidine: Under a protective atmosphere, sulfonamide dimethoxypyrimidine containing methpropylene was dissolved in dimethyl sulfoxide, and then 4-cyano-4-(thiobenzoyl)valerate and azobisisobutyronitrile solution were added to form a second homogeneous mixed reaction system. After freezing to remove oxygen, the mixture was reacted at 70℃ for 24 h to obtain carboxyl-containing polysulfonamide dimethoxypyrimidine. The molar ratio of sulfonamide dimethoxypyrimidine containing methpropylene to 4-cyano-4-(thiobenzoyl)valerate was 1:50. Excess diethyl ether was added dropwise to obtain a white solid, which was collected by filtration, centrifuged, dissolved in dichloromethane, and precipitated with a large amount of diethyl ether. The solid was collected by filtration, dissolved in dichloromethane, and precipitated again with diethyl ether. This process was repeated 3 or more times. The diethyl ether was then removed by rotary evaporation, and the mixture was purified by vacuum drying to obtain carboxyl-containing polysulfonamide dimethoxypyrimidine (COOH-PSDM). Its 1H NMR spectrum is shown below. Figure 2 As shown.

[0103] (3) Preparation of polysulfonamide dimethoxypyrimidine containing cyclodextrin groups: Carboxyl-containing polysulfonamide dimethoxypyrimidine, peraminobetacyclodextrin, and 1-hydroxybenzotriazole were dissolved in dimethylformamide to form a third homogeneous mixing reaction system, and reacted at 20℃ for 24 h to obtain polysulfonamide dimethoxypyrimidine containing cyclodextrin groups. The molar ratio of carboxyl-containing polysulfonamide dimethoxypyrimidine to peraminobetacyclodextrin was 5:1, and the molar ratio of carboxyl-containing polysulfonamide dimethoxypyrimidine to 1-hydroxybenzotriazole was 5:1. The precipitate was collected by centrifugation in a large amount of diethyl ether, dissolved in deionized water, and the pH was adjusted to 8 with sodium hydroxide. Dialysis was performed for three days using a dialysis bag with a molecular weight cutoff of 5000 Da in a 1 μmol / L sodium hydroxide aqueous solution. The precipitate was then freeze-dried to obtain polysulfonamide dimethoxypyrimidine containing cyclodextrin groups (β-CD-PSDM). Its infrared spectrum is shown below. Figure 6 As shown, the synthetic routes in steps (1), (2), and (3) can be represented by the following chemical equations:

[0104]

[0105] (4) Preparation of adamantane molecules containing carboxyl-activated esters: Under a protective atmosphere, 1-adamantane carboxylic acid and pentafluorophenol were dissolved in 1,4-dioxane, and then dicyclohexylcarbodiimide was added to form a fourth homogeneous mixed reaction system. The reaction was carried out at 20°C for 24 h to obtain the adamantane molecules containing carboxyl-activated esters. The molar ratio of pentafluorophenol to 1-adamantane carboxylic acid was 2:1, and the molar ratio of pentafluorophenol to dicyclohexylcarbodiimide was 1:1. After the reaction was completed, the adamantane molecules were purified by silica gel column chromatography using dichloromethane as the eluent. Dichloromethane was then removed by rotary evaporation, and the mixture was purified by vacuum drying to obtain the adamantane molecules containing carboxyl-activated esters (PFP-Ad). The 1H NMR spectrum of PFP-Ad is shown below. Figure 3 As shown.

[0106] (5) Preparation of polyethylene glycol with adamantyl and amino terminal groups: Adamantane molecules containing carboxyl-activated esters and polyethylene glycol with amino and ditert-butyl dicarbonate terminal groups were dissolved in dichloromethane. N,N-diisopropylethylamine was then added to form a fifth homogeneous mixing reaction system. The reaction was carried out at 20°C for 24 h at pH 8 to obtain polyethylene glycol with adamantyl and amino terminal groups. Its 1H NMR spectrum is shown below. Figure 4 As shown. Among them, the molar ratio of N,N-diisopropylethylamine to adamantane molecules containing carboxyl activated ester is 10:1, and the molar ratio of adamantane molecules containing carboxyl activated ester to polyethylene glycol with amino and ditert-butyl dicarbonate-protected amino groups is 5:1. After the reaction is completed, a large amount of trifluoroacetic acid is added and stirred in an ice bath for 2 hours. The obtained solution is concentrated, added dropwise to a large amount of diethyl ether, dissolved in dichloromethane, and precipitated again with diethyl ether. This process is repeated 3 or more times. Then, the diethyl ether is removed by rotary evaporation, and the solution is purified by vacuum drying to obtain ferric oxide nanoparticles (NH2-PEG-Ad) with azido groups partially modified by polyethylene glycol on the surface. The synthetic routes of steps (4) and (5) can be represented by the following chemical equations:

[0107]

[0108] (6) Preparation of ferric oxide nanoparticles with surface partially modified with polyethylene glycol (PEG) adamantyl groups: Ferric oxide nanoparticles with surface modified with carboxyl activated esters at a mass ratio of 1:5 were mixed with polyethylene glycol with terminal groups of adamantyl and amino in chloroform. N,N-diisopropylethylamine was then added to form a sixth homogeneous mixing reaction system, and the mixture was reacted at 20°C for 12 h to obtain the ferric oxide nanoparticles with surface partially modified with PEG adamantyl groups. The molar ratio of N,N-diisopropylethylamine to polyethylene glycol with terminal groups of adamantyl and amino was 100:1. After the reaction, the solution was concentrated and then added dropwise to a large amount of diethyl ether. The solution was collected by centrifugation, dissolved in deionized water, and dialyzed for three days using a dialysis bag with a molecular weight cutoff of 35000 Da. The mixture was then freeze-dried to obtain ferric oxide nanoparticles with surface partially modified with PEG adamantyl groups (ESIONPs-PEG-Ad). The infrared spectrum of this product is shown in the figure below. Figure 5 As shown;

[0109] (7) Preparation of ferric oxide nanoparticles with polysulfonamide dimethoxypyrimidine on the surface: Ferric oxide nanoparticles with polyethylene glycol-modified adamantyl groups at a molar ratio of 1:10 were mixed with polysulfonamide dimethoxypyrimidine containing cyclodextrin groups in an aqueous sodium hydroxide solution and reacted at 20°C for 24 h to obtain the ferric oxide nanoparticles with polysulfonamide dimethoxypyrimidine on the surface. After the reaction, the nanoparticles were dialyzed for three days using a dialysis bag with a molecular weight cutoff of 35000 Da, and then freeze-dried to obtain ferric oxide nanoparticles with polysulfonamide dimethoxypyrimidine on the surface (ESIONPs-PEG-PSDM). The infrared spectrum of the nanoparticles is shown in the figure below. Figure 6 As shown, the synthetic routes in steps (6) and (7) can be represented by the following chemical equations:

[0110]

[0111] Example 2

[0112] This embodiment describes a method for preparing a pH-responsive magnetic resonance imaging contrast agent, comprising the following steps:

[0113] (1) The steps are similar to steps (1) in Example 1, except that the reaction time of sulfadimethoxypyrimidine and methacryloyl chloride is adjusted to 4h, the volume ratio of sodium hydroxide aqueous solution to acetone is adjusted to 2:1, and the molar ratio of sulfadimethoxypyrimidine to methacryloyl chloride is adjusted to 2:1.

[0114] (2) This step is similar to step (2) in Example 1, except that the reaction conditions of sulfadimethoxypyrimidine containing methpropylene, 4-cyano-4-(thiobenzoyl)valerate, and azobisisobutyronitrile solution are adjusted to react at 80°C for 36 h, and the molar ratio of sulfadimethoxypyrimidine containing methpropylene to 4-cyano-4-(thiobenzoyl)valerate is adjusted to 1:40.

[0115] (3) This step is similar to step (3) in Example 1, except that: carboxyl-containing polysulfonamide dimethoxypyrimidine, all-amino beta-cyclodextrin, and 1-hydroxybenzotriazole are dissolved in dimethylformamide to form a third uniform mixed reaction system, and reacted at 25°C for 48 h to obtain polysulfonamide dimethoxypyrimidine containing cyclodextrin. The molar ratio of carboxyl-containing polysulfonamide dimethoxypyrimidine to all-amino beta-cyclodextrin is 10:1, and the molar ratio of carboxyl-containing polysulfonamide dimethoxypyrimidine to 1-hydroxybenzotriazole is 10:1.

[0116] (4) This step is similar to step (4) in Example 1, except that: under a protective atmosphere, 1-adamantane carboxylic acid and pentafluorophenol are dissolved in 1,4-dioxane, and then dicyclohexylcarbodiimide is added to form a fourth homogeneous mixed reaction system, and the reaction is carried out at 23°C for 36 hours to obtain the adamantane molecule containing the carboxyl activated ester. The molar ratio of pentafluorophenol to 1-adamantane carboxylic acid is 8:1, and the molar ratio of pentafluorophenol to dicyclohexylcarbodiimide is 2:1.

[0117] (5) This step is similar to step (5) in Example 1, except that: the pH value is adjusted to 9, the reaction temperature is adjusted to 22°C, the reaction time is adjusted to 36h, the molar ratio of N,N-diisopropylethylamine to adamantane molecules containing carboxyl activated ester is adjusted to 15:1, and the molar ratio of adamantane molecules containing carboxyl activated ester to polyethylene glycol with amino and ditert-butyl dicarbonate-protected amino groups is 8:1.

[0118] (6) This step is similar to step (6) in Example 1, except that: the mass ratio of the surface-modified carboxyl-activated ester iron oxide nanoparticles to polyethylene glycol with terminal groups of adamantyl and amino is adjusted to 1:8, the reaction time at 22°C is adjusted to 20h; and the molar ratio of N,N-diisopropylethylamine to adamantane molecules containing carboxyl-activated ester is adjusted to 150:1.

[0119] (7) This step is similar to step (7) in Example 1, except that the molar ratio of the ferric oxide nanoparticles containing polysulfamine dimethoxypyrimidine on the surface is adjusted to 1:15, and the reaction time at 22°C is adjusted to 40 h. Afterwards, the concentrated solution is added dropwise to a large amount of diethyl ether, collected by centrifugation, dissolved in deionized water, dialyzed for three days using a dialysis bag with a molecular weight cutoff of 30000 Da, and then freeze-dried.

[0120] Example 3

[0121] This embodiment describes a method for preparing a pH-responsive magnetic resonance imaging contrast agent, comprising the following steps:

[0122] (1) The steps are similar to steps (1) in Example 1, except that the reaction time of sulfadimethoxypyrimidine and methacryloyl chloride is adjusted to 6h, the volume ratio of sodium hydroxide aqueous solution to acetone is adjusted to 3:1, and the molar ratio of sulfadimethoxypyrimidine to methacryloyl chloride is adjusted to 1:1.

[0123] (2) This step is similar to step (2) in Example 1, except that the reaction conditions of sulfadimethoxypyrimidine containing methpropylene, 4-cyano-4-(thiobenzoyl)valerate, and azobisisobutyronitrile solution are adjusted to react at 100°C for 48 h, and the molar ratio of sulfadimethoxypyrimidine containing methpropylene to 4-cyano-4-(thiobenzoyl)valerate is adjusted to 1:25;

[0124] (3) This step is similar to step (3) in Example 1, except that: carboxyl-containing polysulfonamide dimethoxypyrimidine, peramino beta-cyclodextrin and 1-hydroxybenzotriazole are dissolved in dimethylformamide to form a third uniform mixed reaction system, and reacted at 25°C for 36 h to obtain polysulfonamide dimethoxypyrimidine containing cyclodextrin. The molar ratio of carboxyl-containing polysulfonamide dimethoxypyrimidine to peramino beta-cyclodextrin is 7:1, and the molar ratio of carboxyl-containing polysulfonamide dimethoxypyrimidine to 1-hydroxybenzotriazole is 7:1. Precipitate in a large amount of diethyl ether, collect the precipitate by centrifugation and dissolve the precipitate in deionized water, adjust the pH to 9 with sodium hydroxide, dialyze for three days in a 10 μmol / L sodium hydroxide aqueous solution using a dialysis bag with a molecular weight cutoff of 7000 Da, and freeze dry;

[0125] (4) This step is similar to step (4) in Example 1, except that: under a protective atmosphere, 1-adamantane carboxylic acid and pentafluorophenol are dissolved in 1,4-dioxane, and then dicyclohexylcarbodiimide is added to form a fourth homogeneous mixed reaction system, and the reaction is carried out at 25°C for 48 hours to obtain the adamantane molecule containing the carboxyl activated ester. The molar ratio of pentafluorophenol to 1-adamantane carboxylic acid is 10:1, and the molar ratio of pentafluorophenol to dicyclohexylcarbodiimide is 3:1.

[0126] (5) This step is similar to step (5) in Example 1, except that: the pH value is adjusted to 10, the reaction temperature is adjusted to 25°C, the reaction time is adjusted to 48h, the molar ratio of N,N-diisopropylethylamine to adamantane molecules containing carboxyl activated ester is adjusted to 20:1, and the molar ratio of adamantane molecules containing carboxyl activated ester to polyethylene glycol with amino and ditert-butyl dicarbonate-protected amino groups is 10:1.

[0127] (6) This step is similar to step (6) in Example 1, except that: the mass ratio of the surface-modified carboxyl-activated ester iron oxide nanoparticles to polyethylene glycol with terminal groups of adamantyl and amino is adjusted to 1:10, and the reaction time at 25°C is adjusted to 24h; the molar ratio of N,N-diisopropylethylamine to adamantane molecules containing carboxyl-activated ester is adjusted to 200:1. After concentrating the solution, it is added dropwise to a large amount of diethyl ether, collected by centrifugation, dissolved in deionized water, dialyzed for three days using a dialysis bag with a molecular weight cutoff of 50000 Da, and then freeze-dried.

[0128] (7) This step is similar to step (6) in Example 1, except that: the molar ratio of the ferric oxide nanoparticles containing polysulfamine dimethoxypyrimidine on the surface is adjusted to 1:20, and the reaction time at 25°C is adjusted to 48h; then, after the solution is concentrated, it is added dropwise into a large amount of ether, collected by centrifugation, dissolved in deionized water, dialyzed for three days using a dialysis bag with a molecular weight cutoff of 50000Da, and then freeze-dried.

[0129] The following demonstrates the advantages of the pH-responsive magnetic resonance imaging contrast agent obtained in Example 1, which is composed of ferric oxide nanoparticles (ESIONPs-PEG-PSDM) with polysulfamine dimethoxypyrimidine on its surface, as a contrast agent through several performance tests.

[0130] Performance Test 1

[0131] The longitudinal relaxation time T1 and T1-weighted imaging of the contrast agent product obtained in Example 1 of this invention were tested on a 0.5T MRI scanner under physiological neutral (pH 7.4) and weakly acidic (pH 6.5) tumor microenvironment conditions. The operation method included:

[0132] Two samples with iron concentrations of 0.60–0.15 mmol / L (mmol / L can be abbreviated as mM) were prepared respectively. After testing on a 0.5T MRI scanner, linear fitting was performed with iron ion concentration as the x-axis and the reciprocal of the longitudinal relaxation time as the y-axis. The longitudinal relaxation rates of the contrast agent of this invention were found to be 3.37 mM at pH values ​​of 7.4 and 6.5. -1 s -1 and 1.24mM -1 s-1 (like Figures 7a-7b As shown in the figure, the longitudinal relaxation rate of the contrast agent of the present invention decreases in a weakly acidic environment.

[0133] It can be seen from the T1-weighted imaging of the two at different concentrations that the T1 contrast agent obtained in Example 1 of the present invention has a much higher imaging brightness under neutral conditions than under weakly acidic conditions.

[0134] Performance Test 2

[0135] The transverse relaxation time T2 and T2-weighted imaging of the contrast agent obtained in Example 1 of this invention were tested on a 0.5T MRI scanner under physiological neutral (pH 7.4) and weakly acidic (pH 6.5) tumor microenvironment conditions. The operation method included:

[0136] Two samples with iron concentrations of 0.60–0.15 mM were prepared and tested on a 0.5T MRI scanner. Linear fitting was performed with iron concentration as the x-axis and the reciprocal of the transverse relaxation time as the y-axis, yielding transverse relaxation rates of 8.72 mM for the contrast agent of this invention before and after incubation with DTT. -1 s -1 and 33.42mM -1 s -1 (like Figures 8a-8b As shown in the figure, the contrast agent of the present invention exhibits a significantly increased transverse relaxation rate in a weakly acidic environment. The ratio of transverse relaxation rate to longitudinal relaxation rate is 2.58 at pH 7.4. The ratio is 26.95 at pH 6.5. The change in these ratios indicates that the contrast agent obtained in this embodiment can respond in a weakly acidic environment and transitions from a T1-type contrast agent to a T2-type contrast agent.

[0137] This embodiment shows a 2.5-fold increase in the ratio of 10.87 compared to that in "CN113616815A-pH-responsive T1 / T2 switching MRI contrast agent and its preparation method and application". This indicates that the pH-responsive magnetic resonance imaging contrast agent effectively improves sensitivity and reduces background signal interference, and has specificity and selectivity for tumor imaging.

[0138] T2-weighted imaging at different concentrations shows that the T2 contrast agent obtained in Example 1 produces significantly darker images under weakly acidic conditions than under neutral conditions.

[0139] Performance Test 3

[0140] The contrast agent obtained in Example 1 of this invention was tested for toxicity to normal non-cancer cells and cancer cells (4T1). The operation method includes:

[0141] The cytotoxicity of the contrast agent obtained in this example in human umbilical vein endothelial cells (HUVEC cells) and mouse breast cancer cells (4T1) was determined using the tetrazolium salt colorimetric method (WST method).

[0142] HUVEC cells or 4T1 cells were seeded into 96-well plates at a density of 5000–8000 cells per well at 100 μL. The 96-well plates were then placed in a CO2 incubator and cultured at 37°C for 24 h. The contrast agent of the present invention was dissolved in complete culture medium and filtered to remove bacteria. The contrast agent of the present invention was then diluted with complete culture medium (culture medium without added contrast agent) to prepare several groups of culture media with concentrations ranging from 0.10 to 4 mM.

[0143] Remove the old culture medium from the 96-well plate, and then add different concentrations of culture medium to the 96-well plate, 100 μL per well. The control group is added with 100 μL of complete culture medium, and the plates are incubated for another 24 h. Finally, remove all culture medium, add 100 μL of fresh complete culture medium to each well, and then add 10 μL of WST-1 (a compound similar to MTT, which, in the presence of electron coupling reagents, can be reduced by some dehydrogenases in mitochondria to produce orange-yellow formazan. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. Its full name is 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, 2-(4-iodophenyl)-3-(4-nitrobenzene)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) to each well. Incubate in an incubator for 2 hours, and measure the absorbance (OD) at 450 nm using a microplate reader. 450nm Four replicates were performed for each contrast agent concentration (referred to as the experimental group) and the control group. The relative cell viability was calculated based on the absorbance values. The blank group consisted of complete culture medium without cells, and the control group consisted of cells without culture medium.

[0144] Relative cell viability (%) = 100 × (Experimental group OD - Blank group OD) - (Control group OD - Blank group OD)

[0145] like Figure 9 As shown, even with an iron concentration of 4 mM, the cell survival rate of HUVEC and 4T1 cells in the ESIONPssystem group was still above 95%, indicating that the prepared pH-responsive magnetic resonance imaging contrast agent ESIONPs-PEG-PSDM has almost no toxicity to normal cells and has good biocompatibility.

[0146] Performance Test 4

[0147] The tissue toxicology test of the contrast agent obtained in Example 1 of this invention includes the following operation method:

[0148] The tissue toxicity of the contrast agent obtained in this example in normal BALB-c mice was determined using hematoxylin-eosin staining (H&E staining).

[0149] Four-week-old normal BALB-c mice were divided into two groups:

[0150] The first group received saline injection via the tail vein as a control group;

[0151] The second group received a tail vein injection of a saline solution containing the contrast agent obtained in this embodiment, wherein the iron ion concentration was 0.1 mmol / kg;

[0152] Two days after being raised under normal conditions, the animals were euthanized by cervical dislocation. Heart, liver, spleen, lung, and kidney were collected for H&E staining and microscopic observation.

[0153] like Figure 10 As shown, the contrast agent obtained in Example 1 caused minimal damage to various organs and tissues, and no significant increase was observed in high-concentration samples. Specifically, the hepatocytes in the liver sections were relatively normal, with no signs of inflammation. No pulmonary fibrosis was observed in the lung sections. No tissue necrosis was observed in any of the other section samples. This indicates that the contrast agent described in this invention does not cause significant pathological changes or damage to vital organs, suggesting that this contrast agent has excellent biocompatibility and safety.

[0154] Performance Test 5

[0155] The in vivo MRI imaging experiment of the contrast agent obtained in Example 1 of this invention includes the following operation method:

[0156] A BALB-c mouse model of 4T1 breast cancer cells was established. First, tumor-bearing mice were divided into two groups: a T1-weighted and a T2-weighted magnetic resonance imaging (MRI) experimental group. The mice were intraperitoneally injected with 20% urethane solution at a dose of 5 mL / kg body weight. After deep anesthesia, blank T1- and T2-weighted MRI scans were performed before contrast agent injection. Then, tumor-bearing mice injected via tail vein with ESIONPs-PEG-PSDM served as the experimental group, with an iron ion dose of 0.1 mmol / kg body weight. The tumor-bearing mice were then fixed and placed in a 1.5T miniature MRI scanner. T1- and T2-weighted MRI images were taken at 2, 4, and 6 hours post-injection.

[0157] like Figure 11As shown, after injecting four groups of contrast agents into tumor-bearing mice via the tail vein at a dose of 0.1 mmol-kg, the two groups exhibited significantly different imaging enhancement effects. To ensure the comparability of the MRI images, the sequence parameters were uniformly set to TE = 60.86 ms and TR = 3000 ms. In the first group of T1 MRI experiments, one hour after tail vein injection of ESIONPs-PEG-PSDM, the MR image of the tumor site was slightly brighter, and the image brightness gradually weakened at the next two time points, indicating that the T1 signal of the contrast agent gradually disappeared at the tumor site. In the second group of T2 MRI experiments, the image of the tumor site was significantly darker after injection, indicating that the contrast agent had a significant T2 signal at the tumor site. Therefore, analysis based on the above results shows that ESIONPs-PEG-PSDM undergoes hydrophilic-hydrophobic conversion in the acidic environment of the tumor, leading to aggregation and conversion from a T1 contrast agent to a T2 contrast agent, thereby activating the T2 MR enhancement effect and achieving high selectivity and specificity at the tumor site.

[0158] Furthermore, the pH-responsive magnetic resonance imaging contrast agent provided by this invention is composed of ferric oxide nanoparticles made of surface-modified stimuli-responsive polymers. The surface-modified stimuli-responsive polymers undergo a hydrophilic-hydrophobic switching response in a weakly acidic environment, resulting in a change from hydrophilic to hydrophobic. Compared to the pH response point of 6.5 in "CN113616815A - pH-responsive T1_T2 switching MRI contrast agent and its preparation method and application," this hydrophobic interaction results in a pH response point of 6.6. The higher weakly acidic pH response point better matches the tumor microenvironment and effectively enhances the aggregation effect of nanoparticles at the tumor site. Through hydrophobic interactions, this pH-responsive magnetic resonance imaging contrast agent aggregates. Therefore, this switching from a T1-type contrast agent to a T2-type contrast agent rapidly activates the T2 contrast enhancement effect at the tumor site, improving the imaging contrast of the tumor site.

[0159] In summary, the pH-responsive magnetic resonance imaging contrast agent provided by this invention exhibits high specificity and selectivity for tumor sites, good biocompatibility, and low toxicity. It is also rapidly metabolized in vivo and can selectively activate the T2 MR signal in the weakly acidic microenvironment of the tumor, converting from a T1 contrast agent to a T2 contrast agent to produce T2 contrast enhancement. This results in excellent imaging contrast performance, avoids interference from background signals, enhances the EPR effect, and activates T2 contrast enhancement. Furthermore, it demonstrates high selectivity and specificity for tumor sites and good biocompatibility.

[0160] In addition, the inventors of this case also conducted corresponding experiments using other raw materials and process conditions listed above to replace the various raw materials and corresponding process conditions in Examples 1-3. The biocompatibility, safety, relaxation rate and imaging contrast performance of the obtained magnetic resonance imaging contrast agent are also quite ideal.

[0161] It should be noted that the specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

[0162] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A pH-responsive magnetic resonance imaging contrast agent, characterized in that, The magnetic resonance imaging contrast agent has the following structural formula: Wherein, X is an assembly formed by adamantane and cyclodextrin; Y is m is 30-50, n is 5-20, and M is ferric oxide nanoparticles with a particle size of less than 5 nm.

2. The method for preparing the pH-responsive magnetic resonance imaging contrast agent as described in claim 1, characterized in that, include: A nucleophilic acyl substitution reaction was carried out on a first homogeneous mixture containing sulfadimethoxypyrimidine and methacryloyl chloride to prepare sulfadimethoxypyrimidine containing methacryloyl chloride. A second homogeneous mixed reaction system comprising sulfadimethoxypyrimidine containing methpropylene, 4-cyano-4-(thiobenzoyl)valerate and azobisisobutyronitrile is subjected to a reversible addition-fracture chain transfer polymerization reaction under a protective atmosphere to obtain carboxyl-containing polysulfadimethoxypyrimidine. A condensation reaction is carried out in a third homogeneous mixture containing the carboxyl-containing polysulfonamide dimethoxypyrimidine, a fully amino betacyclodextrin, and a 1-hydroxybenzotriazole to obtain polysulfonamide dimethoxypyrimidine containing cyclodextrin groups. A fourth homogeneous mixed reaction system containing 1-adamantane carboxylic acid and pentafluorophenol was subjected to a carboxyl activation reaction under a protective atmosphere to obtain adamantane molecules containing carboxyl activated esters. A fifth homogeneous mixing reaction system containing adamantane molecules with carboxyl-activated esters and polyethylene glycol with amino and ditert-butyl dicarbonate-protected amino groups undergoes a condensation reaction under an alkaline environment to obtain polyethylene glycol with adamantane and amino groups at the end. A sixth uniform mixing reaction system containing polyethylene glycol with end groups of adamantyl and amino is reacted with ferric oxide nanoparticles with surface-modified carboxyl activated esters to obtain ferric oxide nanoparticles with surface partially modified with adamantyl groups by polyethylene glycol. The host-guest self-assembly of the seventh homogeneous mixing reaction system of ferric oxide nanoparticles with polyethylene glycol partially modified adamantyl group and polysulfamine dimethoxypyrimidine containing cyclodextrin group was carried out to obtain ferric oxide nanoparticles with polysulfamine dimethoxypyrimidine on the surface, which is the pH-responsive magnetic resonance imaging contrast agent.

3. The preparation method according to claim 2, characterized in that... Specifically, it includes: Sulfamethoxypyrimidine was dissolved in an aqueous sodium hydroxide solution and acetone to form a solution. Then, methacryloyl chloride was added dropwise to the solution under ice bath to form a first homogeneous mixed reaction system, and a nucleophilic acyl substitution reaction was carried out for 2-6 hours to obtain the methacryloyl-containing sulfamethoxypyrimidine. The volume ratio of the aqueous sodium hydroxide solution to acetone was 1-3:

1. The molar ratio of sulfadimethoxypyrimidine to methacryloyl chloride is 3 to 1:

1.

4. The preparation method according to claim 2, characterized in that... Specifically, it includes: Under a protective atmosphere, the methpropylene-containing sulfamethoxypyrimidine was dissolved in dimethyl sulfoxide, and then 4-cyano-4-(thiobenzoyl)valerate and azobisisobutyronitrile solution were added to form a second homogeneous mixed reaction system. After freezing and deoxygenation treatment, a reversible addition-fragmentation chain transfer polymerization reaction was carried out at 70-100°C for 24-48 h to obtain the carboxyl-containing polysulfamethoxypyrimidine.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the methpropylene-containing sulfadimethoxypyrimidine to 4-cyano-4-(thiobenzoyl)valerate is 1:25-50.

6. The preparation method according to claim 2, characterized in that... Specifically, it includes: The carboxyl-containing polysulfamine dimethoxypyrimidine, all-amino beta-cyclodextrin, and 1-hydroxybenzotriazole were dissolved in dimethylformamide to form a third homogeneous mixed reaction system, and a condensation reaction was carried out at 20-25°C for 24-48 hours to obtain the polysulfamine dimethoxypyrimidine containing cyclodextrin groups.

7. The preparation method according to claim 6, characterized in that: The molar ratio of the carboxyl-containing polysulfamine dimethoxypyrimidine to the fully amino betacyclodextrin is 5–10:

1.

8. The preparation method according to claim 6, characterized in that: The molar ratio of the carboxyl-containing polysulfamine dimethoxypyrimidine to 1-hydroxybenzotriazole is 5 to 10:

1.

9. The preparation method according to claim 6, characterized in that... Also includes: After the condensation reaction is completed, the resulting mixture is subjected to dialysis and freeze-drying. The dialysis bags used in the dialysis treatment have a molecular weight cutoff of 5000–8000 Da.

10. The preparation method according to claim 9, characterized in that: The dialysis bags used in the dialysis treatment have a molecular weight cutoff of 5000–7000 Da.

11. The preparation method according to claim 2, characterized in that... Specifically, it includes: Under a protective atmosphere, 1-adamantane carboxylic acid and pentafluorophenol are dissolved in 1,4-dioxane, and then dicyclohexylcarbodiimide is added to form a fourth homogeneous mixed reaction system. The carboxyl group activation reaction is carried out at 20-25°C for 24-48 hours to obtain the adamantane molecule containing the carboxyl group activated ester.

12. The preparation method according to claim 11, characterized in that: The molar ratio of pentafluorophenol to 1-adamantanecarboxylic acid is 2 to 10:

1.

13. The preparation method according to claim 11, characterized in that: The molar ratio of pentafluorophenol to dicyclohexylcarbodiimide is 1 to 3:

1.

14. The preparation method according to claim 2, characterized in that... Specifically, it includes: The adamantane molecule containing carboxyl activated ester and the polyethylene glycol with amino and ditert-butyl dicarbonate end groups were dissolved in dichloromethane, and then N,N-diisopropylethylamine was added to form a fifth homogeneous mixed reaction system. The mixture was subjected to a condensation reaction at 20-25°C for 24-48 hours under alkaline conditions to obtain the polyethylene glycol with adamantyl and amino end groups.

15. The preparation method according to claim 14, characterized in that: The molar ratio of N,N-diisopropylethylamine to adamantane molecules containing carboxyl activated esters is 10–20:

1.

16. The preparation method according to claim 14, characterized in that: The molar ratio of the adamantane molecule containing a carboxyl-activated ester to the polyethylene glycol with an amino group protected by di-tert-butyl dicarbonate is 5–10:

1.

17. The preparation method according to claim 14, characterized in that: The pH value of the alkaline environment is 8 to 10.

18. The preparation method according to claim 2, characterized in that... Specifically, it includes: Ferric oxide nanoparticles with surface-modified carboxyl-activated esters were mixed with polyethylene glycol with terminal groups of adamantyl and amino in chloroform, and then N,N-diisopropylethylamine was added to form a sixth homogeneous mixing reaction system. The mixture was reacted at 20-25°C for 12-24 hours to obtain ferric oxide nanoparticles with surface-modified adamantyl groups by polyethylene glycol. The mass ratio of the ferric oxide nanoparticles with surface-modified carboxyl-activated esters to the polyethylene glycol with terminal groups of adamantyl and amino was 1:5-10.

19. The preparation method according to claim 18, characterized in that: The surface-modified carboxyl-activated ester ferric oxide nanoparticles include surface-modified pentafluorophenol ester ferric oxide nanoparticles.

20. The preparation method according to claim 18, characterized in that: The molar ratio of N,N-diisopropylethylamine to polyethylene glycol with terminal groups of adamantyl and amino is 100-200:

1.

21. The preparation method according to claim 2, characterized in that... Specifically, it includes: The ferric oxide nanoparticles with polyethylene glycol-modified adamantyl groups were dissolved in an aqueous sodium hydroxide solution, and then polysulfamine dimethoxypyrimidine containing cyclodextrin groups was added to form a seventh homogeneous mixing reaction system. The host-guest self-assembly reaction was carried out at 20-25°C for 24-48 hours to obtain the ferric oxide nanoparticles with polysulfamine dimethoxypyrimidine on the surface.

22. The preparation method according to claim 21, characterized in that: The molar ratio of the ferric oxide nanoparticles with polyethylene glycol partially modified adamantyl groups to polysulfamine dimethoxypyrimidine containing cyclodextrin groups is 1:10-20.

23. The preparation method according to claim 21, characterized in that... Also includes: After the host-guest self-assembly reaction is completed, the resulting mixture is subjected to dialysis and freeze-drying; the dialysis bag used for the dialysis treatment has a molecular weight cutoff of 30,000 to 50,000 Da.

24. Use of the pH-responsive magnetic resonance imaging contrast agent of claim 1 in the preparation of a product with tumor detection function.

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