CN104922702A - Superparamagnetic nano-particle and preparation method and application thereof - Google Patents

Abstract

The invention provides a superparamagnetic nanoparticle and its preparation method and application. In the invention, ferrous and ferric iron compounds are reacted with an alkali solution, and then water-soluble citric acid compound is used as a modifier to obtain the superparamagnetic nanoparticle by coprecipitation Iron ferric oxide superparamagnetic nanoparticles coated with water-soluble citric acid compound on the surface. The method of the present invention makes the nucleation and growth stages of ferric oxide particles effectively separated, and obtains superparamagnetic nanoparticles with small and uniform particle size and good dispersion; and utilizes water-soluble citric acid compound to treat ferric oxide nanoparticles Surface modification improves the stability and biocompatibility of superparamagnetic nanoparticles in vivo. The present invention forms a stable dispersion system by dispersing the superparamagnetic nanoparticles in an aqueous solution to obtain a superparamagnetic contrast agent, and the contrast agent can perform nuclear magnetic resonance imaging on lesion tissues of myocardial infarction.

Description

A kind of superparamagnetic nanoparticle and its preparation method and application

technical field

The invention belongs to the field of nanomedicine, and relates to a superparamagnetic nanoparticle, a preparation method and application thereof, and a superparamagnetic contrast agent prepared from the superparamagnetic nanoparticle, and a preparation method and application thereof.

Background technique

Magnetic resonance imaging (Magnetic Resonance Imaging, MRI) is a technology that uses a magnetic field and radio frequency pulses to precess the precessing hydrogen nuclei in human tissue to generate radio frequency signals, which are processed by a computer to draw a structural image inside the object. Compared with other imaging methods, MRI has the following three advantages: 1. It has excellent resolution for soft tissues; 2. Various parameters can be used for imaging, and multiple imaging parameters can provide rich diagnostic information; 3. For the human body No ionizing radiation damage. In the gradual research process, it was found that the overlapping relaxation times of some different tissues severely limited the imaging capability of MRI, so people began to realize that the use of MRI contrast agents can improve the sensitivity and specificity of MRI diagnosis. MRI contrast agents change the relaxation rate of water protons in local tissues and improve the imaging contrast between normal and diseased parts to display the function and status of internal organs.

MRI contrast agents mainly include paramagnetic contrast agents and superparamagnetic contrast agents. At present, the commonly used paramagnetic contrast agent is gadolinium diethylenediamine acetate (Gd-DTPA), but the distribution of this contrast agent in the body is non-specific, and it quickly enters the intercellular space after entering the blood. To maintain a sufficient concentration during the imaging time, it is necessary to Injecting larger doses can only change the T1 signal of the tissue, so its use is relatively limited. The superparamagnetic contrast agent is mainly composed of iron ferric oxide (Fe ₃ O ₄ ) nanoparticles. The magnetic moment of superparamagnetic iron oxide is much larger than that of paramagnetic substances, and the relaxation performance is high. It can be selected by size selection or specific surface molecules. The modification achieves targeting to specific tissues, and has a unique transmembrane mechanism that enables intracellular molecular targeting. The blood half-life and in vivo distribution of superparamagnetic iron oxide are directly related to the size and surface state of its particles. The contrast agent with a smaller particle size has a longer blood circulation time, and can go through the blood circulation to the lesion, resulting in a difference in the imaging signal of the lesion.

According to the reaction conditions, the synthesis of superparamagnetic iron oxide can be divided into two categories: aqueous phase and non-aqueous phase synthesis. The aqueous phase synthesis methods mainly include co-precipitation and hydrothermal synthesis. Among them, the co-precipitation method is simple and easy to implement, and most of the commercial products are obtained by co-precipitation. Co-precipitation synthesized nanoparticles have good size uniformity and excellent dispersion in solution. Hydrothermal reaction uses high temperature and high pressure reaction to make iron oxide crystals maintain good magnetic response ability through aging. Non-aqueous phase synthesis mostly uses non-polar high-boiling point solvents, and iron oxide nanoparticles are obtained by precursor decomposition reaction. The dissolution and recrystallization process of metal ions in high-temperature reactions ensures the crystallinity of nanocrystals. Compared with low-temperature reactions Greatly improve the magnetic properties of iron oxide. At the same time, the nanoparticles synthesized due to the high-temperature pyrolysis reaction are excellent in size control and uniformity. However, the iron oxide surface modifiers synthesized in the non-aqueous phase system are long-chain aliphatic compounds, so that the nanoparticles must be chemically modified and the oil-soluble iron oxide can be transferred to the water phase before they can be used in biological applications.

At present, using citric acid to modify magnetic iron oxide to prepare superparamagnetic iron oxide can improve the water solubility of superparamagnetic iron oxide, which is convenient for biological application. At present, it is generally prepared by co-precipitation method. The co-precipitation method is to use two The aqueous solution of valent iron and ferric compound is mixed with the aqueous solution of citric acid, heated for reaction, and then added with a strong alkali solution for reaction to obtain superparamagnetic nanoparticles of iron ferric oxide. This method cannot separate the nucleation stage and the growth stage of the particles, resulting in The product particles have poor monodispersity.

Therefore, there is a need in the art to develop a superparamagnetic nanoparticle synthesized in an aqueous system that can be directly used in biological applications and has a smaller particle size and good dispersibility.

Contents of the invention

In view of the deficiencies in the prior art, one of the purposes of the present invention is to provide a superparamagnetic nanoparticle and its preparation method and application. The second object of the present invention is to provide a superparamagnetic contrast agent and its preparation method and application.

To achieve this purpose of the invention, the present invention adopts the following technical solutions:

On the one hand, the present invention provides a preparation method of superparamagnetic nanoparticles, the preparation method is: react ferrous iron and ferric iron compound with alkali solution, then use water-soluble citric acid compound as modifier, and co- The precipitation method obtains the ferric iron tetroxide superparamagnetic nanoparticles whose surface is coated with the water-soluble citric acid compound.

In the prior art, the co-precipitation method is used to prepare superparamagnetic nanoparticles of iron ferric oxide, which is to mix the aqueous solution of ferrous iron and ferric compound with the aqueous solution of citric acid, heat the reaction, and then add a strong alkali solution to react to obtain For triferroic superparamagnetic nanoparticles, the reaction occurs very quickly in this process, and the nucleation stage and the growth stage of the particles cannot be separated, resulting in poor monodispersity of the product particles. The present invention adopts the reaction of divalent iron and ferric iron compound with alkali solution to nucleate first, and then reacts with water-soluble citric acid compound to make the particle crystal nucleus grow, effectively separating the nucleation stage and the growth stage, so that the particle has Small particle size, and good dispersion. Moreover, the preparation method of the superparamagnetic nanoparticles described in the present invention is completed in the water phase, has good water solubility, and can be directly used in biological applications.

As a preferred technical solution, the preparation method of superparamagnetic nanoparticles of the present invention comprises the following steps:

(1) Dissolving ferrous iron and ferric iron compounds in water to form a solution a;

(2) Add alkali solution to solution a, and react at 55-65°C under stirring to obtain solution b;

(3) adding an aqueous solution of a citric acid compound to solution b, and reacting at 80-95° C. under stirring to obtain solution c;

(4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet to obtain the superparamagnetic nanoparticles.

On the basis of the technical solution provided by the present invention, the ferrous compound is any one or a mixture of at least two of ferrous chloride, ferrous nitrate or ferrous sulfate.

Preferably, on the basis of the technical solution provided by the present invention, the ferric compound is any one or a mixture of at least two of ferric chloride, ferric nitrate, ferric oxalate or ferric acetate.

On the basis of the technical solution provided by the present invention, the alkaline solution is any one or a mixed solution of at least two of ammonia water, sodium hydroxide or potassium hydroxide.

On the basis of the technical solution provided by the present invention, the water-soluble citric acid compound is citric acid or water-soluble citrate.

Preferably, on the basis of the technical solution provided by the present invention, the water-soluble citric acid compound also includes water-soluble citric acid derivatives and/or water-soluble salts of citric acid derivatives.

Preferably, on the basis of the technical solution provided by the present invention, the water-soluble citrate is any one of sodium citrate, disodium citrate, and trisodium citrate or a mixture of at least two of them.

On the basis of the technical solution provided by the present invention, the concentrations of ferrous iron and ferric iron compounds in solution a in step (1) are all 10-50 mg/mL, such as 12 mg/mL, 15 mg/mL, 18 mg/mL , 24mg/mL, 28mg/mL, 32mg/mL, 36mg/mL, 40mg/mL, 44mg/mL or 48mg/mL.

Preferably, on the basis of the technical scheme provided by the present invention, the molar ratio of the iron element in the ferrous compound to the ferric compound in step (1) is (1-1.2):2, such as 1:2, 1.1: 2 or 1.2:2.

Preferably, on the basis of the technical scheme provided by the present invention, the molar ratio of hydroxide in the alkali solution described in step (2) to the iron element in the ferrous iron compound described in step (1) is ≥ 8:1, for example, 8:1, 8.2:1, 8.5:1, 9:1 or 10:1 etc.

Preferably, on the basis of the technical solution provided by the present invention, the pH of the alkaline solution in step (2) is 13-14.

On the basis of the technical solution provided by the present invention, the reaction temperature in step (2) is 55-65°C, such as 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C or 65°C.

Preferably, on the basis of the technical solution provided by the present invention, the adding method of the alkali solution in step (2) is dropwise, and the dropping rate is 20 to 50 drops/minute, such as 20 drops/minute, 30 drops/minute, 40 drops/min or 50 drops/min.

In the step (2) of the present invention, the divalent iron compound and the ferric compound react with the alkaline solution to nucleate at a temperature of 55 to 65° C., that is, the divalent iron and the ferric compound react with the alkali to generate ferric oxide, Complete the formation of particle nuclei.

On the basis of the technical solution provided by the present invention, the concentration of the citric acid compound aqueous solution in step (3) is 50-90 mg/mL, such as 52 mg/mL, 56 mg/mL, 60 mg/mL, 63 mg/mL, 66 mg/mL , 69mg/mL, 72mg/mL, 76mg/mL, 80mg/mL, 84mg/m or 88mg/mL.

Preferably, relative to 1 g of the ferrous compound, the dosage of the citric acid compound is 100-200 mg, such as 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg or 200 mg.

Preferably, on the basis of the technical solution provided by the present invention, the aqueous solution of the citric acid compound in step (3) is added dropwise, and the dropping rate is 50-80 drops/minute, such as 50 drops/minute, 60 drops /min, 70 drops/min or 80 drops/min.

On the basis of the technical solution provided by the present invention, the reaction temperature in step (3) is 80-95°C, such as 80°C, 85°C, 88°C, 90°C or 95°C.

In the step (3) of the present invention, the iron ferric oxide particles formed in the step (2) are reacted with a water-soluble citric acid compound to modify the surface of the ferric oxide, thereby improving its stability. The reaction at ~95°C is conducive to the growth of crystal nuclei and the formation of morphology, and is conducive to obtaining a small-sized, uniform and dispersed system.

Preferably, the stirring rate in step (2) and step (3) is 800 rpm to 1500 rpm, such as 800 rpm to 1500 rpm, such as 850 rpm, 900 rpm, 950 rpm RPM, 1000 RPM, 1100 RPM, 1200 RPM, 1300 RPM, 1400 RPM or 1500 RPM.

Preferably, the reaction time in step (2) and step (3) is 30 min to 1 h, such as 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 1 h.

Preferably, step (1), step (2) and step (3) are all carried out under protective gas, for example, can be carried out under nitrogen protection.

Preferably, the operation in step (4) is repeated 2 to 5 times, such as 2 times, 3 times, 4 times or 5 times.

As a preferred technical solution of the present invention, the preparation method of the superparamagnetic nanoparticles specifically includes the following steps:

(1) Under the protection of a protective gas, dissolve the ferrous and ferric compounds in water to form a solution a in which the concentrations of the ferrous and ferric compounds are both 10-50 mg/mL;

(2) Under the protection of protective gas, add an alkali solution with a pH of 13 to 14 into solution a, and react at 55 to 65° C. for 30 minutes to 1 hour under stirring at 800 rpm to 1500 rpm to obtain solution b;

(3) Under the protection of protective gas, add an aqueous solution of a citric acid compound with a concentration of 50-90 mg/mL to solution b, and react at 80-95° C. under stirring at 800 rpm to 1500 rpm to obtain a solution c;

(4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet to obtain the superparamagnetic nanoparticles.

In another aspect, the present invention provides superparamagnetic nanoparticles prepared by the preparation method described in the first aspect of the present invention.

In another aspect, the present invention provides a superparamagnetic contrast agent, which comprises superparamagnetic nanoparticles prepared by the preparation method described in the first aspect of the present invention.

The present invention also provides a preparation method of the superparamagnetic contrast agent. The method comprises: dispersing the superparamagnetic nanoparticles in an aqueous solution to form a stable dispersion system to obtain the superparamagnetic contrast agent.

The superparamagnetic contrast agent of the present invention can be used for nuclear magnetic resonance imaging. The contrast agent of the present invention can transport the superparamagnetic iron oxide nanoparticles to the pathological tissue of myocardial infarction through blood circulation, so as to perform nuclear magnetic resonance imaging at the myocardial infarction site.

Myocardial infarction is characterized by coronary artery occlusion, and the blood flow in the heart is fast and the flow rate is large. Large-sized iron oxide nanoparticles are easily phagocytized by macrophages in liver, spleen and other organs, and it is difficult to reach the heart; small-sized surface coatings are high Molecular iron oxide nanoparticles are difficult to accumulate in the myocardium with fast blood flow, while small-sized monodisperse small molecule citrate-modified superparamagnetic iron oxide nanoparticle contrast agents are easier to image in the heart.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention adopts first reacting divalent iron and ferric compound with alkali solution to nucleate (ferric oxide), and then carries out surface modification with water-soluble citric acid compound, and under heating condition, promotes the growth of crystal nuclei to obtain surface-coated Ferric iron tetroxide superparamagnetic nanoparticles of water-soluble citric acid compound. The method of the present invention makes the nucleation and growth stages of ferric oxide particles effectively separated, and obtains superparamagnetic nanoparticles with small and uniform particle size and good dispersion; and utilizes water-soluble citric acid compound to treat ferric oxide nanoparticles Surface modification improves the stability and biocompatibility of superparamagnetic nanoparticles in the body, and can exist stably for a long time in the physiological environment, which is conducive to maintaining long circulation in the body. In the present invention, the superparamagnetic contrast agent obtained by dispersing the superparamagnetic nanoparticles in the aqueous solution to form a stable dispersion system can transport the superparamagnetic iron oxide nanoparticles to the pathological tissue of myocardial infarction through blood circulation, Magnetic resonance imaging at the site of myocardial infarction.

Description of drawings

Fig. 1 is the transmission electron micrograph of the superparamagnetic nanoparticle (A) that embodiment 1 prepares and the superparamagnetic nanoparticle (B) that comparative example 1 prepares;

Fig. 2 is the relaxation rate diagram of the superparamagnetic contrast agent prepared in Example 1;

Fig. 3 is an MRI effect diagram of the superparamagnetic contrast agent prepared in Example 1 at the myocardial infarction site, where A is before injection of the contrast agent, and B is 24 hours after the injection of the contrast agent.

Detailed ways

The technical solutions of the present invention will be further described below through specific embodiments. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

Example 1

In this embodiment, the superparamagnetic contrast agent of the present invention is prepared by the following method, which includes the following steps:

(1) Under the protection of nitrogen, dissolve 1g FeSO ₄ 7H ₂ O and 2g FeCl ₃ 6H ₂ O in a three-necked flask filled with 40mL distilled water to form a ferrous compound with a concentration of 25mg/mL and a ferric compound with a concentration of 50mg/mL solution a;

(2) Add dropwise 30 mL of sodium hydroxide solution of pH=14 to solution a at a rate of 30 drops/min, under nitrogen protection, react at 55° C. for 30 min under mechanical stirring at 1000 rpm to obtain solution b;

(3) Under nitrogen protection, dropwise add 4 mL of an aqueous solution of citric acid with a concentration of 50 mg/mL to solution b at a rate of 50 drops/min, and react at 90° C. for 30 min under stirring at 1000 rpm to obtain solution c;

(4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet, repeating this 3 times to obtain the superparamagnetic nanoparticles;

(5) disperse the superparamagnetic nanoparticles of step (4) in normal saline to form a stable dispersion system, filter and sterilize through a 0.22 micron filter membrane to obtain a superparamagnetic contrast agent, adjust its concentration to 15mg/mL, Store at 4°C until use.

Comparative example 1

In this comparative example, a superparamagnetic contrast agent was prepared by the co-precipitation method in the prior art, and the method included the following steps:

(1) Under the protection of nitrogen, dissolve 1g FeSO ₄ 7H ₂ O and 2g FeCl ₃ 6H ₂ O in a three-necked flask filled with 40mL distilled water to form a ferrous compound with a concentration of 25mg/mL and a ferric compound with a concentration of 50mg/mL solution a;

(2) Dissolve 200mg of citric acid in 40mL of water to make an aqueous solution of citric acid with a concentration of 50mg/mL, add the solution to solution a, stir for 10 minutes, and heat in a water bath at 85°C;

(3) Add dropwise the sodium hydroxide solution of 30mL pH=14 with the speed of 30 drops/min in the solution of step (2), drop complete, under nitrogen protection, 1000 rpm mechanically stirred for 30 minutes, then cooled to room temperature;

(4) the product of step (3) is washed with alcohol, separated by magnet, washed with water, separated by magnet, and so repeated 3 times to obtain the superparamagnetic nanoparticles;

(5) disperse the superparamagnetic nanoparticles of step (4) in normal saline to form a stable dispersion system, filter and sterilize through a 0.22 micron filter membrane to obtain a superparamagnetic contrast agent, adjust its concentration to 15mg/mL, Store at 4°C until use.

Utilize transmission electron microscope (H-7650B, Hitachi, Japan) to characterize the superparamagnetic nanoparticles prepared by the present embodiment and the superparamagnetic nanoparticles prepared by comparative example 1, the results are as shown in Figure 1, as can be seen from Figure 1, The size of the prepared superparamagnetic nanoparticles is about 5-30nm, the particle size is small, the size is uniform, and the dispersibility is good, and the aqueous solution of divalent iron and ferric iron compound is mixed with the aqueous solution of citric acid commonly used in the prior art. , heating and reacting, and then adding a strong alkali solution to carry out the reaction, the dispersibility of the ferric iron tetroxide superparamagnetic nanoparticles obtained is very poor, and many particles gather together, thereby causing the particles to be unstable.

Utilize the magnetic resonance tester (MQ60, Bruker, Germany) to investigate the relaxation rate of the superparamagnetic contrast agent, and Fig. 2 shows the linear relationship diagram between the relaxation rate and the total iron ion concentration of the superparamagnetic contrast agent, by It can be seen from the figure that the relaxation rate of the obtained superparamagnetic contrast agent shows a good linear relationship, and the relaxation rate r ₂ is 173.6321mM ^-1 s ^-1 .

500 μL of the prepared superparamagnetic contrast agent was injected into the model mice through the tail vein, and 24 hours later, the 7-T magnetic resonance imager (ClinScan, Bruker, Germany) was used for imaging under appropriate sequence conditions. Fig. 3 is an MRI effect diagram of the superparamagnetic contrast agent at the myocardial infarction site. It can be seen from the figure that the prepared superparamagnetic contrast agent can reduce the gray value of the myocardial infarction site and realize imaging at the myocardial infarction site.

Example 2

In this embodiment, the superparamagnetic contrast agent of the present invention is prepared by the following method, which includes the following steps:

(1) Under the protection of nitrogen, dissolve 1.2g FeCl ₂ and 4.6g iron nitrate Fe(NO ₃ ) ₃ in a three-necked flask filled with 100mL distilled water to form a ferrous compound with a concentration of 12mg/mL and a ferric compound with a concentration of 12mg/mL. Solution a of 46mg/mL;

(2) Add 80 mL of sodium hydroxide solution with pH=14 dropwise at a rate of 20 drops/min, under nitrogen protection, react at 60° C. for 50 min under mechanical stirring at 800 rpm to obtain solution b;

(3) Under nitrogen protection, add 2 mL of an aqueous solution of sodium citrate with a concentration of 90 mg/mL dropwise to solution b at a rate of 80 drops/min, and react at 80° C. for 1 h under stirring at 1000 rpm to obtain solution c ;

(4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet, repeating this 5 times to obtain the superparamagnetic nanoparticles;

(5) Dispersing the superparamagnetic nanoparticles in step (4) in physiological saline to form a stable dispersion system, and filtering and sterilizing through a 0.22 micron filter membrane to obtain a superparamagnetic contrast agent.

Characterized by electron microscopy, the particle size is 5-40nm, the dispersion is good, and it has good relaxation properties, and can be used for nuclear magnetic resonance imaging in mice with myocardial infarction.

Example 3

In this embodiment, the superparamagnetic contrast agent of the present invention is prepared by the following method, which includes the following steps:

(1) Under the protection of nitrogen, dissolve the mixture of 0.4g FeCl ₂ and 1.2g ferric oxalate and 0.5g FeCl ₃ in a three-necked flask filled with 40mL distilled water to form a ferrous compound with a concentration of 10mg/mL and a ferric compound Solution a with a concentration of 42.5 mg/mL;

(2) Add 260mL of ammonia water with pH=13 dropwise at a rate of 50 drops/min to solution a, under nitrogen protection, react at 55°C for 1h under mechanical stirring at 1500 rpm, to obtain solution b;

(3) Under the protection of nitrogen, add 1 mL of an aqueous solution of disodium citrate with a concentration of 50 mg/mL dropwise to solution b at a rate of 50 drops/min, and react at 95°C for 40 min under stirring at 1500 rpm to obtain solution c ;

(4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet, repeating this 2 times to obtain the superparamagnetic nanoparticles;

(5) Dispersing the superparamagnetic nanoparticles in step (4) in physiological saline to form a stable dispersion system, and filtering and sterilizing through a 0.22 micron filter membrane to obtain a superparamagnetic contrast agent.

Characterized by electron microscopy, the particle size is 5-30nm, the dispersion is good, and it has good relaxation properties, and can be used for nuclear magnetic resonance imaging in mice with myocardial infarction.

Example 4

In this embodiment, the superparamagnetic contrast agent of the present invention is prepared by the following method, which includes the following steps:

(1) Under the protection of nitrogen, dissolve 2.0g Fe(NO ₃ ) ₂ and 3.6g FeCl ₃ in a three-necked flask filled with 80mL distilled water to form a ferrous compound with a concentration of 25mg/mL and a ferric compound with a concentration of 45mg /mL of solution a;

(2) Add dropwise 90 mL of potassium hydroxide solution of pH=14 to solution a at a rate of 50 drops/min, under nitrogen protection, react at 65° C. for 30 min under mechanical stirring at 1000 rpm to obtain solution b;

(3) Under the protection of nitrogen, add 4 mL of an aqueous solution of trisodium citrate with a concentration of 50 mg/mL dropwise to solution b at a rate of 50 drops/min, and react at 95° C. for 40 min under stirring at 1500 rpm to obtain solution c ;

(4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet, repeating this 5 times to obtain the superparamagnetic nanoparticles;

(5) Dispersing the superparamagnetic nanoparticles in step (4) in physiological saline to form a stable dispersion system, and filtering and sterilizing through a 0.22 micron filter membrane to obtain a superparamagnetic contrast agent.

Characterized by electron microscopy, the particle size is 10-40nm, the dispersion is good, and it has good relaxation properties, and can be used for nuclear magnetic resonance imaging in mice with myocardial infarction.

Example 5

In this embodiment, the superparamagnetic contrast agent of the present invention is prepared by the following method, which includes the following steps:

(1) Under the protection of nitrogen, the mixture of 1.0g Fe(NO ₃ ) ₂ and 1.0FeCl ₂ and 1.2g FeCl ₃ were dissolved in a three-necked flask filled with 40mL distilled water to form a ferrous compound with a concentration of 25mg/mL and trivalent Solution a with an iron compound concentration of 30 mg/mL;

(2) Add dropwise 120 mL of potassium hydroxide solution of pH=14 to solution a at a rate of 50 drops/min, under nitrogen protection, react at 65° C. for 30 min under mechanical stirring at 1500 rpm, to obtain solution b;

(3) Under the protection of nitrogen, dropwise add 4 mL of an aqueous solution of citric acid with a concentration of 80 mg/mL to solution b at a rate of 60 drops/min, and react at 95° C. for 30 min under stirring at 1500 rpm to obtain solution c;

(4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet, repeating this 5 times to obtain the superparamagnetic nanoparticles;

(5) Dispersing the superparamagnetic nanoparticles in step (4) in physiological saline to form a stable dispersion system, and filtering and sterilizing through a 0.22 micron filter membrane to obtain a superparamagnetic contrast agent.

Characterized by electron microscopy, the particle size is 5-40nm, the dispersion is good, and it has good relaxation properties, and can be used for nuclear magnetic resonance imaging in mice with myocardial infarction.

Example 6

In this embodiment, the superparamagnetic contrast agent of the present invention is prepared by the following method, which includes the following steps:

(1) Under the protection of nitrogen, 0.51g Fe(NO ₃ ) ₂ ·6H ₂ O and 0.48g FeCl ₃ were dissolved in a three-necked flask filled with 40mL distilled water to form a ferrous compound with a concentration of 12.75mg/mL, ferric iron Solution a with a compound concentration of 12 mg/mL;

(2) Add dropwise 145mL of ammonia solution with pH=13 to solution a at a rate of 70 drops/min, under nitrogen protection, react at 65°C for 30min under mechanical stirring at 1500 rpm, to obtain solution b;

(3) Under nitrogen protection, add 2 mL of an aqueous solution of citric acid with a concentration of 50 mg/mL dropwise to solution b at a rate of 50 drops/min, and react at 95° C. for 30 min under stirring at 1500 rpm to obtain solution c;

(4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet, repeating this 5 times to obtain the superparamagnetic nanoparticles;

(5) Dispersing the superparamagnetic nanoparticles in step (4) in physiological saline to form a stable dispersion system, and filtering and sterilizing through a 0.22 micron filter membrane to obtain a superparamagnetic contrast agent.

Characterized by electron microscopy, the particle size is 5-30nm, the dispersion is good, and it has good relaxation properties, and can be used for nuclear magnetic resonance imaging in mice with myocardial infarction.

Example 7

In this embodiment, the superparamagnetic contrast agent of the present invention is prepared by the following method, which includes the following steps:

(1) Under the protection of nitrogen, 1.9g Fe(NO ₃ ) ₂ ·6H ₂ O and 1.95g FeCl ₃ were dissolved in a three-necked flask filled with 40mL distilled water to form a ferrous compound with a concentration of 47.5mg/mL, ferric iron Solution a with a compound concentration of 48.75 mg/mL;

(2) Add dropwise 55 mL of potassium hydroxide solution of pH=14 to solution a at a rate of 20 drops/min, under nitrogen protection, react at 65° C. for 30 min under mechanical stirring at 800 rpm to obtain solution b;

(3) Under nitrogen protection, add 6 mL of an aqueous solution of citric acid with a concentration of 50 mg/mL dropwise at a rate of 60 drops/min to solution b, and react at 90° C. for 30 min under stirring at 1000 rpm to obtain solution c;

(4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet, repeating this 3 times to obtain the superparamagnetic nanoparticles;

(5) Dispersing the superparamagnetic nanoparticles in step (4) in physiological saline to form a stable dispersion system, and filtering and sterilizing through a 0.22 micron filter membrane to obtain a superparamagnetic contrast agent.

Characterized by electron microscopy, the particle size is 10-30nm, the dispersion is good, and it has good relaxation properties, and can be used for nuclear magnetic resonance imaging in mice with myocardial infarction.

The applicant declares that the present invention illustrates the superparamagnetic nanoparticles of the present invention and its preparation method and use through the above-mentioned examples, but the present invention is not limited to the above-mentioned examples, that is, it does not mean that the present invention must rely on the above-mentioned examples to achieve implement. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. a preparation method of superparamagnetic nanoparticles, characterized in that, the preparation method is: ferrous iron and ferric compound are reacted with alkali solution, then with water-soluble citric acid compound as modifier, with co- The precipitation method obtains the ferric iron tetroxide superparamagnetic nanoparticles whose surface is coated with the water-soluble citric acid compound. 2. the preparation method of superparamagnetic nanoparticles according to claim 1, is characterized in that, described preparation method comprises the steps: (1) Dissolving ferrous iron and ferric iron compounds in water to form a solution a; (2) Add alkali solution to solution a, and react at 55-65°C under stirring to obtain solution b; (3) adding an aqueous solution of a citric acid compound to solution b, and reacting at 80-95° C. under stirring to obtain solution c; (4) washing the solution c with alcohol, separating with a magnet, washing with water, and separating with a magnet to obtain the superparamagnetic nanoparticles. 3. according to the preparation method of claim 1 and 2 described superparamagnetic nanoparticles, it is characterized in that, described ferrous compound is any one or at least in ferrous chloride, ferrous nitrate or ferrous sulfate a mixture of the two; Preferably, the ferric compound is any one or a mixture of at least two of ferric chloride, ferric nitrate, ferric oxalate or ferric acetate. 4. according to the preparation method of the superparamagnetic nanoparticle described in any one in claim 1-3, it is characterized in that, described alkaline solution is any one or at least two in ammoniacal liquor, sodium hydroxide or potassium hydroxide mixed solution of species. 5. according to the preparation method of the superparamagnetic nanoparticle described in any one in claim 1-4, it is characterized in that, described water-soluble citric acid compound is citric acid or water-soluble citrate; Preferably, the water-soluble citric acid compound also includes water-soluble citric acid derivatives and/or water-soluble salts of citric acid derivatives; Preferably, the water-soluble citrate is any one or a mixture of at least two of sodium citrate, disodium citrate, and trisodium citrate. 6. according to the preparation method of the superparamagnetic nanoparticle described in any one in claim 2-5, it is characterized in that, the concentration of ferrous iron and ferric iron compound in the described solution a of step (1) is 10 ~50mg/mL; Preferably, the molar ratio of the ferrous iron compound in step (1) to the iron element in the ferric compound is (1-1.2):2; Preferably, the molar ratio of hydroxide in the alkali solution described in step (2) to iron in the ferrous compound described in step (1) is ≥8:1; Preferably, the pH of the alkaline solution in step (2) is 13-14; Preferably, the alkali solution in step (2) is added dropwise at a rate of 20-50 drops/minute. 7. according to the preparation method of the superparamagnetic nanoparticle described in any one in claim 2-6, it is characterized in that, the concentration of the described citric acid compound aqueous solution of step (3) is 50～90mg/mL; Preferably, the dosage of the citric acid compound is 100-200 mg relative to 1 g of the ferrous compound; Preferably, the aqueous solution of the citric acid compound in step (3) is added dropwise at a rate of 50-80 drops/minute; Preferably, the stirring rate in step (2) and step (3) is 800 rpm to 1500 rpm; Preferably, the reaction time of step (2) and step (3) is 30min～1h; Preferably, step (1), step (2) and step (3) are all carried out under protective gas; Preferably, the operation in step (4) is repeated 2 to 5 times. 8. A superparamagnetic nanoparticle prepared by the preparation method according to any one of claims 1-7. 9. A superparamagnetic contrast agent, characterized in that the superparamagnetic contrast agent comprises the superparamagnetic nanoparticles according to claim 8. 10. the preparation method of superparamagnetic contrast agent according to claim 9, is characterized in that, described method is: the superparamagnetic nano particle described in claim 8 is dispersed in aqueous solution and forms stable dispersion system, obtains The superparamagnetic contrast agent.