CN110755643A - A kind of positive contrast agent and its preparation method and application - Google Patents

Abstract

The invention discloses a positive contrast agent and a preparation method and application thereof. The positive contrast agent includes protein and iron-based nanoparticles. The positive contrast agent prepared by the invention has the advantages of good biocompatibility, smaller size, higher relaxation efficiency and the like, and the preparation method also realizes green production.

Description

A kind of positive contrast agent and its preparation method and application

technical field

The invention belongs to the technical field of magnetic resonance imaging, and particularly relates to a positive contrast agent and a preparation method and application thereof.

Background technique

At present, the commonly used magnetic resonance contrast agents are mainly divided into two categories. One is a small-molecule positive contrast agent represented by Gd-DTPA. By shortening the longitudinal relaxation time (T ₁ ), the lesion is relatively normal tissue The signal becomes brighter; another type of negative contrast agent represented by ferric carboxymeglumine shortens the transverse relaxation time (T ₂ ), making the signal of the lesion area darker than that of normal tissue. However, both types of contrast agents currently have limitations in their use: for example, small-molecule positive contrast agents have a single function, metabolize too quickly, and are prone to cause nephrogenic systemic fibrosis; compared with positive contrast agents, negative contrast agents Contrast agents are often not favored by clinics due to their large size, poor contrast, and low sensitivity. Therefore, there is an urgent need to develop safer and more effective magnetic resonance contrast agents.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a positive contrast agent and its preparation method and application, the prepared positive contrast agent has good biocompatibility, smaller size and higher With the advantages of relaxation efficiency and other advantages, the preparation method also realizes green production.

In order to achieve the above-mentioned purpose or other purposes, the present invention is achieved through the following technical solutions, and the present invention provides a positive contrast agent, comprising:

protein;

Iron-based nanoparticles, wherein the iron-based nanoparticles are grown on the surface of the protein.

In one embodiment, the protein is one or both of serum protein or ferritin.

In one embodiment, the iron-based nanoparticles are ferrous sulfide nanoparticles.

Another object of the present invention is also to provide a method for preparing a positive contrast agent, comprising at least the following steps:

providing a reaction medium;

chelating ferrous salt ions on the protein through the reaction medium to obtain a precursor solution;

Under alkaline conditions, adding a sulfur source to the precursor solution to grow iron-based nanoparticles on the surface of the protein to obtain an intermediate solution;

Dialysis and lyophilization of the intermediate solution were performed to obtain the positive contrast agent.

In one embodiment, the reaction medium is water.

In one embodiment, at a temperature of 25-40° C., adding a sulfur source to the precursor solution and continuing the reaction for 2-4 hours to obtain the intermediate solution.

In one embodiment, the source of the ferrous salt ion includes one or more combinations of ferrous chloride, ferrous nitrate, ferrous sulfate or ferrous acetate.

In one embodiment, the sulfur source is sodium sulfide hydrate.

In one embodiment, the mass ratio of the ferrous salt ion to the sulfur source is (1:1)-(1:8).

In one embodiment, the formation process of the alkaline condition is to adjust the pH value of the precursor solution to 10-12 through a sodium hydroxide solution with a concentration of 1-2 mol/L.

Another object of the present invention is to provide a use of the positive contrast agent, which is applied in magnetic resonance imaging.

The positive contrast agent prepared by the invention is prepared by the protein-mimicking bionic technology, and the technology has the characteristics of simple preparation process, green and mild reaction conditions, low energy consumption, good environmental compatibility, low cost and easy clinical transformation. The positive contrast agent prepared by the invention has good biocompatibility, and can effectively avoid the shortcomings _of the current T1 magnetic resonance contrast agent such as excessive metabolism, easily caused nephrogenic fibrosis and the like. The present invention has certain universality, and other proteins can be used to control the synthesis of other nanoparticles with different shapes, sizes and functions and be used in the field of biotechnology.

Description of drawings

1 is a schematic flowchart of a preparation method in an embodiment;

2 is a schematic diagram of the distribution of iron-based nanoparticles on the protein surface in positive contrast agent A in one embodiment;

FIG. 3 is partial longitudinal relaxation efficiency data of positive contrast agent A in one embodiment;

4 is a schematic diagram of T1 magnetic resonance imaging of positive contrast agent A in _one embodiment;

FIG. 5 is partial longitudinal relaxation efficiency data of positive contrast agent B in one embodiment;

FIG. 6 is partial longitudinal relaxation efficiency data of positive contrast agent C in one embodiment;

FIG. 7 is the elemental composition data of the positive contrast agent D in one example.

Detailed ways

The embodiments of the present invention are described below through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments may be combined with each other under the condition of no conflict. It should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, rather than for limiting the protection scope of the present invention. In the following examples, the test methods without specific conditions are usually in accordance with conventional conditions or in accordance with the conditions suggested by various manufacturers.

When numerical ranges are given in the examples, it is to be understood that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention and those skilled in the art have mastered the prior art and the description of the present invention, and can also use the methods, equipment, and materials described in the embodiments of the present invention. Any methods, devices and materials similar or equivalent to those of the prior art can be used to implement the present invention.

In the present specification, the term "T1 contrast agent" refers to a positive contrast agent that forms an image signal of the body part to be imaged in a relatively high manner compared to the surroundings, thereby brightening the part to be diagnosed.

The invention provides a positive contrast agent, a preparation method and application thereof, and the positive contrast agent is used as a T1 contrast agent in magnetic resonance imaging.

Referring to Figure 1, a method for preparing a positive contrast agent is provided, comprising at least the following steps:

S1, providing a reaction medium;

S2, chelating ferrous salt ions on the protein through the reaction medium to obtain a precursor solution;

S3, adding a sulfur source to the precursor solution under alkaline conditions to grow iron-based nanoparticles on the surface of the protein to obtain an intermediate solution;

S4. Dialyzing and lyophilizing the intermediate solution to obtain the positive contrast agent.

The present invention will be specifically described below from step S1 to step S4.

In step S1, the reaction medium is, for example, water.

In step S2, the source of the ferrous salt ion includes one or more combinations of ferrous chloride, ferrous nitrate, ferrous sulfate or ferrous acetate. The protein is one or both of serum protein or ferritin. The protein is a protein in a protein solution, and the concentration of the protein solution is 6.25-25 mg/mL. The protein concentration of the present application will change the hydrodynamic particle size of the protein nanoparticle, and the protein particle size of the present application is easier to grow iron. based nanoparticles.

In step S3, the pH value of the precursor solution is adjusted to 10-12 by a sodium hydroxide solution with a concentration of 1-2 mol/L under alkaline conditions. Under the condition of a temperature of 25-40° C., a sulfur source is added to the precursor solution and the reaction is continued for 2-4 hours to obtain the intermediate solution. The mass ratio of the ferrous salt ion to the sulfur source is (1:1)-(1:8). The sulfur source is sodium sulfide hydrate.

In step S4, after the end of step S3, the intermediate solution is taken out, added to a dialysis bag with a molecular weight cut-off of 8000-14000, placed in a beaker filled with ultrapure water, and dialyzed for 24-48 hours, changing during Water 5-6 times. The solution after dialysis is collected, stored at -80°C for 3-4 hours, put into a freeze-drying bottle for freeze-drying and concentration treatment, and finally the freeze-dried positive contrast agent is obtained.

Another object of the present invention is to provide a positive contrast agent, comprising: protein and iron-based nanoparticles, wherein the iron-based nanoparticles grow on the surface of the protein.

The present invention is specifically described below with some embodiments.

Please refer to FIG. 2 to FIG. 4 , which are partial data of the positive contrast agent A obtained in an embodiment. The longitudinal relaxation efficiency is 5.35 mM ^-1 s ^-1 . The specific preparation process of the positive contrast agent A is as follows: an embodiment , accurately weigh 200-250 mg of human serum albumin (HSA) powder, add 8-10 mL of ultrapure water, and dissolve by ultrasonic vibration to obtain a human serum albumin solution with a concentration of 20-50 mg/mL; take 0.03-0.05 mmol of HSA FeCl ₂ ·4H ₂ O was added to the human serum albumin solution, and the mixture was continuously stirred for 3-5 minutes at room temperature. Prepare a 1-2 mol/L NaOH solution, add 0.5-1 mL into the reaction flask, and adjust the pH of the system to 11-12. 0.06-0.07 mmol of Na ₂ S·9H ₂ O solution was added to the above reaction system, and the reaction was continued at 35-37° C. for 2-4 hours. After the reaction, the solution was taken out, put into a dialysis bag with a molecular weight cut-off of 8000-14000, placed in a beaker containing ultrapure water, and dialyzed for 24-48 hours, during which the water was changed 5-6 times. The dialyzed solution was collected, stored at -80°C for 3-4 hours, and then put into a freeze-drying bottle for freeze-drying and concentration treatment. Finally, a freeze-dried positive contrast agent was obtained, which was recorded as positive contrast agent A. The size of the positive contrast agent A prepared in this example is 3-4 nm, and the longitudinal relaxation efficiency can reach 5.35-5.5 mM ^-1 s ^-1 . Referring to FIG. 3 is a schematic diagram of the longitudinal relaxation efficiency reaching 5.35 mM ^-1 s ^-1 in an embodiment. Referring to FIG. 4 , the positive contrast agent A has a good T ₁ magnetic resonance imaging effect. Referring to FIG. 2 , the positive contrast agent In A, the iron-based nanoparticles grow uniformly on the surface of the protein, and the dispersibility is very good.

Please refer to FIG. 5 , which is part of the data of the positive contrast agent B prepared in one embodiment. The longitudinal relaxation efficiency can reach 5.0 mM ^-1 s ^-1 . The specific preparation process of the positive contrast agent B is as follows: in one embodiment , accurately weigh, for example, 120-125 mg of human serum albumin (HSA) powder, add 8-10 mL of ultrapure water, and dissolve by ultrasonic vibration to obtain a human serum albumin solution with a concentration of 12-16 mg/mL. Add 0.03-0.05 mmol of FeCl ₂ ·4H ₂ O to the above-mentioned human serum albumin solution, and continue stirring for 3-5 minutes at room temperature. Prepare a 1-2 mol/L NaOH solution, add 0.5-1 mL into the reaction flask, and adjust the pH of the reaction system to 11-12. 0.03-0.05 mmol of Na ₂ S·9H ₂ O solution was added to the above reaction system, and the reaction was continued at 35-37° C. for 2-4 hours. After the reaction, the solution was taken out, put into a dialysis bag with a molecular weight cut-off of 8000-14000, placed in a beaker containing ultrapure water, and dialyzed for 24-48 hours, during which the water was changed 5-6 times. Collect the solution after dialysis, store it at -80°C for 3-4 hours, put it into a freeze-drying bottle for freeze-drying and concentration treatment, and finally obtain a freeze-dried positive contrast agent, denoted as positive contrast agent B, longitudinally. The relaxation efficiencies reached 4.9-5.0 mM ^-1 s ^-1 . Referring to FIG. 5 , it is a schematic diagram illustrating that the longitudinal relaxation efficiency reaches 5.0 mM ^-1 s ^-1 in an embodiment. The contrast agent prepared in this example has a good T ₁ magnetic resonance imaging effect.

Please refer to FIG. 6 , which is part of the data of the positive contrast agent C obtained by another embodiment. The longitudinal relaxation efficiency can reach 4.88mM ^-1 s ^-1 . The specific preparation process of the positive contrast agent C is as follows: in one embodiment , accurately weigh, for example, 60-62.5 mg of human serum albumin (HSA) powder, add 8-10 mL of ultrapure water, and dissolve by ultrasonic vibration to obtain a human serum albumin solution with a concentration of 6-7.81 mg/mL; take 0.03-0.05 mmol of FeCl ₂ ·4H ₂ O was added to the human serum albumin solution, and stirring was continued for 3-5 minutes at room temperature. Prepare a NaOH solution with a concentration of 1 mol/L, add 0.5-1 mL into the reaction flask, and adjust the pH of the system to 11-12. 0.1-0.12 mmol of Na ₂ S·9H ₂ O solution was added to the above reaction system, and the reaction was continued at 35-37° C. for 2-4 hours. After the reaction, the solution was taken out, put into a dialysis bag with a molecular weight cut-off of 8000-14000, placed in a beaker containing ultrapure water, and dialyzed for 24-48 hours, during which the water was changed 5-6 times. Collect the solution after dialysis, store it at -80°C for 3-4 hours, put it into a freeze-drying bottle for freeze-drying and concentration treatment, and finally obtain the freeze-dried solution, which is recorded as positive contrast agent, which is recorded as positive contrast agent C. . The longitudinal relaxation efficiency of the positive contrast agent C prepared in this example is 4.55-4.88 mM ^-1 s ^-1 . Referring ^to FIG. 6 , a schematic diagram of the longitudinal relaxation efficiency of an embodiment reaching 4.88 mM ^-1 s can be obtained. The contrast agent prepared in this example has a good T ₁ magnetic resonance imaging effect.

Please refer to FIG. 7 , which is part of the data of the positive contrast agent D obtained by another embodiment. From FIG. 7 , it is concluded that the basic element composition of the positive contrast agent D is Fe and S. The specific preparation process of positive contrast agent D is: in one embodiment, accurately weigh, for example, 60-62.5mg human serum albumin (HSA) powder, add 8-10mL ultrapure water, and dissolve by ultrasonic vibration to obtain a concentration of 6.0-7.81 mg/mL solution of human serum albumin; take 0.03-0.05 mmol of FeCl ₂ ·4H ₂ O and add it to the solution of human serum albumin, and continue stirring for 3-5 minutes at room temperature. Prepare a 1-2 mol/L NaOH solution, add 0.2-0.25 mL into the reaction flask, and adjust the pH of the system to 9-10. 0.2-0.24 mmol of Na ₂ S·9H ₂ O solution was added to the above reaction system, and the reaction was continued at 80-85° C. for 2-4 hours. After the reaction, the solution was taken out, put into a dialysis bag with a molecular weight cut-off of 8000-14000, placed in a beaker containing ultrapure water, and dialyzed for 24-48 hours, during which the water was changed 5-6 times. Collect the solution after dialysis, store it at -80°C for 3-4 hours, put it into a freeze-drying bottle for freeze-drying and concentration treatment, and finally obtain the freeze-dried solution, which is recorded as positive contrast agent, which is recorded as positive contrast agent D. . Referring to FIG. 7 , it can be obtained that the elemental composition of the contrast agent in an embodiment is Fe and S. The contrast agent prepared in this example has a good T ₁ magnetic resonance imaging effect.

The invention utilizes the protein simulation bionic technology, selects serum albumin or ferritin, and chelates with ferrous ions by means of amino acids rich in amino groups, carboxyl groups or sulfhydryl groups on the protein, and then changes the secondary structure of the protein in an alkaline environment. , after the addition of sodium sulfide, ferrous ions and sulfide ions rapidly nucleate and react, and under the confinement of proteins, an ultra-small protein-mimetic iron-based T ₁ contrast agent is finally formed.

Furthermore, it should be understood that the mention of one or more method steps in the present invention does not exclude that other method steps may also be present before and after said combined step or that other method steps may be inserted between these expressly mentioned steps, unless otherwise There are descriptions; it should also be understood that the combined connection relationship between one or more devices/devices mentioned in the present invention does not exclude that there may be other devices/devices before and after the combined device/device or explicitly mentioned in these Other devices/devices can be inserted between the two devices/devices unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, rather than limiting the arrangement order of each method step or limiting the scope of the present invention. In the case where the technical content is not substantially changed, it should also be regarded as the scope in which the present invention can be implemented.

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims (10)

1. a positive contrast agent, is characterized in that, comprises: protein; Iron-based nanoparticles, wherein the iron-based nanoparticles are grown on the surface of the protein. 2 . The positive contrast agent according to claim 1 , wherein the protein is one or both of serum protein and ferritin. 3 . 3 . The positive contrast agent according to claim 1 , wherein the iron-based nanoparticles are ferrous sulfide nanoparticles. 4 . 4. a preparation method of positive contrast agent, is characterized in that, comprises the following steps at least: providing a reaction medium; chelating ferrous salt ions on the protein through the reaction medium to obtain a precursor solution; Under alkaline conditions, adding a sulfur source to the precursor solution to grow iron-based nanoparticles on the surface of the protein to obtain an intermediate solution; Dialysis and lyophilization of the intermediate solution were performed to obtain the positive contrast agent. 5. The preparation method of a positive contrast agent according to claim 4, wherein the reaction medium is water. 6. the preparation method of a kind of positive contrast agent according to claim 4, is characterized in that, under the condition that temperature is 25-40 ℃, adds sulfur source to described precursor solution and continues to react 2-4 hours , to obtain the intermediate solution. 7. the preparation method of a kind of positive contrast agent according to claim 4, is characterized in that, the source of described ferrous salt ion comprises one in ferrous chloride, ferrous nitrate, ferrous sulfate or ferrous acetate one or more combinations. 8. The preparation method of a positive contrast agent according to claim 4, wherein the sulfur source is sodium sulfide hydrate. 9 . The method for preparing a positive contrast agent according to claim 4 , wherein the mass ratio of the ferrous salt ion to the sulfur source is (1:1)-(1:8). 10 . 10 . The use of the positive contrast agent according to claim 1 , wherein the positive contrast agent is used in magnetic resonance imaging. 11 .

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