A basic responding t1-type magnetic resonance imaging contrast agent and its preparation method and application

Abstract

This invention provides an alkaline-responsive T1-type magnetic resonance imaging contrast agent, its preparation method, and its application. The contrast agent comprises a nanogel carrier formed from borate ester monomers, hydroxyl monomers, and a crosslinking agent, and an imaging substance loaded within the carrier. The borate ester monomers include acryloyloxy polyethylene glycol borate and / or acryloyloxy polyethylene glycol phenylboronic acid ester; the hydroxyl monomers include hydroxypropyl methacrylate and / or hydroxyethyl acrylate. The contrast agent releases the imaging substance under alkaline conditions, achieving a significant change in T1 relaxation rate, specifically enhancing the magnetic resonance imaging signal of the alkaline tumor microenvironment, and delaying the precipitation and metabolic clearance of the imaging substance.

Description

Technical Field

[0001] This invention belongs to the field of medical imaging technology, specifically relating to an alkaline-responsive T1-type magnetic resonance imaging contrast agent, its preparation method, and its application. Background Technology

[0002] Magnetic resonance imaging (MRI) is a non-invasive imaging technique widely used in clinical diagnosis and medical research, offering advantages such as high spatial resolution, no ionizing radiation, and multi-parameter imaging. T1-weighted contrast agents produce bright signal enhancement in images by shortening the longitudinal relaxation time (T1) of water protons, and are commonly used for imaging lesions such as blood vessels, tumors, and inflammation. However, traditional T1 contrast agents (such as gadolinium complexes) suffer from problems such as lack of microenvironment responsiveness, potential toxicity risks, and low targeting, limiting their application in precision medicine.

[0003] In recent years, pH-responsive contrast agents have attracted widespread attention due to their ability to specifically recognize pathological microenvironments. However, most studies have focused on acidic pH response mechanisms (e.g., the acidic environment of tumor microenvironment pH=6.5-7.0), while research on T1 contrast agents targeting alkaline microenvironments (pH>7.4) is relatively limited. Certain tumors (such as some breast cancers and prostate cancers), as well as inflammatory and metabolic diseases, may be accompanied by local alkalization, necessitating the development of contrast agents that can specifically enhance imaging contrast in alkaline regions.

[0004] Currently, the T1 contrast agents used clinically mainly include small molecule gadolinium (Gd). 3+ Gd complexes (such as Gd-DTPA and Gd-DOTA) have high relaxation efficiency but lack microenvironment responsiveness, and long-term use may lead to gadolinium deposition risks. Manganese (Mn) 2+ It has natural T1 enhancement capabilities, and compared to Gd 3+ Manganese (Mn) 2+ Contrast agents based on Mn have better biocompatibility, but free Mn... 2+ Easily cleared by the liver, resulting in a short imaging time window. Nanoparticle carriers (such as liposomes and polymer nanoparticles) can prolong blood circulation time, but most still rely on passive targeting (EPR effect) and lack intelligent response mechanisms.

[0005] Currently reported pH-responsive contrast agents are mainly based on the following mechanism: utilizing an acidic environment to trigger ligand structural changes (such as hydrazone bond breakage and carboxyl protonation), releasing active metal ions (such as Mn). 2+ Fe 3+ Under the action of specific enzymes (such as MMP-2), the carrier degrades and releases the contrast agent. However, these designs are mostly designed for acidic or neutral environments, and alkaline-responsive T1 contrast agents still face the following challenges: (1) It is difficult to regulate the relaxation efficiency. At high pH, ​​Mn 2+(1) Mn(OH)2 precipitate is easily formed; (2) The ligand is not stable enough and is easily hydrolyzed under alkaline conditions, affecting the integrity of the nanogel structure; (3) The signal change amplitude is limited, and the relaxation rate change of existing alkaline response probes is usually <50%, which is difficult to meet the requirements of high contrast imaging. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an alkaline-responsive T1-type magnetic resonance imaging contrast agent, its preparation method, and its application. The contrast agent provided by the present invention has advantages such as alkaline-responsive design, superior imaging effect, strong controllability, good biocompatibility, and wide application fields, providing broad application prospects for magnetic resonance imaging technology in biomedicine and imaging diagnosis.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides an alkaline-responsive T1-type magnetic resonance imaging contrast agent, the contrast agent comprising a nanogel carrier formed of a borate ester monomer, a hydroxyl monomer and a crosslinking agent, and an imaging substance loaded in the carrier; The borate ester monomers include acryloyloxy polyethylene glycol borate and / or acryloyloxy polyethylene glycol phenylborate. The hydroxy monomers include hydroxypropyl methacrylate and / or hydroxyethyl acrylate.

[0008] In this invention, both the borate ester monomer and the hydroxyl monomer possess carbon-carbon unsaturated double bonds, copolymerizing to form the main chain. A crosslinking agent connects the polymer main chain, forming crosslinking nodes and constructing the basic gel framework. The borate ester groups and hydroxyl groups are exposed in the crosslinking network voids or on the gel surface. The hydroxyl groups enhance the hydrophilicity of the gel carrier, and the imaging material binds to the borate ester groups through coordination bonds, distributing within or on the surface of the gel network.

[0009] The contrast agent provided by this invention is pH-responsive. The borate ester groups undergo deprotonation in an alkaline environment, causing the nanogel to swell and disintegrate, increasing the exposure points of the imaging material or releasing the imaging material, thus achieving a significant change in T1 relaxation rate in an alkaline environment. It specifically enhances the T1-mode magnetic resonance imaging signal in alkaline tumor microenvironments, improves the accuracy of identifying the boundaries of inflammatory microenvironments accompanied by local alkalization, and dynamically monitors pH changes in the tumor microenvironment. It possesses good liquid-phase dispersibility and biocompatibility, resolving the contradiction between the stability and relaxation efficiency of imaging materials in alkaline environments.

[0010] Preferably, the molar ratio of the borate ester monomer, hydroxy monomer, crosslinking agent and imaging substance is 1:(5-10):(0.02-0.1):(0.2-0.5).

[0011] The specific point values ​​in (5-10) can be 5, 5.1, 5.2, 5.5, 5.7, 6, 6.3, 6.5, 6.8, 7, 7.5, 8, 8.5, 9, 9.5 or 10, etc.

[0012] The specific point values ​​in (0.02-0.1) can be 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1, etc.

[0013] The specific point values ​​in (0.2-0.5) can be 0.2, 0.21, 0.22, 0.25, 0.27, 0.3, 0.33, 0.35, 0.38, 0.4, 0.45 or 0.5, etc.

[0014] Preferably, the borate ester monomer includes acryloyloxy polyethylene glycol borate and acryloyloxy polyethylene glycol phenylborate.

[0015] In this invention, acryloyloxy polyethylene glycol borate and acryloyloxy polyethylene glycol phenylboronic acid ester are preferred as borate monomers to synergistically construct the polymer backbone. The three-dimensional gel network formed by the combination of the two has better responsiveness to alkaline environments, thereby achieving better imaging results.

[0016] The structural formula of acryloyloxypolyethylene glycol borate is: n is an integer from 1 to 300.

[0017] The structural formula of acryloyloxypolyethylene glycol phenylboronic acid ester is: n is an integer from 1 to 300.

[0018] Preferably, the molar ratio of acryloyloxy polyethylene glycol borate to acryloyloxy polyethylene glycol phenyl borate is (1-10):(1-10).

[0019] The specific point values ​​in the first (1-10) can be 1, 2.2, 3.5, 4.7, 5, 6.3, 7.5, 8.8, 9 or 10, etc.

[0020] The specific point values ​​in the second (1-10) can be 1, 2.2, 3.5, 4.7, 5, 6.3, 7.5, 8.8, 9 or 10, etc.

[0021] Preferably, the hydroxy monomer includes hydroxypropyl methacrylate and hydroxyethyl acrylate.

[0022] In this invention, hydroxypropyl methacrylate and hydroxyethyl acrylate are preferred as hydroxy monomers to synergistically construct the polymer backbone. The three-dimensional gel network formed by the combination of the two has better responsiveness to alkaline environments, thereby achieving better imaging results.

[0023] Preferably, the molar ratio of hydroxypropyl methacrylate to hydroxyethyl acrylate is (1-10):(1-10).

[0024] The specific point values ​​in the first (1-10) can be 1, 2.2, 3.5, 4.7, 5, 6.3, 7.5, 8.8, 9 or 10, etc.

[0025] The specific point values ​​in the second (1-10) can be 1, 2.2, 3.5, 4.7, 5, 6.3, 7.5, 8.8, 9 or 10, etc.

[0026] Preferably, the crosslinking agent includes any one or a combination of at least two of N,N'-(1,2-dihydroxyethylene)bisacrylamide, 2,2'-bipyridine-4,4'-diacrylamide, or citrate-diacrylamide.

[0027] The structural formula of N,N'-(1,2-dihydroxyethylene)bisacrylamide is: .

[0028] The structural formula of 2,2'-bipyridine-4,4'-diacrylamide is: .

[0029] Preferably, the crosslinking agent is N,N'-(1,2-dihydroxyethylene)bisacrylamide.

[0030] The present invention preferably uses N,N'-(1,2-dihydroxyethylene)bisacrylamide as a crosslinking agent, and the resulting three-dimensional gel network has better responsiveness to alkaline environments, thereby achieving better imaging results.

[0031] Preferably, the imaging material comprises metal ions and / or metal oxides.

[0032] Preferably, the metal elements in the metal ions and metal oxides are each independently selected from any one or a combination of at least two of gadolinium, iron, manganese, cobalt, nickel, holmium, europium, terbium, dysprosium, thulium, or ytterbium.

[0033] Preferably, the hydrated particle size of the contrast agent under neutral conditions is 50-200 nm, for example, it can be 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 150 nm, 170 nm or 200 nm, etc.

[0034] Preferably, the hydrated particle size of the contrast agent under alkaline conditions does not exceed 30 nm, for example, it can be 30 nm, 25 nm, 20 nm, 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm or 10 nm, etc.

[0035] Preferably, the pH value of the alkaline response is 8-12, for example, it can be 8, 8.1, 8.2, 8.5, 8.7, 9, 9.3, 9.5, 9.8, 10, 10.5, 11, 11.5 or 12.

[0036] In a second aspect, the present invention provides a method for preparing the contrast agent as described in the first aspect, the method comprising: (1) After the emulsifier is dissolved in water, borate ester monomer, hydroxy monomer, crosslinking agent and imaging substance are added in sequence, mixed and sonicated to form a nanoemulsion; (2) Mix the nanoemulsion and the initiator, react them, and terminate the reaction process with a terminator to obtain the contrast agent.

[0037] Preferably, in step (1), the emulsifier includes any one or a combination of at least two of sodium dodecyl sulfate, Tween 80, Span 80, Span 60 or poloxamer 188.

[0038] Preferably, the ratio of emulsifier to water is (0.5-2) g:(70-100) mL.

[0039] The specific point values ​​in (0.5-2) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2, etc.

[0040] The specific point values ​​in (70-100) can be 70, 72, 75, 77, 80, 83, 85, 88, 90, 95 or 100, etc.

[0041] Preferably, the mixing method is stirring.

[0042] Preferably, the stirring speed is 300-700 rpm, for example, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm or 700 rpm; the stirring time is 5-15 min, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0043] Preferably, the power of the ultrasound is 100-200 W, for example, 100 W, 110 W, 120 W, 130 W, 140 W, 150 W, 160 W, 170 W, 180 W, 190 W or 200 W; the duration is 3-8 min, for example, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min or 8 min.

[0044] Preferably, in step (2), the initiator includes any one or a combination of at least two of ammonium persulfate, sodium persulfate, or potassium persulfate.

[0045] Preferably, the reaction is carried out under nitrogen protection.

[0046] Preferably, the reaction temperature is 60-70℃, for example, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃ or 70℃, etc.; the time is 4-6 h, for example, 4 h, 4.2 h, 4.5 h, 4.7 h, 5 h, 5.3 h, 5.5 h, 5.8 h or 6 h, etc.

[0047] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0048] Preferably, the terminating agent comprises any one or a combination of at least two of hydroquinone, methylhydroquinone, or benzoquinone.

[0049] Preferably, the process further includes a purification step after the reaction process is terminated.

[0050] Preferably, the purification method is dialysis.

[0051] Thirdly, the present invention provides the use of the contrast agent as described in the first aspect in the preparation of markers, imaging agents or tracers for the diagnosis of triple-negative breast cancer.

[0052] The contrast agent provided by this invention hydrolyzes in an alkaline microenvironment, releasing free imaging substances, reducing the local concentration of imaging substances, enhancing negative MRI contrast signals, and achieving precise tumor imaging. Simultaneously, the contrast agent can be further modified with targeting molecules (such as folic acid, RGD peptides, etc.) to achieve lesion-specific enrichment.

[0053] Compared with the prior art, the present invention has the following beneficial effects: (1) The contrast agent provided by the present invention has pH responsiveness. The borate ester group undergoes deprotonation in an alkaline environment, and the nanogel swells and disintegrates, reducing the concentration of local imaging material and enhancing the negative contrast ability of magnetic resonance imaging. (2) The contrast agent provided by the present invention can achieve controllable loading and sustained release of imaging material. The borate ester group forms a dynamic coordination bond with the imaging material and partially dissociates under alkaline conditions, achieving a significant change in T1 relaxation rate. The encapsulation of the nanogel network prevents the imaging material from being precipitated under high alkaline conditions, delays the metabolic clearance of the imaging material, and prolongs the imaging window period. Attached Figure Description

[0054] Figure 1 Transmission electron microscopy image of the T1 type magnetic resonance imaging contrast agent provided in Example 1 under neutral (pH=7.4) conditions; Figure 2 Transmission electron microscopy image of the T1 type magnetic resonance imaging contrast agent provided in Example 1 under alkaline conditions (pH=8.0); Figure 3 Hydration particle size distribution of the T1 type magnetic resonance imaging contrast agent provided in Example 1 under neutral (pH=7.4) and alkaline (pH=8.0) environments; Figure 4 Electromotive force diagrams of the T1 type magnetic resonance imaging contrast agent provided in Example 1 under neutral (pH=7.4) and alkaline (pH=8.0) environments; Figure 5 The curves showing the change in longitudinal relaxation time of the T1 type magnetic resonance imaging contrast agent provided in Example 1 under neutral (pH=7.4) and alkaline (pH=8.0) environments as a function of incubation time; Figure 6 Linear fitting plots of the longitudinal relaxation rate of the T1 type magnetic resonance imaging contrast agent provided in Example 1 under neutral (pH=7.4) and alkaline (pH=8.5) environments; Figure 7 T1-weighted imaging images of the T1-type magnetic resonance imaging contrast agent provided in Example 1 under neutral (pH=7.4) and alkaline (pH=8.5) environments; Figure 8 The cell viability graph is shown after 24 hours of co-culturing the T1 type magnetic resonance imaging contrast agent provided in Example 1 with triple-negative mouse breast cancer cells 4T1. Figure 9 T1-weighted imaging of the T1-type magnetic resonance imaging contrast agent provided in Example 1 and triple-negative mouse breast cancer cells 4T1 cells after co-culturing for 12 hours in neutral (pH=7.4) and alkaline (pH=8.5) environments. Detailed Implementation

[0055] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0056] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0057] 1 mol of hydroxyl-terminated PEG2000 borate was dissolved in anhydrous dichloromethane, and 3 mol of triethylamine was added and mixed thoroughly. 2 mol of acryloyl chloride was then slowly added dropwise under ice bath conditions at 0°C. The reaction was first carried out at 0°C for 2 h, and then at 25°C for 24 h. After the reaction was completed, the mixture was purified to obtain acryloyloxy polyethylene glycol borate.

[0058] 1 mol of hydroxyl-terminated PEG2000 phenylboronic acid ester was dissolved in anhydrous dichloromethane, and 3 mol of triethylamine was added and mixed thoroughly. 2 mol of acryloyl chloride was then slowly added dropwise under ice bath conditions at 0°C. The reaction was first carried out at 0°C for 2 h, and then at 25°C for 24 h. After the reaction was completed, the mixture was purified to obtain acryloyloxy polyethylene glycol phenylboronic acid ester.

[0059] 1 mol of 4,4'-diamino-2,2'-bipyridine was dissolved in anhydrous dichloromethane, and 3 mol of triethylamine was added and mixed thoroughly. 2 mol of acryloyl chloride was then slowly added dropwise under ice bath conditions at 0°C. The reaction was first carried out at 0°C for 2 h, and then at 25°C for 24 h. After the reaction was completed, the mixture was purified to obtain 2,2'-bipyridine-4,4'-diacrylamide.

[0060] Example 1 This embodiment provides a T1 type magnetic resonance imaging contrast agent, the preparation method of which includes: (1) Dissolve 1 g of sodium dodecyl sulfate in 80 mL of water, and then add borate ester monomers (0.005 mol acryloyloxy polyethylene glycol borate and 0.005 mol acryloyloxy polyethylene glycol phenylboronic acid ester), hydroxy monomers (0.03 mol hydroxypropyl methacrylate and 0.02 mol hydroxyethyl acrylate), crosslinking agent (0.0005 mol N,N'-(1,2-dihydroxyethylene)bisacrylamide) and 0.004 mol manganese ions in sequence; stir at 500 rpm for 10 min, and then sonicate at 150 W for 5 min to form a nanoemulsion; (2) Add 10 mL of an aqueous solution containing 1% ammonium persulfate to the nanoemulsion obtained in step (1), react at 65°C for 5 h under nitrogen protection, and then terminate the reaction with hydroquinone; dialyze with a 3.5 kDa dialysis bag for 72 h and then freeze dry to obtain the contrast agent.

[0061] Example 2 This embodiment provides a T1 type magnetic resonance imaging contrast agent, the preparation method of which includes: (1) Dissolve 0.8 g Tween 80 in 80 mL of water, then add borate ester monomers (0.003 mol acryloyloxy polyethylene glycol borate and 0.007 mol acryloyloxy polyethylene glycol phenylboronic acid ester), hydroxy monomers (0.03 mol hydroxypropyl methacrylate and 0.03 mol hydroxyethyl acrylate), crosslinking agent (0.0010 mol N,N'-(1,2-dihydroxyethylene)bisacrylamide) and 0.005 mol gadolinium ions in sequence; stir at 300 rpm for 15 min, then sonicate at 200 W for 3 min to form a nanoemulsion; (2) Add 10 mL of an aqueous solution containing 1% sodium persulfate to the nanoemulsion obtained in step (1), react at 60°C for 6 h under nitrogen protection, and then terminate the reaction with hydroquinone; dialyze with a 3.5 kDa dialysis bag for 72 h and then freeze dry to obtain the contrast agent.

[0062] Example 3 This embodiment provides a T1 type magnetic resonance imaging contrast agent, the preparation method of which includes: (1) Dissolve 1.6 g of Span 80 in 80 mL of water, then add borate ester monomers (0.007 mol acryloyloxy polyethylene glycol borate and 0.003 mol acryloyloxy polyethylene glycol phenylboronic acid ester), hydroxy monomers (0.02 mol hydroxypropyl methacrylate and 0.05 mol hydroxyethyl acrylate), crosslinking agent (0.0002 mol N,N'-(1,2-dihydroxyethylene)bisacrylamide) and 0.002 mol iron ions in sequence; stir at 700 rpm for 5 min, then sonicate at 100 W for 8 min to form a nanoemulsion; (2) Add 10 mL of an aqueous solution containing 1% potassium persulfate to the nanoemulsion obtained in step (1), react at 70°C for 4 h under nitrogen protection, and then terminate the reaction with hydroquinone; dialyze with a 3.5 kDa dialysis bag for 72 h and then freeze dry to obtain the contrast agent.

[0063] Example 4 This embodiment provides a T1 type magnetic resonance imaging contrast agent, which differs from Example 1 only in that: acryloyloxy polyethylene glycol borate is not added to the borate ester monomer, and the reduction is made up by acryloyloxy polyethylene glycol phenylboronic acid ester, while the other raw materials and steps remain unchanged.

[0064] Example 5 This embodiment provides a T1 type magnetic resonance imaging contrast agent, which differs from Example 1 only in that: acryloyloxy polyethylene glycol phenylboronic acid ester is not added to the borate ester monomer, and the reduction is made up by acryloyloxy polyethylene glycol borate ester, while the other raw materials and steps remain unchanged.

[0065] Example 6 This embodiment provides a T1 type magnetic resonance imaging contrast agent, which differs from Example 1 only in that: hydroxypropyl methacrylate is not added to the hydroxy monomer, and the reduction is made up by hydroxyethyl acrylate, while the other raw materials and steps remain unchanged.

[0066] Example 7 This embodiment provides a T1 type magnetic resonance imaging contrast agent, which differs from Example 1 only in that: hydroxyethyl acrylate is not added to the hydroxy monomer, and the reduction is made up by hydroxypropyl methacrylate, while the other raw materials and steps remain unchanged.

[0067] Example 8 This embodiment provides a T1 type magnetic resonance imaging contrast agent, which differs from Example 1 only in that the crosslinking agent N,N'-(1,2-dihydroxyethylene)bisacrylamide is replaced with an equal amount of 2,2'-bipyridine-4,4'-diacrylamide, while the other raw materials and steps remain unchanged.

[0068] Example 9 This embodiment provides a T1 type magnetic resonance imaging contrast agent, which differs from Example 1 only in that the crosslinking agent N,N'-(1,2-dihydroxyethylene)bisacrylamide is replaced with an equal amount of N,N'-methylenebisacrylamide, while the other raw materials and steps remain unchanged.

[0069] Comparative Example 1 This comparative example provides a T1 type magnetic resonance imaging contrast agent, which differs from Example 1 only in that the borate ester monomers "0.005 mol acryloyloxy polyethylene glycol borate and 0.005 mol acryloyloxy polyethylene glycol phenylboronic acid ester" are replaced with "0.005 mol phosphate acrylate and 0.005 mol methacrylate sulfonate", while the other raw materials and steps remain unchanged.

[0070] Comparative Example 2 This comparative example provides a T1 type magnetic resonance imaging contrast agent, which differs from Example 1 only in that the hydroxy monomers "0.03 mol hydroxypropyl methacrylate and 0.02 mol hydroxyethyl acrylate" are replaced with "0.03 mol hydroxyethyl methacrylate and 0.02 mol polyvinyl alcohol", while the other raw materials and steps remain unchanged.

[0071] Test Example 1 Take 1 mg of the T1 type magnetic resonance imaging contrast agent provided in Example 1 and dissolve it in 1 mL of deionized water (pH=7.4) to obtain solution 1; take 1 mg of the T1 type magnetic resonance imaging contrast agent provided in Example 1 and dissolve it in 1 mL of PBS buffer (pH=8.0), and react at 37℃ for 12 h to obtain solution 2.

[0072] Take 5 μL of either solution 1 or solution 2 and drop it onto the carbon support membrane. Blot away excess liquid with filter paper and allow to air dry at 25°C until the solvent has completely evaporated. Then, observe the morphology of the sample under a transmission electron microscope. Solution 1 is as follows... Figure 1 As shown, the alkaline-responsive T1-type magnetic resonance imaging contrast agent is a typical monodisperse spherical nanogel with a relatively smooth surface and uniform morphology. Solution 2 is as follows: Figure 2 As shown, the T1 type magnetic resonance imaging contrast agent disintegrates under alkaline conditions, producing loose, irregular fragments, and has good alkaline response capability.

[0073] Take 5 μL of either solution 1 or solution 2 into the sample cell, and measure the hydrated particle size of the sample using a laser particle size analyzer. Figure 3 As shown, the T1 type magnetic resonance imaging contrast agent has a particle size of 50-200 nm under neutral conditions, while the particle size shrinks to about 15 nm under alkaline conditions. This indicates that the T1 type magnetic resonance imaging contrast agent can achieve hydrolysis and degradation of nanogels through alkaline response, thereby releasing free imaging substances.

[0074] Take 5 μL of either solution 1 or solution 2 into the sample cell, and measure the zeta potential of the sample by dynamic light scattering. Figure 4 As shown, the Zeta potential of the T1 type magnetic resonance imaging contrast agent is -13 mV under neutral conditions and -33 mV under alkaline conditions. This indicates that the borate ester group of the T1 type magnetic resonance imaging contrast agent is deprotonated under alkaline conditions, which enhances the surface negative charge density and promotes the swelling of the nanogel and the release of imaging material.

[0075] Test Example 2 Five mg of the T1-type magnetic resonance imaging contrast agent provided in Example 1 was dissolved in 5 mL of PBS buffer (pH=8.0) and reacted at 37°C. 150 μL of the solution was taken at 0 h, 0.2 h, 0.3 h, 0.5 h, 0.7 h, 0.8 h, 1.0 h, 1.2 h, 1.3 h, 1.5 h, 1.7 h, 1.8 h, 2.0 h, 3.0 h, 5.0 h, 8.0 h, and 20 h, respectively, and the longitudinal relaxation time was measured on a 0.5 T pulsed magnetic resonance imaging system. Simultaneously, the relaxation time of the contrast agent in deionized water (pH=7.4) was tested as a control to assess its variation under alkaline conditions.

[0076] like Figure 5 As shown, the longitudinal relaxation time of the T1 type magnetic resonance imaging contrast agent gradually increases with time under alkaline conditions, while it remains almost unchanged under neutral conditions. This indicates that the longitudinal relaxation rate of the contrast agent decreases after alkaline response, thus improving the negative contrast capability of magnetic resonance imaging.

[0077] Test Example 3 5 mg of the T1-type magnetic resonance imaging contrast agent provided in Examples 1-9 and Comparative Examples 1-2 were dissolved in 5 mL of PBS buffer (pH=8.5) and reacted at 37°C for 12 h. Then, aqueous solutions of different concentrations (3.67 mM, 1.84 mM, 0.92 mM, 0.46 mM, and 0.23 mM for imaging metal ions) were prepared for each group. 150 μL of each solution was added to the sample cell, and T1 tests were performed on a 0.5 T pulsed NMR analyzer. Based on the relaxation times obtained for different concentrations, a linear fit was performed with the reciprocal of the relaxation time (1 / T1) as the x-axis and the imaging ion concentration as the y-axis to obtain the longitudinal relaxation rate (r1) of the contrast agent after alkaline response. The case of T1-type magnetic resonance imaging contrast agent dissolved in deionized water (pH=7.4) was used as a control.

[0078] The linear fitting of the T1-type magnetic resonance imaging contrast agent provided in Example 1 is as follows: Figure 6 As shown, its longitudinal relaxation rate under neutral conditions is 10.92 mM. -1 s -1 The longitudinal relaxation rate under alkaline conditions is 1.70 mM. -1 s -1 This indicates that the T1 signal of the contrast agent weakens after the alkaline response, and the negative contrast ability is enhanced.

[0079] Furthermore, as shown in Table 1, the types of borate ester monomers, hydroxy monomers, and crosslinking agents further affect the three-dimensional network structure of the nanogel, the release effect of imaging substances under alkaline conditions, and thus its imaging ability.

[0080] Table 1

[0081] Test Example 4 The T1-type magnetic resonance imaging contrast agent provided in Example 1 was prepared into solutions of different concentrations (0.46 mM, 0.23 mM, 0.12 mM, 0.06 mM, and 0.03 mM manganese ions) using deionized water (pH=7.4) or PBS buffer (pH=8.5). 150 μL of each solution was added to the sample cell, and T1-weighted solution imaging was performed on a 0.5 T pulsed NMR analyzer.

[0082] like Figure 7 As shown, the T1 type magnetic resonance imaging contrast agent produces darker images and has enhanced negative contrast capabilities after undergoing an alkaline response.

[0083] Test Example 5 Triple-negative mouse breast cancer cells (4T1 cells) were seeded at a density of 8000 cells / well in 96-well plates. After 12 hours, the T1-type magnetic resonance imaging contrast agent provided in Example 1 was prepared into solutions of different concentrations (1 mM, 0.5 mM, 0.25 mM, 0.125 mM, and 0.0625 mM manganese ions) using culture medium and co-cultured with the cells. CCK8 toxicity was tested after 24 hours.

[0084] like Figure 8 As shown, even at a relatively high concentration of 1.0 mM, the T1-type magnetic resonance imaging contrast agent still exhibits good cell activity in triple-negative mouse breast cancer cells, indicating that the contrast agent has good biosafety and can be used in biomedical research and clinical practice.

[0085] Test Example 6 Triple-negative mouse breast cancer cells (4T1 cells) were seeded at a density of 8000 / well in 96-well plates. After 12 hours, the T1-type magnetic resonance imaging contrast agent provided in Example 1 was dissolved in culture medium at pH 7.4 or pH 8.5 (0.23 mM manganese ions) and co-cultured with the cells for 12 hours. The cells were then collected and T1-weighted imaging was performed on a 0.5 T pulsed nuclear magnetic resonance analyzer. Untreated cells served as blank controls.

[0086] like Figure 9 As shown, under neutral conditions, the brightness of cells labeled with T1-type magnetic resonance imaging contrast agent is much higher than that of cells without contrast agent labeling. However, under alkaline conditions, the brightness of contrast agent-labeled cells is significantly reduced, showing a negative MRI signal. This indicates that an alkaline environment can induce changes in the T1 signal of the contrast agent, thereby causing the image to darken from bright to dark, thus achieving responsiveness to an alkaline environment.

[0087] This invention illustrates an alkaline-responsive T1-type magnetic resonance imaging contrast agent, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials in the product, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0088] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0089] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A basic responsive T1 -weighted magnetic resonance imaging contrast agent, characterized in that, The contrast agent includes a nanogel carrier formed from borate ester monomers, hydroxy monomers and crosslinking agents, and an imaging substance loaded in the carrier; The borate ester monomers include acryloyloxy polyethylene glycol borate and / or acryloyloxy polyethylene glycol phenylborate. The hydroxy monomers include hydroxypropyl methacrylate and / or hydroxyethyl acrylate.

2. The contrast agent of claim 1, wherein, The molar ratio of the borate ester monomer, hydroxyl monomer, crosslinking agent and imaging material is 1:(5-10):(0.02-0.1):(0.2-0.5).

3. The contrast agent of claim 1, wherein, The borate ester monomers include acryloyloxy polyethylene glycol borate and acryloyloxy polyethylene glycol phenylboronic acid ester.

4. The contrast agent of claim 1, wherein, The hydroxy monomers include hydroxypropyl methacrylate and hydroxyethyl acrylate.

5. The contrast agent of claim 1, wherein, The crosslinking agent includes any one or a combination of at least two of N,N'-(1,2-dihydroxyethylene)bisacrylamide, 2,2'-bipyridine-4,4'-diacrylamide, or citrate-diacrylamide.

6. The contrast agent of claim 1, wherein, The imaging material includes metal ions and / or metal oxides; The metal elements in the metal ions and metal oxides are each independently selected from any one or at least a combination of two of gadolinium, iron, manganese, cobalt, nickel, holmium, europium, terbium, dysprosium, thulium, or ytterbium.

7. The method of claim 1-6, wherein, The method includes: (1) After the emulsifier is dissolved in water, borate ester monomer, hydroxy monomer, crosslinking agent and imaging substance are added in sequence, mixed and sonicated to form a nanoemulsion; (2) Mix the nanoemulsion and the initiator, react them, and terminate the reaction process with a terminator to obtain the contrast agent.

8. The method of claim 7, wherein, In step (1), the emulsifier includes any one or a combination of at least two of sodium dodecyl sulfate, Tween 80, Span 80, Span 60 or poloxamer 188; The mixing method is stirring, with a stirring speed of 300-700 rpm and a stirring time of 5-15 min; The ultrasound power is 100-200 W, and the duration is 3-8 min.

9. The method of claim 7, wherein, In step (2), the initiator includes any one or a combination of at least two of ammonium persulfate, sodium persulfate, or potassium persulfate; The reaction was carried out under nitrogen protection at a temperature of 60-70°C for 4-6 hours. The terminating agent includes any one or a combination of at least two of hydroquinone, methylhydroquinone, or benzoquinone.

10. Use of the contrast agent according to any one of claims 1-6 in the preparation of a marker, imaging agent or tracer for the diagnosis of triple-negative breast cancer.

Images