Magnetic resonance contrast agent, preparation method therefor, and use thereof

Abstract

The present invention relates to a magnetic resonance contrast agent, a preparation method therefor, and use thereof. The magnetic resonance contrast agent comprises a lanthanide-binding protein mutant. The lanthanide-binding protein mutant is formed by mutating the 108th-position asparagine of the lanthanide-binding protein to aspartic acid. In the magnetic resonance contrast agent, the lanthanide-binding protein mutant is formed by mutating the 108th-position asparagine of the lanthanide-binding protein to aspartic acid, which is beneficial to improving the relaxation degree. Tests have proved that the effective relaxation degree of the magnetic resonance contrast agent (about 52 mM-1s-1) is 10 times that of Magnevist (about 5 mM-1s-1), so that the magnetic resonance contrast agent can be applied to magnetic resonance imaging detection.

Description

Magnetic resonance contrast agent and its preparation method and application Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to a magnetic resonance contrast agent and a preparation method and application thereof. Background Art

[0002] The kidneys perform critical metabolic functions, regulating water, electrolyte, and acid-base balances, producing erythropoietin, and influencing bone and mineral metabolism. Due to their diverse functions, the kidneys are vulnerable to factors such as toxins, infections, genetic factors, and hemodynamic changes, which can lead to dysfunction. Kidney disease can progress to chronic kidney disease and renal failure, making early diagnosis of kidney disease crucial. Current diagnostic methods, such as blood tests measuring urea nitrogen and serum creatinine, fail to provide real-time information on the kidney's status. While biopsy provides a definitive diagnosis, it is an invasive procedure and carries the risk of damaging adjacent organs. Several non-invasive imaging techniques, including single-photon emission computed tomography (SPECT), positron emission tomography (PET), and computed tomography (CT), emit radiation. Fluorescence techniques, however, are limited in their tissue penetration, making them difficult to apply in humans. Therefore, there is an urgent need for non-invasive, radiation-free imaging methods for clinical assessment of renal dysfunction.

[0003] Magnetic resonance imaging (MRI), a key tool in medical imaging, plays a significant role in the diagnosis of kidney diseases. Imaging sensitivity can be further improved by injecting contrast agents to enhance imaging contrast. Currently, commonly used MRI contrast agents include Magnevist, Dotarem, and Gadavist. However, these lanthanide-based gadolinium (Gd) contrast agents still face several challenges in clinical application: i) relatively low relaxivities (<10 mM⁻¹ s⁻¹); ii) short circulation times (<30 minutes); and iii) toxic side effects such as gadolinium deposition, skin fibrosis, worsening renal function, and patient allergies. Therefore, the development of novel contrast agents with renal imaging capabilities is of great significance.

[0004] Lanmodulins, discovered in methylotrophic bacteria, have three binding sites for lanthanides (Gd, Nd, Eu, etc.) with picomolar affinity, making them a newly discovered class of molecules with lanthanide selectivity. Currently, they are primarily used for rare earth element separation, with limited research in magnetic resonance imaging applications. Several studies have disclosed metal-binding proteins with highly selective affinities for certain trivalent and / or tetravalent cations derived from rare earth elements or their ions (e.g., lanthanides) and actinides or their ions, as well as hafnium and zirconium or their compounds, as well as sensors containing these metal-binding proteins and methods for using them to capture and separate such trivalent and / or tetravalent cations. Several studies have also described rare earth element (REE) binding proteins (e.g., lanthanide binding proteins), host cells expressing these REE-binding proteins, and methods for recovering REEs. Some studies have prepared a bovine serum albumin-loaded gadolinium-based magnetic resonance contrast agent (BSA-Gd), which is metabolized by the liver. This synthesized albumin-Gd-based magnetic resonance contrast agent not only effectively visualizes blood vessels but also avoids renal metabolism, effectively resolving existing clinical challenges. Some studies have disclosed a low-nephrotoxic protein-iron oxide composite nano-MRI contrast agent, its preparation method, and its application. This contrast agent, with an iron oxide core coated with a functionalized protein, exhibits high uniformity, hydrophilicity, and monodispersity, as well as low nephrotoxicity and good biocompatibility, making it a safe magnetic resonance contrast agent. Some studies have disclosed an MRI contrast agent using an endogenous human protein as a chelating agent and its preparation. The contrast agent comprises a chelating agent and a paramagnetic metal ion coordinated to the chelating agent, with the chelating agent being an endogenous human protein. Some studies have disclosed a protamine-stabilized manganese dioxide nanoparticle complex (PS@MnO2) and its preparation method. This invention uses the positively charged protein protamine to prepare PS@MnO2 through a one-step redox method. The prepared PS@MnO2 is spherical with a particle size of 90-350nm. The magnetic resonance imaging contrast agents disclosed in these studies have low relaxivities and are difficult to meet the demand.

[0005] Summary of the Invention

[0006] Based on this, the present application provides a magnetic resonance contrast agent with higher relaxivity, and its preparation method and application.

[0007] A magnetic resonance contrast agent comprises a lanthanide binding protein mutant, wherein the 108th asparagine of the lanthanide binding protein is mutated to aspartic acid.

[0008] In the above-mentioned magnetic resonance contrast agent, the lanthanide binding protein mutant is mutated from asparagine at position 108 of the lanthanide binding protein to aspartic acid, which is beneficial to improve the relaxivity. The effective relaxivity of the above-mentioned magnetic resonance contrast agent (about 52mM -1 s -1 ) is Magnevist (about 5mM -1 s -1 ) and can be used in magnetic resonance imaging detection.

[0009] In one embodiment, the amino acid sequence of the lanthanide binding protein is shown as SEQ ID No. 1.

[0010] In one embodiment, the lanthanide binding protein mutant binds to a lanthanide ion, and the lanthanide ion includes a gadolinium ion.

[0011] A method for preparing a magnetic resonance contrast agent comprises the following steps: preparing the magnetic resonance contrast agent using genetic engineering technology, wherein the magnetic resonance contrast agent comprises a lanthanide binding protein mutant, wherein the lanthanide binding protein mutant is obtained by mutating the 108th asparagine of the lanthanide binding protein to aspartic acid.

[0012] In one embodiment, the step of preparing the magnetic resonance contrast agent using genetic engineering technology includes:

[0013] preparing the lanthanide-binding protein mutant gene fragment;

[0014] introducing the lanthanide-binding protein mutant gene fragment into a host for expression, and isolating and collecting the lanthanide-binding protein mutant;

[0015] The lanthanide binding protein mutant is mixed with lanthanide ions and incubated to obtain the magnetic resonance contrast agent.

[0016] In one embodiment, overlapping PCR amplification technology is used to mutate the asparagine at position 108 of the lanthanide-binding protein to aspartic acid to obtain the lanthanide-binding protein mutant gene fragment.

[0017] In one embodiment, the amino acid sequence of the lanthanide binding protein is shown as SEQ ID No. 1.

[0018] In one embodiment, the host is Escherichia coli, and the steps of introducing the lanthanide-binding protein mutant gene fragment into the host for expression and isolating and collecting the lanthanide-binding protein mutant include: introducing the lanthanide-binding protein mutant gene fragment into the Escherichia coli host for expression, disrupting the bacteria, isolating and purifying to obtain the lanthanide-binding protein mutant.

[0019] In one embodiment, the lanthanide ions include gadolinium ions;

[0020] And / or, the molar ratio of the lanthanide ion to the lanthanide binding protein mutant is 1-4.

[0021] The use of the magnetic resonance contrast agent or the magnetic resonance contrast agent prepared by the method for preparing the magnetic resonance contrast agent in magnetic resonance imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is an SDS-PAGE image of lanthanide protein before and after mutation;

[0023] FIG2 is a graph showing the evaluation results of the binding performance of lanthanide binding protein with gadolinium ions before and after mutation;

[0024] FIG3 is a photograph and SDS-PAGE image of the lanthanide protein before and after mutation and binding to gadolinium ions;

[0025] FIG4 is a graph showing the results of the magnetic resonance imaging capability assessment of point gene mutation lanthanide binding protein-gadolinium;

[0026] FIG5 is a diagram showing the in vivo magnetic resonance imaging capability and metabolic assessment results of the point gene mutation lanthanide binding protein-gadolinium. DETAILED DESCRIPTION

[0027] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, the following detailed description of the specific embodiments of the present invention is provided in conjunction with specific embodiments and accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] One embodiment of the present application provides a magnetic resonance contrast agent, which includes a lanthanide-binding protein mutant, wherein the lanthanide-binding protein mutant is mutated from asparagine at position 108 of the lanthanide-binding protein to aspartic acid.

[0029] In one embodiment, the amino acid sequence of the lanthanide binding protein is shown in SEQ ID No. 1. Specifically, the sequence shown in SEQ ID No. 1 is MAFRLSSAVLLAALVAAPAYAAPTT TTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGR VSEADLKKLDPDNDGTLDKKEYLAAVEAQFKAANPDNDGTIDARELASPA GSALVNLIR.

[0030] In some embodiments, the lanthanide-binding protein mutants bind to lanthanide ions. The lanthanide ions include gadolinium ions. It should be noted that the lanthanide ions are not limited to gadolinium ions and may also be other lanthanide ions, such as lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, and lutetium.

[0031] In the above-mentioned magnetic resonance contrast agent, the lanthanide binding protein mutant is mutated from asparagine at position 108 of the lanthanide binding protein to aspartic acid, which is beneficial for improving the relaxivity and can be used in magnetic resonance imaging. The effective relaxivity of the above-mentioned magnetic resonance contrast agent (about 52mM -1 s -1 ) is 10 times that of Magnevist (about 5mM-1s-1).

[0032] Furthermore, this MRI contrast agent modifies a lanthanide-binding protein through a single-point genetic mutation, enhancing its binding to gadolinium ions. This property of the binding protein suppresses the molecular rotational correlation time, improving the agent's relaxivity and prolonging its in vivo metabolism. This overcomes the low relaxivity and short imaging window of existing gadolinium-based contrast agents. Furthermore, the lanthanide-binding protein-gadolinium can be metabolized by the kidneys, making it useful for assessing renal function.

[0033] One embodiment of the present application also provides a method for preparing the aforementioned magnetic resonance contrast agent, comprising the following steps: preparing the magnetic resonance contrast agent using genetic engineering techniques. The magnetic resonance contrast agent comprises a lanthanide-binding protein mutant, wherein the lanthanide-binding protein mutant comprises a mutation of asparagine at position 108 of the lanthanide-binding protein to aspartic acid.

[0034] The detailed description of magnetic resonance contrast agents is given above and will not be repeated here.

[0035] In some embodiments, the steps of preparing the magnetic resonance contrast agent using genetic engineering technology include S110-S130:

[0036] S110. Prepare a lanthanide-binding protein mutant gene fragment.

[0037] Among them, overlapping PCR amplification technology was used to mutate the 108th asparagine of the lanthanide binding protein to aspartic acid, and the lanthanide binding protein mutant gene fragment was obtained.

[0038] In one specific example, the amino acid sequence of the lanthanide binding protein is shown in SEQ ID No. 1. Specifically, the sequence shown in SEQ ID No. 1 is MAFRLSSAVLLAALVAAPAYAAPTT TTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGR VSEADLKKLDPDNDGTLDKKEYLAAVEAQFKAADPDNDGTIDARELASPA GSALVNLIR.

[0039] S120. Introduce the lanthanide-binding protein mutant gene fragment into the host for expression, and isolate and collect the lanthanide-binding protein mutant.

[0040] In some embodiments, the host is Escherichia coli. S120 includes the following steps: introducing the lanthanide binding protein mutant gene fragment into the Escherichia coli host for expression, disrupting the cells, and isolating and purifying the cells to obtain the lanthanide binding protein mutant.

[0041] The method of disrupting the cells is not limited, for example, ultrasonic disruption can be used. The method of separation and purification is not limited, for example, Ni column affinity chromatography can be used.

[0042] S130. Mixing the lanthanide binding protein mutant with lanthanide ions and incubating the mixture to obtain a magnetic resonance imaging contrast agent.

[0043] In one embodiment, the lanthanide ions include gadolinium ions.

[0044] In some embodiments, the molar ratio of the lanthanide ion to the lanthanide binding protein mutant is 1-4.

[0045] In the preparation method of the above-mentioned magnetic resonance contrast agent of the present application, the engineered lanthanide-binding protein is expressed by introducing a constructed point mutation gene into Escherichia coli through genetic engineering technology, making full use of synthetic biology technology. The preparation method is simple and has the prospect of large-scale production. In terms of purification and separation, compared with the separation of small molecule chelates and unchelated gadolinium ions, the separation of protein macromolecules and unchelated gadolinium ions is easier to achieve due to the large difference in molecular weight and size.

[0046] Existing protein-based magnetic resonance imaging contrast agents (such as albumin-gadolinium, protein-iron oxide, and protein-manganese dioxide) are difficult to metabolize through the kidneys. The present invention discloses a method for preparing a magnetic resonance imaging contrast agent that produces a genetically modified lanthanide protein with high relaxivity, long circulation time, and high affinity for gadolinium ions. This genetically modified lanthanide protein-gadolinium magnetic resonance contrast agent exhibits renal metabolism and can be used in magnetic resonance imaging.

[0047] Traditional small molecule contrast agents require complex coupling and purification to construct contrast agents with active targeted imaging capabilities. However, the engineered lanthanide-binding proteins constructed through biological methods in this application can be customized through underlying genetic design to synthesize molecules with different targeting functions, laying the foundation for expanding the application range of lanthanide-binding proteins.

[0048] The following are specific examples.

[0049] Unless otherwise specified, the drugs and instruments used in the examples are all conventionally selected in the art. Experimental methods without specific conditions specified in the examples are generally carried out under conventional conditions, such as those described in literature or books, or methods recommended by the kit manufacturer.

[0050] Example 1

[0051] 1. Design point mutation primers and clone the lanthanide protein with asparagine mutated to aspartic acid at position 108 by overlapping PCR amplification. Sequence and compare the PCR fragments.

[0052] 2. The mutant lanthanide binding protein was introduced into the expression-competent Escherichia coli BL21 (DE3) strain. The bacteria were then evenly spread on LB agar plates containing 50 μg / mL kanamycin and cultured at 37°C. A single colony was selected for inoculation and cultured at 37°C at a shaking speed of 200 rpm for about 16 hours. The mixture was then inoculated for large-scale culture. When the optical density at 600 nm (OD 600 ) reached 0.6, the culture temperature was set to 16°C, and IPTG was added at a final concentration of 0.6 mM to induce recombinant protein expression. After further overnight incubation on a shaking platform, the bacterial cells were harvested by centrifugation at 6000 rpm for 20 minutes at 4°C.

[0053] 3. Bacteria were lysed by sonication in a buffer containing 150 mM NaCl, 10 mM Tris·HCl (pH 7.5), and 100 μM PMSF. The cell lysate was then centrifuged at 15,000 rpm for 30 minutes at 4°C. The supernatant was collected and incubated with Ni-NTA agarose 6FF beads pre-equilibrated in 150 mM NaCl, 10 mM Tris·HCl (pH 7.5) (Buffer A) for 1 hour at 4°C. It is important to note that the Ni-NTA beads were pre-equilibrated in Buffer A containing 150 mM NaCl, 10 mM Tris·HCl (pH 7.5). The mixed solution was then transferred to a column and sequentially eluted with Buffer A containing 20 mM imidazole. Proteins bound to the beads were subsequently eluted with Buffer A containing 200 mM imidazole and further concentrated using a 3-kD centrifugal filter unit.

[0054] 4. In order to 3+ To load the protein, GdCl3 was added to the purified protein at a molar ratio of 5:1, and then they were incubated at 4°C for 30 minutes to ensure that the metal ions were fully bound to the protein. Unbound Gd was removed from the mixture by ultrafiltration centrifugation. 3+ and quantitative determination of Gd in proteins by inductively coupled plasma-optical emission spectrometry (ICP-OES) 3+ content.

[0055] 5. Prior to the competition experiment, buffer A for the point-mutated lanthanide-binding protein (LanND) and the unmutated lanthanide-binding protein (LanM) was replaced with a buffer containing 20 mM MOPS, 100 mM NaCl, pH 6.0 (buffer B). LanM and LanND were diluted to the same concentration (100 μM) in buffer B. GdCl₃ and xylenol orange (XO) were also diluted to 100 μM in buffer B. For the competition assay, three groups were set up: control, LanM, and LanND. First, 10 μL of XO was added to the 96-well plate, followed by 10 μL of buffer B, LanM, or LanND mixed with XO. Subsequently, GdCl₃ was added dropwise to the wells using alternating 10 μM steps, resulting in final concentrations ranging from 0 to 70 μM. Various volumes of buffer B were added to ensure a final volume of 100 μL in each well. After all components are thoroughly mixed, the plate is transferred to a microwell absorbance reader and the absorbance is read at 570 nm. 3+ The concentrations of Gd at the point where XO saturation is 10% are plotted and fitted using Boltzmann fitting. 3+ The concentration was taken as an estimate of the point at which tight binding sites within the protein were saturated.

[0056] 6. Lanthanide protein-gadolinium solutions of varying concentrations were prepared to measure T1-weighted relaxation time. The T1WI and T2WI scanning parameters at 3 T (uMR 790, United Imaging Healthcare) were as follows: the RF coil was a human 48-channel receiving head coil; TR was 500 ms; TE was 20 ms; FOV was 150 mm × 250 mm; FA was 145°; matrix size was 512 × 308; bandwidth / pixel was 200 Hz / pixel; and slice thickness was 3 mm. For T1-weighted imaging of animals, a 3.0-T preclinical magnetic resonance imaging system was used with the following specifications: a dedicated small-animal coil (mouse coil) for radiofrequency coils; TR, 600 msec; TE, 12.16 msec; FOV, 60 mm × 30 mm; FA, 145°; voxel size, 0.47 mm × 0.23 mm × 0.6 mm; matrix size, 256 × 256; bandwidth / pixel, 260 Hz / pixel; slice thickness, 0.6 mm; number of averages, 8; and number of slices, 10.

[0057] The test results are shown in Figures 1 to 5. Figure 1 shows the SDS-PAGE images of the lanthanide protein before and after mutation; Figure 2 shows the results of the evaluation of the binding performance of the lanthanide binding protein to gadolinium ions before and after mutation; Figure 3 shows photographs and SDS-PAGE images of the lanthanide protein before and after mutation and before and after binding to gadolinium ions; Figure 4 shows the results of the evaluation of the magnetic resonance imaging ability of the point gene mutation lanthanide binding protein-gadolinium; and Figure 5 shows the results of the in vivo magnetic resonance imaging ability and metabolism evaluation of the point gene mutation lanthanide binding protein-gadolinium.

[0058] As shown in Figures 1 to 3, weakly bound ions in one of the binding sites of lanthanide binding proteins may easily dissociate from the protein, leading to potential toxicity. Therefore, in this example, a single point mutation N108D was introduced into the fourth EF-hand structure to form a mutant engineered lanthanide binding protein (LanND). SDS-PAGE gel bands showed that the purified wild-type LanM and mutant LanND had similar molecular weights of approximately 12 kDa. We used xylene orange as an indicator of free Gd3+ ions to evaluate their Gd 3+ The results showed that a single point mutation in LanND increased its Gd binding capacity compared with LanM. 3+ The number of binding sites. The stoichiometric ratio of Gd3+ increased from about 2.1 to 3.8. 3+ In none of the cases did the mutants induce any abnormal aggregation or degradation.

[0059] As shown in Figures 4 and 5, at a magnetic field strength of 3.0 T, LanND-Gd exhibits a T1 relaxivity (r1) of 13.25 mM. -1 s -1It is Magnevist (4.97mM) used clinically -1 s -1 ) is 2.7 times that of the Gd molecule. Moreover, a single LanND-Gd molecule contains four Gd 3+ Therefore, the effective relaxivity of LanND-Gd (about 52mM -1 s -1 ) is 10 times that of Magnevist (about 5mM-1s-1). In order to develop contrast agents with clinical translation prospects, its metabolic pathway in the body must first be determined. Traditional nanoparticle-based contrast agents usually circulate in the blood for too long and may accumulate in organs, causing potential side effects or toxicity. Therefore, this application studies the metabolism of engineered protein contrast agents. In this example, LanND-Gd was injected intravenously into mice at a drug dose of 2.5μmol / kg. MRI images showed obvious MRI signals in the kidneys and bladder, indicating that the protein was filtered through the kidneys and excreted through the urethra. Within 0-75 minutes, the bladder MRI signals from transverse and longitudinal abdominal imaging sections increased significantly, confirming that the engineered protein was cleared by the kidneys through the urine. Compared with the clinical Magnevist, the in vivo metabolism time of LanND-Gd was significantly prolonged.

[0060] In summary, this application discloses a magnetic resonance imaging contrast agent and its preparation method. This contrast agent modifies a lanthanide-binding protein through a single-point genetic mutation, enhancing its binding properties to gadolinium ions. This binding protein's properties suppress the molecular rotational correlation time, increasing the contrast agent's relaxivity and prolonging its in vivo metabolism. This overcomes the low relaxivity and short imaging window of existing gadolinium-based contrast agents. Furthermore, the lanthanide-binding protein-gadolinium can be metabolized by the kidneys, making it useful for assessing renal function.

[0061] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0062] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that variations and improvements are possible within the scope of the present invention, as would be apparent to one skilled in the art. These variations and improvements fall within the scope of the present invention. Therefore, the scope of the present invention is governed by the appended claims.

Claims

1. A magnetic resonance contrast agent, characterized in that, Comprising a lanthanide-binding protein mutant, wherein the lanthanide-binding protein mutant is obtained by mutating asparagine at position 108 of the lanthanide-binding protein to aspartic acid.

2. The magnetic resonance contrast agent according to claim 1, wherein The amino acid sequence of the lanthanide-binding protein is shown as SEQ ID No.

1.

3. The magnetic resonance contrast agent according to any one of claims 1-2, characterized in that, The lanthanide-binding protein mutant binds to lanthanide ions, and the lanthanide ions include gadolinium ions.

4. A method for preparing a magnetic resonance contrast agent, characterized in that, Comprising the following steps: preparing the magnetic resonance contrast agent by genetic engineering technology, wherein the magnetic resonance contrast agent comprises a lanthanide-binding protein mutant, and the lanthanide-binding protein mutant is obtained by mutating asparagine at position 108 of the lanthanide-binding protein to aspartic acid.

5. The preparation method according to claim 4, characterized in that, The steps of preparing the magnetic resonance contrast agent by genetic engineering technology include: Preparing the gene fragment of the lanthanide-binding protein mutant; Introducing the gene fragment of the lanthanide-binding protein mutant into a host for expression, and separating and collecting the lanthanide-binding protein mutant; Mixing and incubating the lanthanide-binding protein mutant with lanthanide ions to obtain the magnetic resonance contrast agent.

6. The preparation method according to claim 5, characterized in that, Using the overlapping PCR amplification technology to mutate asparagine at position 108 of the lanthanide-binding protein to aspartic acid to obtain the gene fragment of the lanthanide-binding protein mutant.

7. The preparation method according to claim 6, characterized in that, The amino acid sequence of the lanthanide-binding protein is shown as SEQ ID No.

1.

8. The preparation method according to claim 5, characterized in that, The host is Escherichia coli, and the steps of introducing the gene fragment of the lanthanide-binding protein mutant into the host for expression, and separating and collecting the lanthanide-binding protein mutant include: introducing the gene fragment of the lanthanide-binding protein mutant into the Escherichia coli host for expression, disrupting the bacterial cells, and separating and purifying to obtain the lanthanide-binding protein mutant.

9. The preparation method according to any one of claims 5-7, characterized in that, The lanthanide ions include gadolinium ions; And / or, the molar ratio of the lanthanide ions to the lanthanide-binding protein mutant is 1 to 4.

10. Use of the magnetic resonance contrast agent according to any one of claims 1-3, or the magnetic resonance contrast agent prepared by the preparation method of the magnetic resonance contrast agent according to any one of claims 4-9 in magnetic resonance imaging.

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