CN101642579B - Preparation method of chitosan modified paramagnetic metal ion magnetic resonance imaging contrast agent - Google Patents

Abstract

The invention provides a chitosan modified paramagnetic metal ion magnetic resonance imaging contrast agent preparation method, belonging to the technical field of magnetic resonance imaging contrast agent. The method comprises the following steps: firstly preparing acid anhydride with polyamine and polycarboxylic acid, adopting the acid anhydride to react with water soluble chitosan or chitosan with narrow molecular weight distribution in the presence of catalyst, using absolute alcohol to precipitate and obtain solids, purifying the solids with a Sephadex G-25 type gel column to obtain chitosan ligand, adopting the ligand to react with paramagnetic metal inorganic salt, using absolute alcohol to precipitate and finally dying the sediment to obtain the magnetic resonance imaging contrastagent solid powder. The invention combines the acid anhydride with polyamine and polycarboxylic acid with the amino of chitosan through covalent bond and adopts paramagnetic rare earth or transition metal ions for complexing so as to ensure the obtained product to be a new magnetic resonance imaging contrast agent with a certain targeting effect, proper drug duration in vivo, higher relaxivity than magnevist and low toxicity. The invention has simple processing and producing technology and low facility request, thus being easy for processing and producing.

Description

Preparation method of chitosan modified paramagnetic metal ion magnetic resonance imaging contrast agent

technical field

The invention relates to the technical field of magnetic resonance imaging, in particular to a preparation method of a novel magnetic resonance imaging contrast agent.

Background technique

Magnetic Resonance Imaging (MRI) is a medical imaging technology that uses different magnetic resonance signals generated by different tissues of the body under the influence of an external magnetic field for imaging. MRI has no ionizing radiation, can realize multi-nuclear and multi-parameter imaging, can scan in any orientation without changing the body position, has high spatial resolution and contrast without bone artifacts, and can reflect the water proton surrounding environment of the examined tissue With the advantages of obtaining relevant physiological and biochemical information, it has become one of the most effective clinical diagnostic methods. In order to ensure the accuracy of clinical diagnosis, more than 30% of MRI needs magnetic resonance imaging contrast agent (abbreviated as contrast agent or called contrast agent) to improve image contrast. More than 30 tons, it is a high value-added pharmaceutical product. An excellent contrast agent should have: i) sufficient structural stability; ii) low self-toxicity; iii) high relaxation efficiency; iv) selectivity to tissues and organs (targeting); v) proper retention in the body time and is easily excreted from the body. Gadolinium-based contrast agents play an important role in magnetic resonance imaging contrast agents, and the corresponding ligands are: diethylenetriaminepentaacetic acid (DTPA); 1,4,7,10-tetraazacyclododecane; 1 , 4,7,10-tetraacetic acid (DOTA) and ethylenediaminetetraacetic acid (EDTA) and their derivatives. Gd-DTPA (Magnevist, Magnevist), Gd-DTPA-BMA, Gd-DO3A-HP (gadotirol, Prohance) and Gd-DOTA (Dotarem) have entered clinical application. However, it has been discovered since 1987 that gadolinium-based contrast agents can cause direct damage to renal tubules, and in recent years it has been confirmed that traditional gadolinium-based imaging contrast agents can cause renal cell fibrosis. Therefore, how to avoid additional toxic and side effects is the research and development direction of gadolinium-based contrast agents in the future. The current international development trend is still to pursue the design and synthesis of contrast agent molecules with high selectivity and strong targeting for specific tissues and organs on the basis of ensuring the stability, low toxicity and high relaxation rate of contrast agent molecules, including gadolinium-based Chemical structure modification of magnetic resonance imaging contrast agents and exploration of new superparamagnetic (Fe ₃ O ₄ -based) contrast agents and water-soluble paramagnetic contrast agents. These compounds can be further chemically and physically modified with albumin, dextran, polystyrene, monoclonal antibodies, etc. to enhance targeting. However, since most of them are in the research and development stage, there is no final conclusion. At the same time, it is noted that seeking contrast agents with lower toxic and side effects is also the direction of future research.

As a kind of natural polysaccharide, chitosan is an amino straight-chain polysaccharide, which is a pure natural high-yield renewable green resource. Chitosan is selected to modify the contrast agent because it has many excellent properties, such as: i) its own zero toxicity, good water solubility and affinity with heavy metal ions; ii) it is easy to pass through the polyamine polyacid contrast agent Covalently bonded, after the combination, the long-chain winding characteristics of chitosan are used to wrap the chelated paramagnetic metal ions in the core, and at the same time increase the relaxation efficiency; iii) chitosan will degrade after entering the body, but it will take a certain time, Ensure that the metal ions of the contrast agent are in a packaged state and do not leak out when they remain in the body; iv) when the degree of polymerization of chitosan is 12-6, the binding ability to transport proteins such as human serum albumin is the strongest, and the untreated protein can be removed after the imaging measurement. The completely degraded contrast agent is metabolized and excreted from the body; v) carbohydrate compounds have different affinities to different organs or lesion organs, so as to improve the targeting of the contrast agent; vi) chitosan itself can also improve the Immunity, and has a certain anticancer effect, which is expected to enhance the synergistic effect; vii) Chitosan is easy to process into microcapsules, so it also has advantages in the preparation of dosage forms. These features fit well with the prerequisites for use as an MRI contrast agent. In addition, judging from the current research on the contrast performance of dextran and other carbohydrate compounds, chitosan is extremely expected to become a new type of MRI contrast agent with excellent performance.

In this method, the anhydride of the polyamine polyacid compound is combined with the amino group of chitosan through a covalent bond, and then complexed with paramagnetic ions such as rare earths or transition metals, such as: gadolinium, iron, manganese, etc., to make it a certain target. It is a new type of MRI contrast agent with tropism, suitable residence time in vivo, higher relaxation efficiency than Magnevist, and less toxicity. Its advantages are: i) amide bond-linked chitosan itself is non-toxic, which can reduce the drug application process and is easy to market; ii) sugar molecules can recognize different organs and lesions, improving the corresponding selectivity; iii) The same imaging contrast effect can be achieved by using 1/4 to 2/3 of the gadolinium content of Magnevist, which can further reduce the risk of metal ion toxicity in the body; iv) The processing and preparation process is simple, the equipment requirements are low, and it is easy to use Processing.

Contents of the invention

The purpose of the present invention is to reduce the toxic and side effects of traditional gadolinium-based contrast agents on organs, provide a potential new functional nuclear magnetic resonance imaging contrast agent and its preparation method, improve the relaxation efficiency and prolong the relaxation time in vivo Selectivity of different organ tissues or lesions, and reduce toxic side effects.

The preparation method of chitosan modified paramagnetic metal ion magnetic resonance imaging contrast agent of the present invention is as follows:

(1) In the reaction vessel, add a certain amount of polyamine polycarboxylic acid solid, and then add a certain volume of anhydrous acetic anhydride, wherein said polyamine polycarboxylic acid can be diethylenetriaminepentaacetic acid (DTPA ) or ethylenediaminetetraacetic acid (EDTA), wherein the ratio (mass: volume) of polyamine polycarboxylic acid to anhydrous acetic anhydride is 1:1 to 3, then add a certain volume of anhydrous pyridine as a catalyst, and pass dry nitrogen protection, stirred and reacted at 55-60°C for 24h, then suction-filtered, washed with anhydrous acetic anhydride, anhydrous N,N'-dimethylformamide (DMF) and anhydrous ether respectively to obtain polyamine polycarboxylic acid The corresponding acid anhydride, the obtained anhydride of polyamine polycarboxylic acid is diethylenetriaminepentaacetic anhydride (DTPAA) or ethylenediaminetetraacetic anhydride (EDTAA).

(2) Add water-soluble chitosan or narrow molecular weight distribution water-soluble chitosan to anhydrous N, N'-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), and then add 4-bis Methylaminopyridine or triethylamine or pyridine are used as a catalyst, and the polyamine polycarboxylic acid anhydride prepared in step (1) is added under normal temperature stirring, wherein the mol ratio of chitosan and polyamine polycarboxylic acid anhydride is 1: 1～ 5. Stir at room temperature for 36-72 hours, add distilled water after cooling in an ice bath and continue stirring for 12-120 hours. Concentrate in vacuo to a small volume, add absolute ethanol to precipitate a pale yellow solid, and dry in vacuo.

(3) Weigh the Sephadex G-25 gel, activate it and pack it into a column. Dissolve the solid obtained in step (2) in a small amount of distilled water, wherein the ratio of solid to distilled water (mass: volume) is 1:2 to 100, put it on the column, use distilled water as the eluting solvent, and use 0.1% ninhydrin n-butyl Alcohol solution is used for color development, and it is detected by an ultraviolet analyzer (detection wavelength is 254nm). After cooling, absolute ethanol was added to obtain a pale yellow precipitate, which was filtered and vacuum-dried to obtain a ligand powder.

(4) Weigh the ligand powder obtained in step (3) and dissolve it in distilled water, wherein the ratio (mass: volume) of the ligand powder to distilled water is 1: 5～20, add metal inorganic salt under stirring, the ligand and The molar ratio of the metal-inorganic salt is 1:1-5, continuously stirred at room temperature for 2-24 hours, concentrated the solution in vacuum, added absolute ethanol to precipitate after cooling, washed and filtered, and dried to obtain a solid powder of magnetic resonance imaging contrast agent 0.3 ~3.0g. The metal-inorganic salts described herein are all paramagnetic rare earth and transition metal ion salts, including hydrochloride, nitrate, acetate, sulfate or perchlorate containing different crystal water, preferably Gd ³⁺ Salts, Mn ²⁺ salts, Fe ³⁺ salts.

Compared with the prior art, the present invention has the following significant advantages:

1) Reaction at normal temperature, simple reaction operation and required equipment, low cost, and easy to realize large-scale production; 2) The product has good water solubility and a relaxation rate of 10-14L·mmol ^-1 ·s ^-1 , which is Gd- 1 to 3 times the relaxation rate of DTPA; 3) It has a targeted selection for liver and kidney tissue, which can effectively improve the clarity of contrast contrast between liver and kidney; 4) In vivo experiments in mice compared with Magnevist The results showed that the residues of gadolinium ions in the liver and kidney could be reduced by 2/5-2/3 30 days after drug withdrawal; It is non-toxic and harmless.

Specific implementation examples:

Example 1

The preparation method of water-soluble chitosan is as follows: take the high molecular weight chitosan of certain quality in there-necked flask, add the acetic acid solution that the volume fraction of certain volume is 0.5%, wherein the ratio of chitosan and acetic acid (mass: volume ) is 1:50, under stirring, add a certain volume of hydrogen peroxide with a mass fraction of 10%, wherein the ratio (mass:volume) of chitosan to hydrogen peroxide is 1:1, and stir for about 10min. The obtained solution was subjected to microwave treatment, and the microwave parameters were set (microwave time: 30 min, microwave temperature: 60° C.). After the microwave was finished, it was allowed to stand for cooling, and the pH was adjusted to neutral. Then the resulting solution was concentrated in vacuo to a certain volume, ethanol was added to precipitate the solid, washed with ether, and dried in vacuo to obtain an average molecular weight of 1980 (the degree of polymerization was 11, and the molecular weight distribution index (DPI or _Mw / _Mn ) =1.9) of water-soluble chitosan.

(1) In a three-necked flask, add a certain quality of diethylenetriaminepentaacetic acid (DTPA) solid, and then add a certain volume of anhydrous acetic anhydride, wherein the ratio (mass: volume) of DTPA to anhydrous acetic anhydride is 1 : 1, then add a certain volume of anhydrous pyridine as a catalyst, protect it with dry nitrogen, stir and react at 55-60°C for 24 hours, then filter it with suction, and use anhydrous acetic anhydride, anhydrous N,N'-dimethylformaldehyde respectively Amide (DMF) and anhydrous ether were washed to obtain a white solid powder of anhydride DTPAA of polyamine polycarboxylic acid. (2) add the chitosan prepared in step (1) in anhydrous N, N'-dimethylformamide (DMF), then add 4-dimethylaminopyridine as a catalyst, add step ( The polyamine polycarboxylic anhydride prepared in 1), wherein the molar ratio of chitosan and polyamine polyanhydride is 1:1, stirred at room temperature for 36h, cooled in an ice bath, added distilled water and continued to stir for 12h. Concentrate in vacuo to a small volume, add absolute ethanol to precipitate a pale yellow solid, and dry in vacuo. (3) Weigh the Sephadex G-25 gel, activate it and pack it into a column. Dissolve the solid obtained in step (2) in a small amount of distilled water, wherein the ratio of solid to distilled water (mass: volume) is 1:2, put it on the column, use distilled water as the eluting solvent, 0.1% ninhydrin n-butanol solution Color development, detection with a UV analyzer (wavelength is 254nm), two elution peaks appear during the elution process, the elution peak of the ligand first goes out of the column, it is collected and concentrated in a vacuum to a small volume, and after cooling Add absolute ethanol to obtain a pale yellow precipitate, filter and dry in vacuo to obtain a ligand powder. (4) Weigh the ligand powder obtained in step (3) and dissolve it in distilled water, wherein the ratio of ligand powder to distilled water (mass:volume) is 1:5, and add metal inorganic salt GdCl ₃ 6H ₂ O while stirring , wherein the molar ratio of the ligand to GdCl ₃ 6H ₂ O is 1:1, continuously stirred at room temperature for 2 hours, concentrated the solution in vacuum, added absolute ethanol to precipitate after cooling, washed and filtered, and dried to obtain the magnetic resonance imaging contrast agent The solid powder is about 1.0g.

Example 2

(1) In a three-necked flask, add a certain quality of diethylenetriaminepentaacetic acid (DTPA) solid, and then add a certain volume of anhydrous acetic anhydride, wherein the ratio (mass: volume) of DTPA to anhydrous acetic anhydride is 1 : 3, then add a certain volume of anhydrous pyridine as a catalyst, protect it with dry nitrogen, stir and react at 55～60°C for 24h, then suction filter, use anhydrous acetic anhydride, anhydrous N,N'-dimethylformamide respectively (DMF) and anhydrous ether washing to obtain the white solid powder of the anhydride DTPAA of polyamine polycarboxylic acid. (2) Add a water-soluble shell with an average molecular weight of 1460 (degree of polymerization 8, molecular weight distribution index (DPI or M _w /M _n ) = 1.06) in anhydrous N, N'-dimethylformamide (DMF) Polysaccharide (this raw material is provided by the Key Laboratory of Fine Chemical Industry of Hainan Province, and the preparation method is the same as example 1), then add 4-dimethylaminopyridine as a catalyst, add the polyamine polycarboxylic acid prepared in step (1) under normal temperature stirring anhydride, wherein the molar ratio of chitosan to polyamine polyanhydride DTPAA is 1:5, stirred at room temperature for 72 hours, cooled in an ice bath, added distilled water and continued to stir for 120 hours. Concentrate in vacuo to a small volume, add absolute ethanol to precipitate a pale yellow solid, and dry in vacuo. (3) Weigh the Sephadex G-25 gel, activate it and pack it into a column. Dissolve the solid obtained in step (2) in a small amount of distilled water, wherein the ratio of solid to distilled water (mass:volume) is 1:100, put it on the column, use distilled water as the eluting solvent, 0.1% ninhydrin n-butanol solution Color development, detection with a UV analyzer (wavelength is 254nm), two elution peaks appear during the elution process, the elution peak of the ligand first goes out of the column, it is collected and concentrated in a vacuum to a small volume, and after cooling Add absolute ethanol to obtain a pale yellow precipitate, filter and dry in vacuo to obtain a ligand powder. (4) Weigh the ligand powder obtained in step (3) and dissolve it in distilled water, wherein the ratio of ligand powder to distilled water (mass:volume) is 1:20, and add metal inorganic salt MnCl ₂ ·4H ₂ O under stirring , wherein the molar ratio of the ligand to MnCl ₂ ·4H ₂ O is 1:5, continuously stirred at room temperature for 24 hours, concentrated the solution in vacuum, added absolute ethanol to precipitate after cooling, washed and filtered, and dried to obtain the magnetic resonance imaging contrast agent 1.0g of solid powder.

Example 3

(1) In a three-necked flask, add diethylenetriaminepentaacetic acid (DTPA) solid, then add a certain volume of anhydrous acetic anhydride, wherein the ratio (mass: volume) of DTPA to anhydrous acetic anhydride is 1: 1, Then add a certain volume of anhydrous pyridine as a catalyst, protect it with dry nitrogen, stir and react at 55-60°C for 24 hours, then filter with suction, and use anhydrous acetic anhydride, anhydrous N,N'-dimethylformamide (DMF ) and anhydrous ether washing to obtain the white solid powder of the anhydride DTPAA of polyamine polycarboxylic acid. (2) in anhydrous dimethyl sulfoxide (DMSO), add the water-soluble chitosan (this raw material) that average molecular weight is 1080 (polymerization degree is 6, molecular weight distribution index (DPI or _Mw / _Mn )=1.03) Provided by Hainan Provincial Key Laboratory of Fine Chemical Industry, the preparation method is the same as example 1), then add triethylamine as a catalyst, add the polyamine polycarboxylic acid anhydride prepared in step (1) under normal temperature stirring, wherein chitosan and poly The molar ratio of amino polyacid anhydride DTPAA is 1:1, stirred at room temperature for 36 hours, cooled in an ice bath, added distilled water and continued to stir for 12 hours. Concentrate in vacuo to a small volume, add absolute ethanol to precipitate a pale yellow solid, and dry in vacuo. (3) Weigh the Sephadex G-25 gel, activate it and pack it into a column. Dissolve the solid obtained in step (2) in a small amount of distilled water, wherein the ratio of solid to distilled water (mass: volume) is 1:2, put it on the column, use distilled water as the eluting solvent, 0.1% ninhydrin n-butanol solution Color development, detection with a UV analyzer (wavelength is 254nm), two elution peaks appear during the elution process, the elution peak of the ligand first goes out of the column, it is collected and concentrated in a vacuum to a small volume, and after cooling Add absolute ethanol to obtain a pale yellow precipitate, filter and dry in vacuo to obtain a ligand powder. (4) Weigh the ligand powder obtained in step (3) and dissolve it in distilled water, wherein the ratio of ligand powder to distilled water (mass:volume) is 1:5, and add metal inorganic salt GdCl ₃ 6H ₂ O while stirring , wherein the molar ratio of the ligand to GdCl ₃ 6H ₂ O is 1:1, continuously stirred at room temperature for 2 hours, concentrated the solution in vacuum, added absolute ethanol to precipitate after cooling, washed and filtered, and dried to obtain the magnetic resonance imaging contrast agent The solid powder is about 0.5g.

Example 4

(1) In a three-necked flask, add ethylenediaminetetraacetic acid (EDTA) solid, then add a certain volume of anhydrous acetic anhydride, wherein the ratio (mass:volume) of EDTA to anhydrous acetic anhydride is 1:3, and then Add a certain volume of anhydrous pyridine as a catalyst, protect it with dry nitrogen, stir and react at 55-60°C for 24 hours, then filter with suction, and use anhydrous acetic anhydride and anhydrous N,N'-dimethylformamide (DMF) respectively Wash with anhydrous ether to obtain white solid powder of the anhydride EDTAA of polyamine polycarboxylic acid. (2) Add a water-soluble shell with an average molecular weight of 1080 (polymerization degree of 6, molecular weight distribution index (DPI or M _w /M _n ) = 1.03) in anhydrous N, N'-dimethylformamide (DMF) Polysaccharide (this raw material is provided by the Key Laboratory of Fine Chemical Industry of Hainan Province, and the preparation method is the same as example 1), then add 4-dimethylaminopyridine as a catalyst, add the polyamine polycarboxylic acid prepared in step (1) under normal temperature stirring anhydride, wherein the molar ratio of chitosan to polyamine polyanhydride EDTAA is 1:5, stirred at room temperature for 72 hours, cooled in an ice bath, added distilled water and continued to stir for 120 hours. Concentrate in vacuo to a small volume, add absolute ethanol to precipitate a pale yellow solid, and dry in vacuo. (3) Weigh the Sephadex G-25 gel, activate it and pack it into a column. The solid obtained in step (2) is dissolved with a small amount of distilled water, wherein the ratio of solid to distilled water (mass: volume) is 1: 2, put on the column, use distilled water as the eluting solvent, and 0.1% ninhydrin n-butanol solution is obviously The color is detected by an ultraviolet analyzer (wavelength is 254nm), and two elution peaks appear during the elution process. The elution peak of the ligand first goes out of the column, which is collected and concentrated in a vacuum to a small volume. After cooling, add Absolute ethanol gave a pale yellow precipitate, which was filtered and vacuum-dried to obtain a ligand powder. (4) Weigh the ligand powder obtained in step (3) and dissolve it in distilled water, wherein the ratio of ligand powder to distilled water (mass:volume) is 1:20, and add metal inorganic salt GdCl ₃ 6H ₂ O while stirring , wherein the molar ratio of the ligand to GdCl ₃ 6H ₂ O is 1:5, continuously stirred at room temperature for 24 hours, concentrated the solution in vacuo, added absolute ethanol to precipitate after cooling, washed and filtered, and dried to obtain the magnetic resonance imaging contrast agent The solid powder is about 1.5g.

Example 5

(1) In a three-necked flask, add ethylenediaminetetraacetic acid (EDTA) solid, then add a certain volume of anhydrous acetic anhydride, wherein the ratio (mass:volume) of EDTA to anhydrous acetic anhydride is 1:3, and then Add a certain volume of anhydrous pyridine as a catalyst, protect it with dry nitrogen, stir and react at 55-60°C for 24 hours, then filter with suction, and use anhydrous acetic anhydride and anhydrous N,N'-dimethylformamide (DMF) respectively Wash with anhydrous ether to obtain white solid powder of the anhydride EDTAA of polyamine polycarboxylic acid. (2) Add a water-soluble shell with an average molecular weight of 1460 (degree of polymerization 8, molecular weight distribution index (DPI or M _w /M _n ) = 1.06) in anhydrous N, N'-dimethylformamide (DMF) Polysaccharide (this raw material is provided by the Key Laboratory of Fine Chemical Industry of Hainan Province, and the preparation method is the same as Example 1), then add pyridine as a catalyst, add the polyamine polycarboxylic acid anhydride prepared in step (1) under normal temperature stirring, wherein chitosan The molar ratio of sugar to polyamine polyacid anhydride EDTAA was 1:5, stirred at room temperature for 72 hours, cooled in an ice bath, added distilled water and continued to stir for 120 hours. Concentrate in vacuo to a small volume, add absolute ethanol to precipitate a pale yellow solid, and dry in vacuo. (3) Weigh the Sephadex G-25 gel, activate it and load it into a column. Dissolve the solid obtained in step (2) in a small amount of distilled water, wherein the ratio of solid to distilled water (mass:volume) is 1:100, put it on the column, use distilled water as the eluting solvent, 0.1% ninhydrin n-butanol solution Color development, detection with a UV analyzer (wavelength is 254nm), two elution peaks appear during the elution process, the elution peak of the ligand first goes out of the column, it is collected and concentrated in a vacuum to a small volume, and after cooling Add absolute ethanol to obtain a pale yellow precipitate, filter and dry in vacuo to obtain a ligand powder. (4) Weigh the ligand powder obtained in step (3) and dissolve it in distilled water, wherein the ratio of ligand powder to distilled water (mass:volume) is 1:20, and add metal inorganic salt MnCl ₂ ·4H ₂ O under stirring , wherein the molar ratio of the ligand to MnCl ₂ ·4H ₂ O is 1:5, continuously stirred at room temperature for 24 hours, concentrated the solution in vacuum, added absolute ethanol to precipitate after cooling, washed and filtered, and dried to obtain the magnetic resonance imaging contrast agent The solid powder is about 1.5g.

Example 6

(1) In a three-necked flask, add diethylenetriaminepentaacetic acid (DTPA) solid, then add a certain volume of anhydrous acetic anhydride, wherein the ratio (mass: volume) of DTPA to anhydrous acetic anhydride is 1: 1, Then add a certain volume of anhydrous pyridine as a catalyst, protect it with dry nitrogen, stir and react at 55-60°C for 24 hours, then filter with suction, and use anhydrous acetic anhydride, anhydrous N,N'-dimethylformamide (DMF ) and anhydrous ether washing to obtain the white solid powder of the anhydride DTPAA of polyamine polycarboxylic acid. (2) in anhydrous dimethyl sulfoxide (DMSO), add the water-soluble chitosan (this raw material) that average molecular weight is 1980 (polymerization degree is 11, molecular weight distribution index (DPI or _Mw / _Mn )=1.08) Provided by the Key Laboratory of Fine Chemical Industry of Hainan Province, the preparation method is the same as example 1), then add 4-dimethylaminopyridine as a catalyst, add the polyamine polycarboxylic acid anhydride prepared in step (1) under normal temperature stirring, wherein chitosan The molar ratio of sugar to polyamine polyacid anhydride DTPAA was 1:1, stirred at room temperature for 36 hours, cooled in an ice bath, added distilled water and continued to stir for 12 hours. Concentrate in vacuo to a small volume, add absolute ethanol to precipitate a pale yellow solid, and dry in vacuo. (3) Weigh the Sephadex G-25 gel, activate it and pack it into a column. Dissolve the solid obtained in step (2) in a small amount of distilled water, wherein the ratio of solid to distilled water (mass: volume) is 1:2, put it on the column, use distilled water as the eluting solvent, 0.1% ninhydrin n-butanol solution Color development, detection with a UV analyzer (wavelength is 254nm), two elution peaks appear during the elution process, the elution peak of the ligand first goes out of the column, it is collected and concentrated in a vacuum to a small volume, and after cooling Add absolute ethanol to obtain a pale yellow precipitate, filter and dry in vacuo to obtain a ligand powder. (4) Weigh the ligand powder obtained in step (3) and dissolve it in distilled water, wherein the ratio of ligand powder to distilled water (mass:volume) is 1:5, and add metal inorganic salt GdCl ₃ 6H ₂ O while stirring , wherein the molar ratio of the ligand to GdCl ₃ 6H ₂ O is 1:1, continuously stirred at room temperature for 2 hours, concentrated the solution in vacuum, added absolute ethanol to precipitate after cooling, washed and filtered, and dried to obtain the magnetic resonance imaging contrast agent The solid powder is about 1.2g.

Example 7

(1) Take a certain quality of high molecular weight chitosan in a three-necked flask, add a certain volume of acetic acid solution with a volume fraction of 0.5%, wherein the ratio (mass: volume) of chitosan to acetic acid is 1: 50, in Under stirring, add a certain volume of 10% hydrogen peroxide, wherein the ratio (mass:volume) of chitosan to hydrogen peroxide is 1:1, and stir for about 10 minutes. The obtained solution was subjected to microwave treatment, and the microwave parameters were set (microwave time: 30 min, microwave temperature: 60° C.). After the microwave was finished, it was allowed to stand for cooling, and the pH was adjusted to neutral. Then the resulting solution was concentrated in vacuo to a small volume, ethanol was added to precipitate the solid, washed with ether, and dried in vacuo to obtain an average molecular weight of 1980 (the degree of polymerization was 11, and the molecular weight distribution index (DPI or M _w /M _n ) =1.9) of water-soluble chitosan. (2) In the three-necked flask, add ethylenediaminetetraacetic acid (EDTA) solid, then add a certain volume of anhydrous acetic anhydride, wherein the ratio (mass:volume) of EDTA to anhydrous acetic anhydride is 1:1, and then Add a certain volume of anhydrous pyridine as a catalyst, protect it with dry nitrogen, stir and react at 55-60°C for 24 hours, then filter with suction, and use anhydrous acetic anhydride and anhydrous N,N'-dimethylformamide (DMF) respectively Wash with anhydrous ether to obtain white solid powder of the anhydride EDTAA of polyamine polycarboxylic acid. (3) add the chitosan prepared in step (1) in anhydrous N, N'-dimethylformamide (DMF), then add 4-dimethylaminopyridine as a catalyst, add step ( 2) The polyamine polycarboxylic acid anhydride prepared in 2), wherein the molar ratio of chitosan and polyamine polycarboxylic acid anhydride EDTAA is 1:1, stirred at room temperature for 36h, cooled in an ice bath, added distilled water and continued to stir for 12h. Concentrate in vacuo to a small volume, add absolute ethanol to precipitate a pale yellow solid, and dry in vacuo. (4) Weigh the Sephadex G-25 gel, activate it and pack it into a column. Dissolve the solid obtained in step (3) in a small amount of distilled water, wherein the ratio of solid to distilled water (mass:volume) is 1:100, put it on the column, use distilled water as the eluting solvent, 0.1% ninhydrin n-butanol solution Color development, detection with a UV analyzer (wavelength is 254nm), two elution peaks appear during the elution process, the elution peak of the ligand first goes out of the column, it is collected and concentrated in a vacuum to a small volume, and after cooling Add absolute ethanol to obtain a pale yellow precipitate, filter and dry in vacuo to obtain a ligand powder. (5) Weigh the ligand powder obtained in step (4) and dissolve it in distilled water, wherein the ratio of ligand powder to distilled water (mass:volume) is 1:5, and add metal inorganic salt GdCl ₃ 6H ₂ O while stirring , wherein the molar ratio of the ligand to GdCl ₃ 6H ₂ O is 1:1, continuously stirred at room temperature for 2 hours, concentrated the solution in vacuum, added absolute ethanol to precipitate after cooling, washed and filtered, and dried to obtain the magnetic resonance imaging contrast agent The solid powder is about 1.0g.

Claims (1)

1. the preparation method of chitosan modified paramagnetic metal ion magnetic resonance imaging contrast agent is characterized in that it is carried out in the following steps: (1) In the reaction vessel, add a certain mass of polyamine polycarboxylic acid solids and a certain volume of anhydrous acetic anhydride, then add anhydrous pyridine as a catalyst, protect it with dry nitrogen, and stir and react at 55-60°C for 24 h After suction filtration, wash with anhydrous acetic anhydride, anhydrous N,N'-dimethylformamide and anhydrous ether respectively to obtain the corresponding anhydride of polyamine polycarboxylic acid, wherein polyamine polycarboxylic acid and anhydrous acetic anhydride The ratio of mass to volume is 1 : 1 to 3; the polyamine polycarboxylic acid is diethylenetriaminepentaacetic acid or ethylenediaminetetraacetic acid; (2) Add water-soluble chitosan or narrow molecular weight distribution water-soluble chitosan to anhydrous N,N'-dimethylformamide or dimethyl sulfoxide, then add 4-dimethylaminopyridine or triethylamine Amine or pyridine are used as catalyst, add the polyamine polycarboxylic acid anhydride prepared in step (1) under stirring at normal temperature, wherein the mol ratio of chitosan and polyamine polycarboxylic acid anhydride is 1 : 1～5, stir at room temperature 36-72 h, after cooling in an ice bath, add distilled water and continue stirring for 12-120 h, concentrate in vacuo to a small volume, add anhydrous ethanol to precipitate a pale yellow solid, and dry in vacuo; (3) take by weighing Sephadex G-25 type gel, pack column after activation, the solid obtained in step (2) is dissolved in a small amount of distilled water, wherein solid and distilled water ratio is counted as 1 : 2～2 in mass and volume ratio 100, put on the column, use distilled water as the eluting solvent, 0.1% ninhydrin n-butanol solution is used for color development, detect with an ultraviolet analyzer, the detection wavelength is 254nm, two elution peaks appear in the process of elution, and match The elution peak of the body first goes out of the column, collects it and concentrates it in vacuum to a small volume, adds absolute ethanol after cooling to obtain a light yellow precipitate, filters, and vacuum-dries to obtain a ligand powder; (4) Weigh the ligand powder obtained in step (3) and dissolve it in distilled water, wherein the ratio of the ligand powder to distilled water is 1 : 5 to 20 in terms of mass to volume ratio, and add the metal inorganic salt under stirring, and the ligand The molar ratio to the metal inorganic salt is 1 : 1-5, continuously stirred at room temperature for 2-24 h, concentrated the solution in vacuum, added absolute ethanol to precipitate after cooling, washed and filtered, and dried to obtain the solid of the magnetic resonance imaging contrast agent 0.3-3.0g powder; wherein the metal inorganic salts are Gd ³⁺ salts, Mn ²⁺ salts, Fe ³⁺ salts.