CN110787307A - Magnetic resonance imaging nano contrast agent and preparation method and application thereof - Google Patents

Abstract

The invention discloses a magnetic resonance imaging nano-contrast agent and a preparation method and application thereof. The preparation method comprises: 1) mixing iron source, citrate and water and adjusting pH to acidity, then adding urea and heating and refluxing treatment In order to obtain Fe ₂ O ₃ super nano particles Fe ₂ O ₃ SPs; 2) The Fe ₂ O ₃ SPs and bovine serum albumin (BSA) are subjected to a contact reaction in dissolution to obtain a magnetic resonance imaging nano-contrast agent. The magnetic resonance imaging nano _- contrast agent belongs to T1 contrast agent, and has the advantages of excellent imaging effect, biosafety and easy metabolism, so that it can be applied in magnetic resonance imaging, and the preparation method has the advantages of easily available raw materials and simple operation. and mild conditions.

Description

Magnetic resonance imaging nano-contrast agent and preparation method and application thereof

technical field

The present invention relates to a contrast agent, in particular, to a magnetic resonance imaging nano-contrast agent and a preparation method and application thereof.

Background technique

Magnetic resonance imaging technology has become a powerful tool for current medical diagnosis due to its non-invasiveness and non-radiation. In order to achieve better imaging results, about 40-50% of contrast agents are currently used for MRI detection. Magnetic resonance contrast agents are generally classified into longitudinal (T ₁ ) and transverse (T ₂ ) contrast agents according to their effect on longitudinal or transverse relaxation.

Commonly used T1 contrast agents are Gd- and Mn _- type contrast agents, which are positive signals, show bright images, and have good imaging effects, but they are very dangerous. Once dissociated and accumulated in the body, they will lead to serious diseases and even death. For example, the use of Gd contrast agent can cause nephrogenic systemic fibrosis (NSF) in patients with liver and kidney insufficiency. Most T ₂ contrast agents are superparamagnetic iron oxide nanoparticles, and Fe is an important constituent element of hemoglobin, which is safe and non-toxic. But _T2 contrast agent is a negative signal, showing a dark image, susceptible to interference by some blood pooling, calcification, and metal deposition. Therefore, everyone focuses on the small-sized Fe _- based T1 contrast agent. However, the small size makes the potential high and easy to aggregate. If the aggregation is prevented by the method of loading, it is easy to cause difficulty in excretion.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a magnetic resonance imaging nano _- contrast agent and its preparation method and application. The invention can be applied in magnetic resonance imaging, and the preparation method has the advantages of easy availability of raw materials, simple operation and mild conditions.

In order to achieve the above purpose, the present invention provides a preparation method of a magnetic resonance imaging nano-contrast agent, the preparation method comprising:

1) Mixing iron source, citrate, water and adjusting pH to acidity, then adding urea and heating under reflux to obtain Fe ₂ O ₃ super nanoparticles Fe ₂ O ₃ SPs;

2) Contacting Fe ₂ O ₃ SPs and bovine serum albumin BSA in dissolution to obtain a magnetic resonance imaging nano-contrast agent.

The present invention also provides a magnetic resonance imaging nano-contrast agent, which is prepared by the above-mentioned preparation method.

The present invention further provides the application of the above-mentioned magnetic resonance imaging nano-contrast agent in non-medical magnetic resonance imaging.

Through the above technical solution, the present invention firstly prepares Fe ₂ O ₃ super nanoparticles under reflux conditions, and then adsorbs BSA to the surface of Fe ₂ O ₃ SPs to obtain BSA-modified Fe ₂ O ₃ SPs, The BSA-modified Fe ₂ O ₃ SPs have excellent magnetic resonance imaging imaging effects, present bright images, and belong to T ₁ contrast agents. At the same time, the T ₁ contrast agents are nontoxic to biological tissues and are easy to be metabolized and excreted, thus making the magnetic The resonance imaging nano-contrast agent combines the advantages of T ₁ contrast agent and T ₂ contrast agent, and its application range is guaranteed.

Other features and advantages of the present invention will be described in detail in the detailed description that follows.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification, and together with the following specific embodiments, are used to explain the present invention, but do not constitute a limitation to the present invention. In the attached image:

Fig. 1 is a large-scale transmission electron microscope image in Test Example 1

Fig. 2 is the small-scale transmission electron microscope picture in the detection example 1;

Fig. 3 is the high-resolution transmission electron microscope image in the detection example 1;

Fig. 4 is the electron diffraction pattern of Fe ₂ O ₃ SPs in Detection Example 2;

Fig. 5 is the X-ray powder diffraction pattern of Fe ₂ O ₃ SPs in Test Example 2;

Figure 6 is the X-ray absorption near-edge structure diagram of Fe ₂ O ₃ SPs in Test Example 3 (sample refers to Fe ₂ O ₃ SPs synthesized in this application, Fe foil refers to pure iron, Fe ₂ O ₃ Refers to Fe ₂ O ₃ standard);

Fig. 7 is the expanded X-ray absorption fine structure diagram of Fe ₂ O ₃ SPs in Test Example 3 (sample refers to Fe ₂ O ₃ SPs synthesized in this application, Fe foil refers to pure iron, Fe ₂ O ₃ Refers to Fe ₂ O ₃ standard);

Figure 8 DLS detection diagram in detection example 4;

Figure 9 is the magnetic resonance imaging images of tumor-bearing mice at different time points of tumor, liver and kidney by intravenous injection of BSA-modified Fe ₂ O ₃ SPs;

Figure 10 is a data analysis diagram of the corresponding tumor and liver and kidney parts in Figure 9 (pre refers to before administration);

Fig. 11 is the magnetic resonance imaging images of BSA-modified Fe ₂ O ₃ SPs at different time points in the bladder of tumor-bearing mice by tail vein injection;

Figure 12 is a data analysis diagram of the corresponding tumor and liver and kidney parts in Figure 11 (pre refers to before administration);

Fig. 13 is the urea nitrogen index data analysis figure in the detection example 6 (contrl refers to the non-administration group);

Figure 14 is an analysis diagram of serum creatinine index data in Test Example 6 (contrl refers to the non-administration group);

Fig. 15 is a graph of histopathological analysis in Test Example 6 (contrl refers to the non-administration group).

Detailed ways

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

The endpoints of ranges and any values disclosed herein are not limited to the precise ranges or values, which are to be understood to encompass values proximate to those ranges or values. For ranges of values, the endpoints of each range, the endpoints of each range and the individual point values, and the individual point values can be combined with each other to yield one or more new ranges of values that Ranges should be considered as specifically disclosed herein.

The invention provides a preparation method of a magnetic resonance imaging nano-contrast agent, the preparation method comprising:

1) Mixing iron source, citrate, water and adjusting pH to acidity, then adding urea and heating under reflux to obtain Fe ₂ O ₃ super nanoparticles Fe ₂ O ₃ SPs;

2) Contacting Fe ₂ O ₃ SPs and bovine serum albumin BSA in dissolution to obtain a magnetic resonance imaging nano-contrast agent.

In step 1) of the above preparation method, the amount of each material can be selected within a wide range, but in order to further improve the imaging effect of the prepared contrast agent, preferably, in step 1), iron source, citrate The dosage ratio of , water and urea is 1.6×10 ^-4 mol: 3.0×10 ^-4 -4.0×10 ^-4 mol: 80-150mL: 3×10 ^-4 mol-5×10 ^-4 mol.

In step 1) of the above preparation method, the conditions of the thermal reflux treatment can be selected in a wide range, but in order to further improve the yield of Fe ₂ O ₃ SPs, preferably, in step 1), the thermal reflux treatment satisfies the following Conditions: The temperature is 110-130°C, and/or the treatment time is 20-30h.

In step 1) of the above preparation method, the pH adjustment range of the system can be selected in a wide range, but in order to further improve the yield of Fe ₂ O ₃ SPs, preferably, in step 1), before urea is added, The pH of the system was adjusted to 5.5-6.5. There are various options for the pH adjustment of the system, but considering the cost, preferably, the adjuster used in the pH adjustment of the system is selected from at least one of sodium hydroxide, potassium hydroxide and aqueous ammonia.

In step 1) of the above preparation method, the type of iron source and/or citrate can be selected in a wide range, but considering the cost and yield, preferably, in step 1), the iron source is selected from At least one of ferric chloride hexahydrate, ferric perchlorate and ferric sulfate; and/or, citrate is selected from at least one of trisodium citrate and potassium citrate.

In step 2) of the above preparation method, the amount of each material can be selected within a wide range, but in order to further improve the imaging effect of the prepared contrast agent, preferably, in step 2), Fe ₂ O ₃ SPs, The mass ratio of BSA is 1:0.8-1.2.

In step 2) of the above preparation method, the conditions of the contact reaction can be selected in a wide range, but in order to further improve the yield, preferably, in step 2), the contact reaction satisfies the following conditions: carry out under the condition of shaking , the reaction temperature is 30-37°C, and/or the reaction time is 4-8h.

On the basis of the above embodiment, in order to further improve the purity of the prepared Fe ₂ O ₃ SPs, preferably, before step 2), the preparation method further includes: mixing the system and isopropanol, and then centrifuging to get the precipitate ; More preferably, the volume ratio of the system and isopropanol is 1:3-5;

On the basis of the above embodiment, in order to further improve the purity of the prepared BSA-modified Fe ₂ O ₃ SPs, preferably, after step 2), the preparation method further includes: dialyzing the mixed solution for 20-30 hours to remove excess BSA.

The present invention also provides a magnetic resonance imaging nano-contrast agent, which is prepared by the above-mentioned preparation method.

The present invention further provides the application of the above-mentioned magnetic resonance imaging nano-contrast agent in non-medical magnetic resonance imaging.

The present invention will be described in detail below by means of examples.

Example 1

1) Preparation and purification of Fe ₂ O ₃ super nanoparticles (SPs):

Under the condition of constant stirring, into a 250mL three-necked flask containing 100mL of tertiary water, add 3.4×10 ^-4 mol trisodium citrate, 1.6×10 ^-4 mol ferric chloride hexahydrate in sequence, and use 0.1 mol· The pH was adjusted to 6 with L ^-1 sodium hydroxide, fully shaken, 4×10 ^-4 mol urea was added after mixing evenly, and the mixture was boiled and refluxed for 24 hours at 120°C. After the reaction was completed, the solution was cooled to 25°C and a sample was collected. Take 1 mL of the Fe ₂ O ₃ SPs stock solution prepared above, add 3 mL of isopropanol, mix well, centrifuge at 10,000 rpm for 10 min, discard the supernatant, and redisperse the obtained precipitate in water or freeze-dry it for later use.

2) Preparation and purification of BSA-modified Fe ₂ O ₃ SPs:

BSA solution was added to the purified Fe ₂ O ₃ SPs precipitation (the mass ratio of Fe to BSA was 1:1, the solvent in the BSA solution was water, and the dosage ratio of Fe ₂ O ₃ SPs precipitation to BSA solution was 1 mg: 1 mL), Redispersed; after mixing evenly, put it in a shaker (swinging frequency of 100rpm) and shake at 37°C for 6h to ensure that BSA is fully adsorbed, and finally dialyze for 24h to remove excess BSA (the molecular weight cut-off of the dialysis bag is 100KD).

Example 2

Under the condition of constant stirring, into a 250 mL three-necked flask containing 100 mL of tertiary water, add 3.0×10 ^-4 mol trisodium citrate, 1.6×10 ^-4 mol ferric chloride hexahydrate in sequence, and use 0.1 mol· Adjust the pH to 5.5 with L ^-1 sodium hydroxide, fully shake it, add 3×10 ^-4 mol urea after mixing evenly, and boil and reflux for 30h at 110°C. After the reaction was completed, the solution was cooled to 25°C and a sample was collected. Take 1 mL of the Fe ₂ O ₃ SPs stock solution prepared above, add 3 mL of isopropanol, mix well, centrifuge at 10,000 rpm for 10 min, discard the supernatant, and redisperse the obtained precipitate in water or freeze-dry it for later use.

2) Preparation and purification of BSA-modified Fe ₂ O ₃ SPs:

BSA solution was added to the purified Fe ₂ O ₃ SPs precipitation (the mass ratio of Fe to BSA was 1:0.8, the solvent in the BSA solution was water, and the dosage ratio of Fe ₂ O ₃ SPs precipitation to BSA solution was 1 mg: 1 mL), Re-dispersed; after mixing evenly, put it in a shaker (swing frequency of 100 rpm) and shake at 30 °C for 8 hours to ensure that BSA is fully adsorbed, and finally dialyze for 24 hours to remove excess BSA (the molecular weight cut-off of the dialysis bag is 100KD).

Example 3

Under the condition of constant stirring, into a 250mL three-necked flask containing 100mL of tertiary water, add 4.0×10 ^-4 mol trisodium citrate, 1.6×10 ^-4 mol ferric chloride hexahydrate in sequence, and use 0.1 mol· Adjust the pH to 6.5 with L ^-1 sodium hydroxide, fully shake it, add 5×10 ^-4 mol urea after mixing evenly, boil and reflux at 130°C for 20h. After the reaction was completed, the solution was cooled to 25°C and a sample was collected. Take 1 mL of the Fe ₂ O ₃ SPs stock solution prepared above, add 3 mL of isopropanol, mix well, centrifuge at 10,000 rpm for 10 min, discard the supernatant, and redisperse the obtained precipitate in water or freeze-dry it for later use.

2) Preparation and purification of BSA-modified Fe ₂ O ₃ SPs:

BSA solution was added to the purified Fe ₂ O ₃ SPs precipitation (the mass ratio of Fe to BSA was 1:1.2, the solvent in the BSA solution was water, and the dosage ratio of Fe ₂ O ₃ SPs precipitation to BSA solution was 1 mg: 1 mL), Redispersed; after mixing evenly, put it in a shaker (swinging frequency of 100rpm) and shake at 37°C for 4h to ensure that BSA is fully adsorbed, and finally dialyze for 24h to remove excess BSA (the molecular weight cut-off of the dialysis bag is 100KD).

Test Example 1

The Fe ₂ O ₃ SPs prepared in Example 1 were tested by TEM, and the results are shown in Figures 1-3. Among them, Figure 1 is a large-scale TEM image (the inset is a photo of the corresponding SPs solution), and Figure 2 is a small-scale transmission Electron microscope image, Figure 3 is a high-resolution transmission electron microscope image. It can be seen from Figure 1 that Fe ₂ O ₃ SPs has the advantages of uniform size and good monodispersity. From Figure 2-3, it can be seen that Fe ₂ O ₃ SPs is composed of several small particles and has a topological layered structure. It can be seen from the figure that the average particle size of Fe ₂ O ₃ SPs nanoparticles is 15 nm)

Test example 2

The Fe ₂ O ₃ SPs prepared in Example 1 were detected by electron diffraction and X-ray powder diffraction. The results are shown in Figures 4-5, wherein Figure 4 is the electron diffraction pattern of Fe ₂ O ₃ SPs, and Figure 5 is Fe ₂ The X-ray powder diffraction pattern of O ₃ SPs shows that Fe ₂ O ₃ SPs has a quasi-amorphous structure.

Test Example 3

The Fe ₂ O ₃ SPs prepared in Example 1 were subjected to X-ray absorption detection, and the results are shown in Figures 6-7, wherein Figure 6 is the X-ray absorption near-edge structure diagram of Fe ₂ O ₃ SPs, and Figure 7 is Fe ₂ The extended X-ray absorption fine structure diagram of O ₃ SPs, it can be seen from the figure that the product of Example 1 is quasi-amorphous Fe ₂ O ₃ SPs with good monodispersity and topological layered structure.

Test Example 4

The Fe ₂ O ₃ SPs and BSA-modified Fe ₂ O ₃ SPs in Example 1 were detected by dynamic light scattering DLS, and the results are shown in Figure 8. Figure 8 is the DLS detection. It can be seen from the figure that BSA has been successfully modified to Fe ₂ O ₃ SPs.

Test Example 5

BSA-modified Fe ₂ O ₃ SPs enable in vivo magnetic resonance imaging (MRI) of small tumors:

BSA-modified Fe ₂ O ₃ SPs (100 μL, 3.75 mg·mL ^-1 ) were injected into the tail vein of 4T1 tumor-bearing mice with a tumor volume of about 5 mm ³ , and the differences between before and after administration were tested using Bruker’s small animal nuclear magnetic resonance spectrometer. The MRI effect of the site in different time periods was combined with 1.5% isoflurane anesthesia during the experiment. The specific results are shown in Figure 9-12.

Figure 9 shows the magnetic resonance of BSA-modified Fe ₂ O ₃ SPs in tumor, liver and kidney sites (tumor: bottom circle, liver: top circle, kidney: middle circle) at different time points of tumor-bearing mice after intravenous injection of BSA-modified Fe 2 O 3 SPs Resonance imaging diagram; FIG. 10 is the data analysis diagram of the tumor and liver and kidney parts corresponding to FIG. 9 , and the tumor volume is about 5 mm ³ . Fig. 11 is the magnetic resonance imaging images at different time points of the bladder site (indicated by circles) of BSA-modified Fe ₂ O ₃ SPs injected into the tail vein of tumor-bearing mice, and Fig. 12 is the data analysis diagram of the corresponding tumor and liver and kidney sites in Fig. 11 , The tumor volume was approximately 5 mm ³ .

It can be seen from the above figure that BSA-modified Fe ₂ O ₃ SPs can effectively detect small tumors as T ₁ contrast agent, and has excellent imaging effect. It can also be seen that BSA-modified Fe ₂ O ₃ SPs can be quickly excreted from the body. , high biological safety.

Test Example 6

Biosafety test of BSA-modified Fe ₂ O ₃ SPs (no kidney damage):

Healthy female BALB/c mice were divided into four groups, two in each group. One group was injected with PBS (100 μL, the control group in the figure) through the tail vein, and the other three groups of mice (named 1, 7, and 14 day groups, respectively) were intravenously injected with BSA-modified Fe ₂ O ₃ SPs (100 μL, 3.75 mg· mL ^-1 ). Blood was collected from mouse eyeballs at the corresponding time points. The blood was placed in a centrifuge tube, left standing at 37°C for 2h, centrifuged at 3000rpm for 15min, and the upper serum was taken and stored at -80°C. Blood biochemical data (urea nitrogen, urea nitrogen, urea nitrogen, etc.) Serum creatinine), the results are shown in Figures 13-14. The mice were sacrificed after blood collection, and the kidneys were washed with PBS, fixed with 4% paraformaldehyde tissue fixative, embedded in paraffin block, sectioned, stained with H&E, and subjected to histopathological analysis. The results are shown in Figure 15.

It can be seen from Figure 13-14 that the renal function indicators urea nitrogen and serum creatinine levels did not change significantly, and there was no obvious blood toxicity. It can be seen from Figure 15 that there is no obvious histological abnormality in the mice, and Fe ₂ O ₃ SPs does not cause renal damage through renal metabolism, and the biological safety is high.

The product of Example 2-3 was detected according to the above method, and the detection result showed that Example 2-3 also successfully prepared BSA-modified Fe ₂ O ₃ SPs.

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. In order to avoid unnecessary repetition, the present invention provides The combination method will not be specified otherwise.

In addition, the various embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the spirit of the present invention, they should also be regarded as the contents disclosed in the present invention.

Claims (10)

1. A preparation method of a magnetic resonance imaging nano contrast agent is characterized by comprising the following steps:

1) mixing iron source, citrate, water and adjusting pH to acidity, then adding urea and heating reflux treatment to obtain Fe₂O₃Super nanoparticle Fe₂O₃SPs；

2) Subjecting said Fe to₂O₃And performing contact reaction on the SPs and the bovine serum albumin BSA in the solution to obtain the magnetic resonance imaging nano contrast agent.

2. The method according to claim 1, wherein the iron source, the citrate, the water and the urea are used in a ratio of 1.6 x 10 in step 1)^-4mol：3.0×10^-4-4.0×10^-4mol：80-150mL：3×10^-4mol-5×10^-4mol。

3. The production method according to claim 1, wherein, in step 1), the heat reflux treatment satisfies the following condition: the temperature is 110-130 ℃, and/or the treatment time is 20-30 h.

4. The process according to claim 1, wherein in step 1), the pH of the system is adjusted to 5.5-6.5 before the addition of the urea;

preferably, the adjusting agent used in the pH adjustment of the system is selected from at least one of sodium hydroxide, potassium hydroxide and ammonia water.

5. The production method according to claim 1, wherein, in step 1), the iron source is selected from at least one of ferric trichloride hexahydrate, ferric perchlorate and ferric sulfate; and/or the presence of a gas in the gas,

the citrate is at least one of trisodium citrate and potassium citrate.

6. The production process according to claim 1, wherein,wherein, in step 2), the Fe₂O₃The mass ratio of SPs to BSA is 1: 0.8-1.2.

7. The production method according to claim 1, wherein, in step 2), the contact reaction satisfies the following condition: under the condition of shaking, the reaction temperature is 30-37 ℃, and/or the reaction time is 4-8 h.

8. The preparation method according to claim 1, wherein, prior to step 2), the preparation method further comprises: mixing the system and isopropanol, and centrifuging to obtain a precipitate;

preferably, the volume ratio of the system to the isopropanol is 1: 3-5;

preferably, after step 2), the preparation method further comprises: the mixed solution was dialyzed for 20-30h to remove excess BSA.

9. A magnetic resonance imaging nano-contrast agent, which is prepared by the preparation method of any one of claims 1 to 8.

10. Use of a magnetic resonance imaging nano-contrast agent as defined in claim 9 in non-medical magnetic resonance imaging.

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