A hyperpolarization 129 Xe magnetic resonance molecular probe and its preparation method

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a hyperpolarized... 129 Xe magnetic resonance molecular probe and its preparation method. This probe uses ZIF-8 as a template and 2-aminoimidazole as a ligand exchange component. The molecular probe is formed by the interaction of a monomer with a ligand in a methanol solution. The monomer is synthesized from 2-methylimidazole and zinc nitrate hexahydrate. 129 Xe molecular cage ZIF-8, wherein the monomer ZIF-8 is modified by post-synthetic modification with a 2-aminoimidazolium ligand. 129 Xe molecular cage, i.e., hyperpolarization 129 Xe magnetic resonance molecular probe. The principle of this molecular probe is as follows: ZIF-8 is a Zn... 2+ The metal-organic framework (MOF) formed by coordination with the N atom on the 2-methylimidazolium ring exhibits acid sensitivity and pH responsiveness. Through post-synthetic modification (PSM) of ZIF-8, ligand exchange between 2-aminoimidazolium and ZIF-8 is achieved, resulting in a novel ZIF-8-AIM molecular probe for hyperpolarization. 129 Xe chemical exchange saturation transfer will produce a new signal different from the original ZIF-8, which can be used for magnetic resonance molecular probes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a hyperpolarized... 129 Xe magnetic resonance molecular probe and its preparation method. Background Technology

[0002] 129 Xe is a non-toxic inert gas, highly sensitive to chemical environments, exhibiting large chemical shift changes. It is soluble in many solvents and can interact with various proteins, phospholipids, and spores, making it well-suited for biomedical applications. When spin-exchange optical pumping (SEOP) technology is used to... 129 After Xe undergoes hyperpolarization 129 The Xe nuclear magnetic resonance signal was greatly enhanced, with sensitivity increased by 10,000 times compared to the thermally polarized state, making it suitable as a contrast agent for MRI of the lungs or brain in animals and humans. Hyperpolarization 129 Xe technology inherits the non-destructive advantage of magnetic resonance imaging (MRI) while overcoming the low sensitivity of traditional MRI, providing a highly promising technique for detecting ultra-low concentrations of chemical or biomolecules. However, hyperpolarization... 129 Using Xe directly as a molecular probe has drawbacks such as lack of targeting and excessively fast exchange rates, making it difficult to detect. To overcome these shortcomings and fully utilize the advantages of Xe, in 2001, the Pines group first proposed a method based on... 129 Xe's molecular probe design strategy: utilizing a cage-like compound called cavitary as... 129 The main molecule of Xe is used to functionalize the cage-like compound cavitary molecule. After modification, the functionalized cavitary molecule can achieve specific recognition of the target analyte. 129 Xe is particularly sensitive to chemical environments; when the probe interacts with the target, the cavitation cage... 129 The chemical shift of Xe changes, and this change can be used to monitor the recognition of probe molecules and targets. ZIF-8, a widely used cage-like molecule, has been shown to have an affinity for Xe. Studies at Zhoulab have confirmed that ZIF-8 can also be used as a... 129 The main molecule of Xe can be used to develop ZIF-8-based [molecules]. 129 Xe molecular probe. Summary of the Invention

[0003] The existing ZIF-8 is composed of 2-methylimidazole coordinated with zinc ions, lacking hydrophilic sites, resulting in poor water solubility. Introducing hydrophilic groups such as carboxyl and amino groups is expected to improve its water solubility, enabling its use in cell and in vivo research. Building upon previous research, the applicant uses ZIF-8 as... 129Molecular probes were prepared by exchanging the host molecule of Xe with a 2-aminoimidazole ligand, and then tested using Hyper-CEST technology, which revealed good performance. 129 Xe signal. Specifically, using ZIF-8 as the monomer, ZIF-8 with amino functional groups was synthesized. During a single test, dual signals appeared and changed, originating from the six-membered and four-membered pores of ZIF-8, respectively. ZIF-8 has two pore structures. The Xe signal mainly originates from the Xe chemical exchange in the six-membered pores. The four-membered pores are generally smaller, making it difficult for Xe to enter. However, after modification with aminoimidazole, the four-membered pores become larger, thus leading to new chemical exchanges.

[0004] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0005] A hyperpolarization 129 The Xe magnetic resonance molecular probe uses ZIF-8 as a template and 2-aminoimidazole as the ligand. Further, the molecular probe is synthesized by the interaction of a monomer with the ligand in a methanol solution; the monomer is 2-methylimidazole synthesized from zinc nitrate hexahydrate. 129 Xe molecular cage ZIF-8, wherein the monomer ZIF-8 is obtained by post-synthetic modification with a 2-aminoimidazolium ligand. 129 Xe molecular cage, i.e., hyperpolarization 129 Xe magnetic resonance molecular probe.

[0006] The present invention also provides the above-mentioned hyperpolarization 129 The preparation method of Xe magnetic resonance molecular probes specifically includes the following steps:

[0007] 1) Synthesis of ZIF-8:

[0008] With stirring, the 2-methylimidazole solution was added to the zinc nitrate solution. After mixing evenly, the mixture was sealed and allowed to stand at room temperature. Then, it was centrifuged and washed to remove unreacted 2-methylimidazole. Finally, the precipitate was dried under vacuum to obtain ZIF-8.

[0009] 2) Synthesis of ZIF-8-AIM probes via post-synthesis modification method:

[0010] The dried ZIF-8 obtained in step 1) was uniformly dispersed in an organic solvent to obtain a ZIF-8 suspension. The ligand 2-aminoimidazole was added to the ZIF-8 suspension, and the mixture was reacted at 40-80°C for 1-24 hours (preferably at 50°C for 1 hour) under a nitrogen atmosphere. The mixture was then centrifuged and washed to remove unreacted ligands. Finally, the resulting precipitate was vacuum dried to obtain the ZIF-8-AIM probe, i.e., the hyperpolarized probe. 129 Xe magnetic resonance molecular probe.

[0011] Furthermore, in step 1), the molar ratio of zinc nitrate to 2-methylimidazole is 1:(3-5);

[0012] Furthermore, in step 2), the mass ratio of dried ZIF-8 to 2-aminoimidazole is 1:(2-5), preferably 1:2.89.

[0013] Furthermore, the organic solvent mentioned in step 2) is methanol.

[0014] Furthermore, the vacuum drying described in steps 1) and 2) is performed at 50°C for 12 hours.

[0015] The above hyperpolarization 129 Xe magnetic resonance molecular probes can be used as molecular cages for the detection of Xe signals in serum.

[0016] The principle of the molecular probe of this invention is as follows: ZIF-8 is a probe based on Zn... 2+ The metal-organic framework (MOF) formed by coordination with the N atom on the 2-methylimidazole ring exhibits acid sensitivity and pH responsiveness. Through post-modification synthesis (PSM) of ZIF-8, ligand exchange between 2-aminoimidazole and ZIF-8 is achieved, resulting in a novel ZIF-8-AIM molecular probe with hyperpolarization. 129 The Xe chemical exchange saturation transfer signals are 86 ppm and 145 ppm, which, to some extent, are achieved through hyperpolarization. 129 Xe was used to detect Xe signals in serum.

[0017] A novel hyperpolarization of the present invention 129 Compared with existing technologies, the advantages and benefits of Xe magnetic resonance molecular probes are as follows:

[0018] This molecular probe has extremely high... 129 Xe sensitivity, along with good dispersibility and stability, makes it suitable for subsequent cell and in vivo studies. In hyperpolarization... 129 In Xe magnetic resonance imaging, a single sampling will have 129 The appearance of the Xe signal, when tested using chemical exchange saturation transfer technology, will produce another new signal. The emergence of this new signal in chemical exchange saturation transfer technology can avoid false positives, thus representing a novel approach. 129 Xe molecular probe.

[0019] This molecular probe exhibits hyperpolarization. 129 Xe chemical exchange saturation transfer effect.

[0020] A magnetic resonance spectrometer coupled with a hyperpolarization device was used to detect the probe. 129 The Xe chemical exchange saturation transfer signal is used to study Xe contrast agents by utilizing changes in this signal. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the synthesis of the molecular probe of the present invention;

[0022] Figure 2 Transmission electron microscope image and mapping of the nanoparticle structure in Example 1;

[0023] Figure 3 The image shows the powder X-ray diffraction pattern of the nanoparticles in Example 1.

[0024] Figure 4 This is a nitrogen adsorption curve of the nanoparticles in Example 1;

[0025] Figure 5 The nanoparticles obtained at different reaction times in Example 2 in aqueous solution 129 Xe spectrum;

[0026] Figure 6 The nanoparticles obtained at different reaction times in Example 3 were in serum. 129 Xe spectrum;

[0027] Figure 7 The nanoparticles in Example 4 were at different concentrations in aqueous solution. 129 Xe CEST spectrum;

[0028] Figure 8 This is a schematic diagram showing the dispersibility and stability of nanoparticles and ZIF-8 in Example 1 after being sonicated in an aqueous solution for 30 minutes and left to stand for two days. ZIF-8 is on the left and ZIF-8-AIM is on the right. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the claims. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0030] The following describes specific embodiments and... Figures 1 to 8 The technical solution of the present invention will be described in detail below.

[0031] The main reagents and materials used in Examples 1-3 below are sourced as follows:

[0032] Zinc nitrate hexahydrate, 2-methylimidazole, 2-aminoimidazole, hydrochloric acid, and methanol were all purchased from Sinopharm Group and were all of analytical grade.

[0033] Fetal bovine serum is premium grade fetal bovine serum (HYCEZMBIO) TM ), produced in South America.

[0034] Unless otherwise specified, all reagents above are used directly without further purification.

[0035] All water used is deionized water.

[0036] Example 1: Synthesis of nanoparticles, the specific steps are as follows:

[0037] 1) Zinc nitrate hexahydrate (734.4 mg, 2.469 mmol) was placed in a 250 mL Erlenmeyer flask, and 50 mL of methanol was added. 2-Methylimidazole (810.6 mg, 9.874 mmol) was placed in another 250 mL Erlenmeyer flask, and 50 mL of methanol was added. The solutions in both flasks were sonicated until clear and transparent. Then, while stirring, the 2-methylimidazole solution was added to the zinc nitrate solution. After thorough mixing, stirring was stopped, the magnetic stir bar was removed, the flasks were sealed, and the mixture was allowed to stand at room temperature for 24 hours. After 24 hours, the mixture was centrifuged at 10,000 rpm to separate the precipitate. The supernatant was removed to obtain a white precipitate. The white precipitate was then washed with methanol to remove unreacted 2-methylimidazole ligand. After washing, the supernatant was removed by centrifugation. This washing and centrifugation process was repeated three times. Finally, the white precipitate was placed in a double-row tube and vacuum dried at 50 °C for 12 hours to remove residual organic solvent from the cavity, yielding ZIF-8.

[0038] 2) Synthesis of ZIF-8-AIM probe by post-synthesis modification method

[0039] 100 mg of ZIF-8 obtained in step 1) was uniformly dispersed in 50 mL of methanol and sonicated for 30 min to obtain a 2 mg / mL ZIF-8 suspension. 289.06 mg of 2-aminoimidazole was added to the ZIF-8 suspension, and the reaction was carried out at 50 °C for 1 h under a nitrogen atmosphere. The product was then centrifuged at 10,000 rpm to remove the supernatant, yielding a yellow precipitate. The precipitate was then washed with methanol to remove unreacted 2-aminoimidazole ligands. After washing, the supernatant was removed by centrifugation. This washing and centrifugation process was repeated three times. Finally, the resulting yellow precipitate was placed in a double-row tube and vacuum-dried at 50 °C for 12 h to obtain the final product – nanoparticles, namely the ZIF-8-AIM probe.

[0040] 3) Add ZIF-8-AIM to distilled water and sonicate for 30 minutes to obtain an aqueous solution of the target nanoparticles for later use;

[0041] In Example 1, the nanoparticles and ZIF-8 were sonicated in an aqueous solution for 30 minutes and left to stand for two days. The diagram shows their dispersibility and stability. The left side is ZIF-8 and the right side is ZIF-8-AIM. It can be seen that the ZIF-8 group has obvious sedimentation, while the ZIF-8-AIM group has no obvious sedimentation.

[0042] The aqueous solution of ZIF-8-AIM nanoparticles (monomer concentration 1 mg / mL) prepared in this embodiment was scanned using a high-resolution transmission electron microscope (TEM). The resulting TEM image is shown below. Figure 2 As shown, from Figure 2 It can be seen that the particle size of the nanoparticles is around 90±15nm.

[0043] Figure 3 and Figure 4 The images show the powder X-ray diffraction pattern and nitrogen adsorption curve of the ZIF-8-AIM nanoparticles prepared in this embodiment. Figure 3 As can be seen, the PXRD data of ZIF-8-AIM nanoparticles and ZIF-8 are unchanged, and both correspond one-to-one with the simulated peaks of ZIF-8 single crystal. This indicates that the structure of ZIF-8 remains unchanged after amino ligand exchange, preserving the original crystal structure of ZIF-8. Figure 4 As can be seen, the nitrogen adsorption curve of ZIF-8-AIM shows a sharp increase in the high-pressure region (the range of P / P0 from 0.9 to 1.0), indicating that the amount of nitrogen adsorbed in this range increases sharply. This means that the adsorption between nanoparticles is very large, which is similar to the nitrogen adsorption curve of ZIF-8 in the high-pressure region, thus retaining the gas adsorption performance of ZIF-8.

[0044] Example 2: Nanoparticles prepared in Example 1 in aqueous solution 129 The specific steps for testing the Xe spectrum are as follows:

[0045] 129 Xe nuclear magnetic resonance and magnetic resonance imaging experiments were conducted on a 400 MHz (9.4 T) wide-aperture Bruker AV400 spectrometer (Bruker Biospin, Ettlingen, Germany) equipped with microimaging gradient coils, with an RF pulse frequency of 110.7 MHz for Xe nuclei. 129 Xe NMR spectroscopy was performed using a 10mm dual-resonance probe. 129 Xe and 1H, PA BBO 400 W1 / S2 BB-HD-10Z), rectangular pulses with a flip angle (90°). Hyperpolarized pulses are generated by spin-exchange optical pumping using a continuous flow polarization device. 129 Xe gas. Nuclear spin polarization is approximately 20%. It is enriched by 10% N2, 88% He, and 2% Xe (Xe is 86% enriched by volume).129 Xe or natural abundance 129 Xe, the natural abundance used in this embodiment. 129 A mixture of Xe and other gases was directly passed into a 10 mm NMR tube for 20 seconds, followed by a 3-second wait to ensure the bubbles had completely burst before signal acquisition. The sample temperature was set to 300 K on the NMR spectrometer.

[0046] By changing the reaction time of step 2) in Example 1 to 8 h and 24 h, ZIF-8-AIM with different reaction times was prepared. Then, 20 mg of ZIF-8-AIM prepared at different reaction times was dissolved in 2 ml of distilled water, sonicated for 30 min, and then subjected to the above process. 129 Xe nuclear magnetic resonance and magnetic resonance imaging experiments. Continuous (CW) saturated pulse scans were performed with chemical shifts ranging from δ = 30 to 250 ppm.

[0047] This embodiment measures the nanoparticles in aqueous solution. 129 The Xe spectrum results are as follows: Figure 5 As shown, from top to bottom, these are ZIF-8-AIM nanoparticles obtained after reactions of 1 h, 8 h, and 24 h. 129 The Xe spectrum shows two signals. One signal is attributed to the dissolved state. 129 Xe is directly saturated, with a chemical shift at δ = 193.5 ppm, while another signal, which the applicant desired, is a novel signal at 86 ppm originating from the six-membered ring of ZIF-8-AIM nanoparticles. 129 Chemical transfer of Xe.

[0048] Example 3: Nanoparticles prepared in Example 1 in serum 129 The specific steps for testing the Xe spectrum are as follows:

[0049] 129 Xe nuclear magnetic resonance and magnetic resonance imaging experiments were conducted on a 400 MHz (9.4 T) wide-aperture Bruker AV400 spectrometer (Bruker Biospin, Ettlingen, Germany) equipped with microimaging gradient coils, with an RF pulse frequency of 110.7 MHz for Xe nuclei. 129 Xe NMR spectroscopy was performed using a 10mm dual-resonance probe. 129 Xe and 1H, PA BBO 400 W1 / S2 BB-HD-10Z), rectangular pulses with a flip angle (90°). Hyperpolarized pulses are generated by spin-exchange optical pumping using a continuous flow polarization device. 129 Xe gas. Nuclear spin polarization is approximately 20%. It is enriched by 10% N2, 88% He, and 2% Xe (Xe is 86% enriched by volume). 129Xe or natural abundance 129 Xe, the natural abundance used in this embodiment. 129 A mixture of Xe and other gases was directly passed into a 10 mm NMR tube for 20 seconds, followed by a 3-second wait to ensure the bubbles had completely burst before signal acquisition. The sample temperature was set to 300 K on the NMR spectrometer.

[0050] 20 mg of ZIF-8-AIM prepared in Example 2 with reaction times of 1 h and 8 h were dissolved in 2 ml of fetal bovine serum, and sonicated for 30 min, respectively. 129 Xe nuclear magnetic resonance and magnetic resonance imaging experiments. Continuous (CW) saturated pulse scans were performed with chemical shifts ranging from δ = 30 to 250 ppm.

[0051] This embodiment measured the concentration of nanoparticles in fetal bovine serum. 129 The Xe spectrum results are as follows: Figure 6 As shown, from top to bottom, these are ZIF-8-AIM nanoparticles obtained after reacting for 1 h and 8 h. 129 The Xe spectrum shows two signals. One signal is attributed to the dissolved state. 129 Xe is directly saturated, with a chemical shift at δ = 193.5 ppm, while another signal, which the applicant desired, is a novel signal at 86 ppm originating from the six-membered ring of ZIF-8-AIM nanoparticles. 129 Chemical transfer of Xe.

[0052] Example 4: Nanoparticles prepared in Example 1 in aqueous solution 129 The specific steps for testing the Xe CEST spectrum are as follows:

[0053] 129 Xe nuclear magnetic resonance and magnetic resonance imaging experiments were conducted on a 400 MHz (9.4 T) wide-aperture Bruker AV400 spectrometer (Bruker Biospin, Ettlingen, Germany) equipped with microimaging gradient coils, with an RF pulse frequency of 110.7 MHz for Xe nuclei. 129 Xe NMR spectroscopy was performed using a 10mm dual-resonance probe. 129 Xe and 1H, PA BBO 400 W1 / S2 BB-HD-10Z), rectangular pulses with a flip angle (90°). Hyperpolarized pulses are generated by spin-exchange optical pumping using a continuous flow polarization device. 129 Xe gas. Nuclear spin polarization is approximately 20%. It is enriched by 10% N2, 88% He, and 2% Xe (Xe is 86% enriched by volume). 129 Xe or natural abundance 129 Xe, the natural abundance used in this embodiment.129 A mixture of Xe and other gases was directly passed into a 10 mm NMR tube for 20 seconds, followed by a 3-second wait to ensure the bubbles had completely burst before signal acquisition. The sample temperature was set to 300 K on the NMR spectrometer.

[0054] 200 μg, 250 μg, 300 μg, 350 μg, and 400 μg of ZIF-8-AIM prepared in Example 1 after reacting for 1 hour were dissolved in 2 ml of distilled water, and sonicated for 30 min before proceeding with the reaction. 129 Xe CEST spectrum analysis. Continuous (CW) saturated pulse scans were performed with chemical shifts ranging from δ = 30 to 250 ppm.

[0055] This embodiment measures the nanoparticles in aqueous solution. 129 The Xe spectrum results are as follows: Figure 7 As shown. In nanoparticles 129 The Xe spectrum shows three CEST signals. One signal is attributed to the dissolved state. 129 Xe is directly saturated, with a chemical shift at δ = 193.5 ppm. The other two signals, which the applicant desired, are novel signals at 86 ppm and 145 ppm, originating from the six-membered and four-membered rings of the ZIF-8-AIM nanoparticles. 129 Chemical transfer of Xe.

[0056] Conclusion: When nanoparticles dissolved in serum, a unique precipitate was observed at 86 ppm. 129 Xe directly samples the signal, and its signal strength is stronger than other existing molecular cage signals such as xanthannae, indicating that it can achieve [signal processing] in a relatively complex blood environment. 129 Xe sampling has extremely high 129 Xe sensitivity is an excellent hyperpolarization 129 Xe-modified probes can be used for cell and in vivo research.

Claims

1. A hyperpolarization 129 Xe magnetic resonance molecular probe, wherein the molecular probe uses ZIF-8 as a template and 2-aminoimidazole as a ligand, is characterized in that... The hyperpolarization 129 The preparation method of Xe magnetic resonance molecular probes specifically includes the following steps: 1) Synthesis of ZIF-8: With stirring, the 2-methylimidazole solution was added to the zinc nitrate solution. After mixing evenly, the mixture was sealed and allowed to stand at room temperature. Then, it was centrifuged and washed to remove unreacted 2-methylimidazole. Finally, the precipitate was dried under vacuum to obtain ZIF-8. The molar ratio of zinc nitrate to 2-methylimidazole is 1:(3-5). 2) Synthesis of ZIF-8-AIM probes via post-synthesis modification method: The dried ZIF-8 obtained in step 1) was uniformly dispersed in an organic solvent to obtain a ZIF-8 suspension. The ligand 2-aminoimidazole was added to the ZIF-8 suspension, and the mixture was reacted at 40-80℃ for 1-24 h under a nitrogen atmosphere. The mixture was then centrifuged and washed to remove unreacted ligands. Finally, the resulting precipitate was vacuum dried to obtain the ZIF-8-AIM probe, i.e., the hyperpolarized probe. 129 Xe magnetic resonance molecular probe; The mass ratio of the dried ZIF-8 to the 2-aminoimidazole is 1:(2-5).

2. The hyperpolarization according to claim 1 129 Xe magnetic resonance molecular probe, characterized in that... The organic solvent mentioned in step 2) is methanol; the vacuum drying mentioned in both steps 1) and 2) is vacuum drying at 50°C for 12 h.

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