Water-soluble melanin CEST contrast agent, its preparation and application

Abstract

This invention discloses a water-soluble melanin CEST contrast agent, its preparation method, and its application, relating to the field of magnetic resonance imaging contrast agent technology. This contrast agent, its preparation method, and its application aim to solve the technical problem that existing contrast agents containing paramagnetic metals can damage liver and kidney function. This water-soluble melanin CEST contrast agent comprises the following components by weight: 2-3 parts ammonia, 40 parts ethanol, 100 parts deionized water, and 0.5 parts reaction substrate. The chemical shift corresponding to this contrast agent preparation method is 4.0 ppm, exhibiting good adaptability to acidic environments and suitability for use in the tumor microenvironment. It is polymerized using dopamine hydrochloride or 3,4-dihydroxyphenylalanine, with inexpensive, simple, and readily available raw materials, simple synthesis, and easy large-scale preparation. It achieves tumor imaging without the need for paramagnetic metal ions, avoiding the damage to liver and kidney function caused by heavy metal ions.

Description

Technical Field

[0001] This invention belongs to the field of magnetic resonance imaging contrast agent technology, specifically relating to a water-soluble melanin CEST contrast agent, its preparation method and application. Background Technology

[0002] Magnetic Resonance Imaging (MRI) has high spatial resolution and is safe without radiation damage, and has been widely used in clinical medical diagnosis. However, due to the low sensitivity of MRI, the contrast between different tissues or tumors is not obvious, making diagnosis difficult. Currently, contrast agents used in clinical practice mainly improve imaging contrast by changing the local relaxation properties of tissues, thereby improving diagnostic accuracy. These contrast agents often require high doses of heavy metal ions, such as gadolinium, iron, and manganese ions. The introduction of heavy metal ions will increase the metabolic burden on the patient's liver and kidneys, and is especially unsuitable for patients with liver and kidney dysfunction. Therefore, there is an urgent need to develop a metal ion-free contrast agent.

[0003] Chemical exchange saturation transfer (CEST) is a novel MRI imaging mechanism. Its imaging principle involves using selective saturation pulses to pre-saturate exchangeable protons with specific chemical shifts. As the saturated protons exchange chemically with surrounding water protons, saturation transfer occurs onto free water, reducing the signal intensity of the free water. Therefore, detecting changes in the water signal can indirectly reflect information about this substance. Compared to currently used T1 and T2 contrast agents, CEST imaging does not require paramagnetic heavy metal ions such as Gd3+, Fe3+, or Mn3+. It only requires diamagnetic exchangeable protons to achieve magnetic resonance imaging. Biomolecules such as glycoproteins, glucose, glycogen, inositol, glutamate, and polypeptides all contain a large number of exchangeable protons and can be used for CEST imaging. However, most current contrast agents utilize compounds such as sugars and polypeptides; there are no reports of using water-soluble melanin as a corresponding CEST MRI contrast agent. Furthermore, the addition of paramagnetic metals to the contrast agent for MRI raises potential drug contraindications, and heavy metal ions can damage liver and kidney function.

[0004] Therefore, the problem of heavy metal ions damaging liver and kidney function urgently needs to be addressed in order to improve the application scenarios of contrast agents. Summary of the Invention

[0005] (1) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a water-soluble melanin CEST contrast agent, its preparation method, and its application. This contrast agent, its preparation method, and its application are intended to solve the technical problem that the addition of paramagnetic metals to existing contrast agents can damage liver and kidney function.

[0007] (2) Technical solution

[0008] To solve the above-mentioned technical problems, the present invention provides a water-soluble melanin CEST contrast agent, which comprises the following components in parts by weight: 2-3 parts ammonia, 40 parts ethanol, 100 parts deionized water and 0.5 parts reaction substrate.

[0009] Furthermore, the reaction substrate is 3,4-dihydroxyphenylalanine or dopamine hydrochloride.

[0010] A method for preparing a water-soluble melanin CEST contrast agent, comprising the water-soluble melanin CEST contrast agent as described above, and the steps are as follows:

[0011] Step 1: Mix ammonia, ethanol and deionized water in a certain proportion to obtain a mixed solvent;

[0012] Step 2: Dissolve the reaction substrate in deionized water and add it to the mixed solvent from Step 1 to obtain a mixed solution;

[0013] Step 3: Depending on the substrate, perform the following treatments: If the substrate is 3,4-dihydroxyphenylalanine, stir at room temperature to allow it to react fully until the color of the mixed solution gradually changes from yellow to dark brown; if the substrate is dopamine hydrochloride, heat the solution and stir to allow it to react fully until the color of the mixed solution gradually changes from yellow to dark brown.

[0014] Step 4: Transfer the above mixed solution to a centrifuge tube, separate it using a centrifuge, then wash the precipitate with deionized water, centrifuge again, repeat 3 times, and finally place it at the working temperature for 2 hours and freeze dry it in a freeze dryer to obtain the target melanin.

[0015] Furthermore, in step one, the mixture is stirred for 30 minutes at room temperature.

[0016] Furthermore, in step one, the volume ratio of ammonia, ethanol, and deionized water is 1-2:20:45.

[0017] Furthermore, in step three, the solution is heated to 45-50°C, and the reaction is stirred for 24-26 hours.

[0018] Furthermore, the operating temperature in step four is -70℃.

[0019] Furthermore, the average particle size of the target melanin in step four is 160 nm.

[0020] The application of a water-soluble melanin CEST contrast agent in a tumor model is not for the purpose of disease diagnosis and treatment.

[0021] (3) Beneficial effects

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: the contrast agent of the present invention has a chemical shift of 4.0 ppm, is well adapted to acidic environments, and is suitable for use in the tumor microenvironment. It is polymerized from dopamine hydrochloride or 3,4-dihydroxyphenylalanine, the raw materials are cheap, simple and easy to obtain, the synthesis is simple, and it is easy to prepare in large quantities. It can achieve tumor contrast without the use of paramagnetic metal ions, thus avoiding the damage of heavy metal ions to liver and kidney function. Attached Figure Description

[0023] Figure 1 This is the synthetic route for the water-soluble melanin CEST contrast agent of the present invention;

[0024] Figure 2 This is a particle size distribution diagram of the water-soluble melanin in Experimental Example 2 of the present invention;

[0025] Figure 3 This is the zeta potential diagram of water-soluble melanin in Experimental Example 2 of the present invention;

[0026] Figure 4 The magnetic susceptibility transfer spectra under different pH conditions in Experimental Example 2 of this invention are shown.

[0027] Figure 5 The magnetization transfer rate spectra are shown in Experimental Example 3 of this invention under different saturation power conditions. Detailed Implementation

[0028] This specific embodiment describes a water-soluble melanin CEST contrast agent, its preparation method, and its application. The synthetic route of the water-soluble melanin CEST contrast agent is as follows: Figure 1 As shown, the particle size distribution diagram of water-soluble melanin in Experiment Example 2 is as follows. Figure 2 As shown, the zeta potential diagram of water-soluble melanin in Experimental Example 2 is as follows. Figure 3 As shown, the magnetic susceptibility transfer spectra under different pH conditions in Experiment Example 2 are as follows: Figure 4 As shown, the magnetization transmissivity spectra under different saturation power conditions in Experiment Example 3 are as follows: Figure 5 As shown, the contrast agent, the water-soluble melanin CEST contrast agent, comprises the following components in parts by weight: 2-3 parts ammonia, 40 parts ethanol, 100 parts deionized water, and 0.5 parts reaction substrate.

[0029] Furthermore, the reaction substrate is 3,4-dihydroxyphenylalanine or dopamine hydrochloride.

[0030] A method for preparing a water-soluble melanin CEST contrast agent, comprising the water-soluble melanin CEST contrast agent as described above, and the steps are as follows:

[0031] Step 1: Mix ammonia, ethanol and deionized water in a certain proportion to obtain a mixed solvent;

[0032] Step 2: Dissolve the reaction substrate in deionized water and add it to the mixed solvent from Step 1 to obtain a mixed solution;

[0033] Step 3: Depending on the substrate, perform the following treatments: If the substrate is 3,4-dihydroxyphenylalanine, stir at room temperature to allow it to react fully until the color of the mixed solution gradually changes from yellow to dark brown; if the substrate is dopamine hydrochloride, heat the solution and stir to allow it to react fully until the color of the mixed solution gradually changes from yellow to dark brown.

[0034] Step 4: Transfer the above mixed solution to a centrifuge tube, separate it using a centrifuge, then wash the precipitate with deionized water, centrifuge again, repeat 3 times, and finally place it at the working temperature for 2 hours and freeze dry it in a freeze dryer to obtain the target melanin.

[0035] Furthermore, in step one, the mixture is stirred for 30 minutes at room temperature.

[0036] Furthermore, in step one, the volume ratio of ammonia, ethanol, and deionized water is 1-2:20:45.

[0037] Furthermore, in step three, the solution is heated to 45-50°C, and the reaction is stirred for 24-26 hours.

[0038] Furthermore, the operating temperature in step four is -70℃.

[0039] Furthermore, the average particle size of the target melanin in step four is 160 nm.

[0040] The application of a water-soluble melanin CEST contrast agent in a tumor model is not for the purpose of disease diagnosis and treatment.

[0041] Example 1

[0042] This embodiment provides a method for preparing a water-soluble melanin CEST contrast agent, the steps of which are as follows:

[0043] Step 1: Mix 2 parts ammonia, 40 parts ethanol and 90 parts deionized water in a volume ratio of 1:20:45, and stir at room temperature for 30 minutes to obtain a mixed solvent;

[0044] Step 2: Dissolve 0.5 parts of 3,4-dihydroxyphenylalanine in 10 parts of deionized water, and add it to the mixed solvent in Step 1 to obtain a mixed solution;

[0045] Step 3: Stir at room temperature for 24 hours to allow the reaction to proceed fully, until the color of the mixed solution gradually changes from yellow to dark brown;

[0046] Step 4: Transfer the above mixed solution to a centrifuge tube, separate it using a centrifuge, then wash the precipitate with deionized water, centrifuge again, repeat 3 times, and finally place it at a working temperature of -70℃ for 2 hours and freeze dry it in a freeze dryer to obtain the target melanin with an average particle size of 160nm.

[0047] Example 2

[0048] This embodiment provides a method for preparing a water-soluble melanin CEST contrast agent, the steps of which are as follows:

[0049] Step 1: Mix 3 parts ammonia, 40 parts ethanol and 90 parts deionized water in a volume ratio of 1.5:20:45, and stir at room temperature for 30 minutes to obtain a mixed solvent;

[0050] Step 2: Dissolve 0.5 parts of dopamine hydrochloride in 10 parts of deionized water, and add it to the mixed solvent in Step 1 to obtain a mixed solution;

[0051] Step 3: Heat to 45℃ and stir for 24 hours to allow the reaction to proceed fully, until the color of the mixed solution gradually changes from yellow to dark brown;

[0052] Step 4: Transfer the above mixed solution to a centrifuge tube, separate it using a centrifuge, then wash the precipitate with deionized water, centrifuge again, repeat 3 times, and finally place it at a working temperature of -70℃ for 2 hours and freeze dry it in a freeze dryer to obtain the target melanin with an average particle size of 160nm.

[0053] Experimental Example 1

[0054] Five milligrams of the water-soluble melanin obtained in Example 2 were dissolved in 15 mL of phosphate-buffered saline (PBS). The pH was adjusted with a high concentration of hydrochloric acid or sodium hydroxide solution to prepare 1 mL of a pH 7.0 solution. The particle size and zeta potential were measured using dynamic light scattering (DLS), and the results are as follows: Figures 2-3 As shown.

[0055] The results show that: Figures 2-5 It can be seen that the average particle size of water-soluble melanin is about 141 nm and the zeta potential is -30.38 mV.

[0056] Experiment Example 2

[0057] 7.2 mg of the water-soluble melanin obtained in Example 1 was dissolved in 1.5 mL of phosphate-buffered saline (PBS) to prepare a solution containing 10.6 mmol / L melanin. The pH was adjusted with a higher concentration of hydrochloric acid or sodium hydroxide solution, and different pH solutions were prepared and placed in 0.3 mm diameter capillary tubes. The sample tubes were vertically fixed in a 20 cm diameter circular tube, and the relative positions were recorded. The circular tube was then inserted into a magnetic resonance imaging probe. The temperature control unit was turned on, and the temperature was maintained at 37 °C for 30 min. CEST imaging was performed every 0.25 ppm in the range of 15 to -15 ppm. Sampling parameters: slice thickness (3 mm), repetition time TR = 8 s, echo time TE = 5.1 ms, sampling rectangular matrix. With a size of 128*96, a rarefactor of 8, a saturation irradiation power of ω1 of 5.4 μT, and a saturation irradiation time of 3 s, B0 field inhomogeneity correction was performed every 0.15 ppm in the range of 1.6 to -1.6 ppm. The correction parameters were: layer thickness (3 mm), repetition time TR of 5 s, echo time TE of 6.4 ms, sampling matrix size of 128*96, a rarefactor of 8, a saturation irradiation power of ω1 of 0.5 μT, and a saturation irradiation time of 0.3 s. The CEST spectra of water-soluble melanin nanoparticles at different pH values ​​were obtained by data processing using Matlab.

[0058] MRI results showed:

[0059] As can be seen from the CEST spectrum, the water-soluble melanin provided by this invention has a strong CEST signal at 4 ppm, and the CEST signal gradually weakens as the pH increases.

[0060] Experimental Example 3

[0061] 7.2 mg of the water-soluble melanin obtained in Example 1 was dissolved in 1.5 mL of phosphate-buffered saline (PBS) to prepare a solution containing 10.6 mmol / L melanin. The pH was adjusted to 7.3 with a higher concentration of hydrochloric acid or sodium hydroxide solution. The solution was placed in a 5.0 mm diameter capillary tube and inserted into a magnetic resonance imaging (MRI) probe. The temperature control unit was turned on, and the temperature was maintained at 37 °C for 30 min. CEST imaging was performed every 0.25 ppm in the range of 15 to -15 ppm. Sampling parameters: slice thickness (3 mm), repetition time TR = 8 s, echo time TE = 5.1 ms, sampling matrix size = 128*96, and acceleration factor rare. With factor = 8, saturation irradiation power ω1 = 1.2, 2.4, 3.6, 5.4, 7.2, and 10.8 μT, and saturation irradiation time t = 3 s, B0 field inhomogeneity correction was performed every 0.15 ppm in the range of 1.6 to -1.6 ppm. Correction parameters were: layer thickness (3 mm), repetition time TR = 5 s, echo time TE = 6.4 ms, sampling matrix size = 128*96, acceleration factor = 8, saturation irradiation power ω1 = 0.5 μT, and saturation irradiation time t = 0.3 s. Data processing using Matlab was used to obtain the CEST spectra of water-soluble melanin nanoparticles under different saturation powers. The results are as follows: Figure 5 As shown.

[0062] MRI experimental results:

[0063] From CEST spectrum ( Figure 4 As can be seen from the data, the water-soluble melanin provided by this invention has a strong CEST signal at around 4 ppm, using a saturation pulse of 5.4 μT. Furthermore, the sensitivity of the CEST signal of water-soluble melanin to acidity can also be used to detect the physiological environment of living tissues (such as the slightly acidic environment of tumor tissue).

Claims

1. The use of water-soluble melanin in the preparation of CEST contrast agents, characterized in that, The water-soluble melanin CEST contrast agent comprises the following components in parts by weight: 2-3 parts ammonia, 40 parts ethanol, 100 parts deionized water, and 0.5 parts reaction substrate; The reaction substrate is 3,4-dihydroxyphenylalanine; The preparation steps are as follows: Step 1: Mix ammonia, ethanol and deionized water in a certain proportion to obtain a mixed solvent; Step 2: Dissolve the reaction substrate in deionized water and add it to the mixed solvent from Step 1 to obtain a mixed solution; Step 3: Stir at room temperature to allow the mixture to react fully until the color of the mixed solution gradually changes from yellow to dark brown; Step 4: Transfer the above mixed solution to a centrifuge tube, separate it using a centrifuge, then wash the precipitate with deionized water, centrifuge again, repeat 3 times, and after finishing, place it at the working temperature for 2 hours, and freeze dry it in a freeze dryer to obtain the water-soluble melanin CEST contrast agent.

2. The use of the water-soluble melanin according to claim 1 in the preparation of CEST contrast agent, characterized in that, In step one, stir for 30 minutes at room temperature.

3. The use of the water-soluble melanin according to claim 1 in the preparation of CEST contrast agent, characterized in that, In step one, the volume ratio of ammonia, ethanol, and deionized water is 1-2:20:

45.

4. The use of the water-soluble melanin according to claim 1 in the preparation of CEST contrast agent, characterized in that, In step three, the solution is heated to 45-50°C and stirred for 24-26 hours.

5. The use of the water-soluble melanin according to claim 1 in the preparation of CEST contrast agent, characterized in that, The operating temperature in step four is -70℃.

6. The use of the water-soluble melanin according to claim 1 in the preparation of CEST contrast agent, characterized in that, The average particle size of the target melanin in step four is 160 nm.

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