Magnetic nanoprobe with small size and high magnetic particle imaging performance as well as preparation method and application of magnetic nanoprobe

Abstract

The invention discloses a magnetic nanoprobe with small size and high magnetic particle imaging performance as well as a preparation method and application of the magnetic nanoprobe. The method comprises the following steps: dissolving ferric chloride in an ethylene glycol solution, and magnetically stirring to form a uniform transparent solution; adding manganese chloride with different concentrations into the solution, and stirring for dissolving; adding sodium acetate, and uniformly stirring to form a transparent solution; finally, dissolving sodium citrate in the solution, uniformly stirring, and reacting the mixed solution through a solvothermal method; after the reaction is completed, a reaction product is purified through centrifugation and dialysis; and finally collecting the supernatant and concentrating to obtain the magnetic nano-probe. The magnetic nanoprobe prepared by the method disclosed by the invention is small in size, has high-sensitivity imaging performance and excellent hydrophilic performance, and is expected to be used for diagnosing tiny focuses.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and particularly relates to a magnetic nanoprobe with small size and high magnetic particle imaging performance, a preparation method and an application thereof. Background Art

[0002] Non-invasive imaging techniques enable the visualization of biological processes with molecular characteristics, significantly promoting the role of medical imaging in disease diagnosis, monitoring and treatment. In particular, the targeted imaging strategy targeting endogenous molecules shows higher sensitivity and specificity in non-invasive imaging, and is expected to improve disease diagnosis and accurately evaluate the therapeutic effect. This strategy utilizes the functions of the prepared probes and has been effectively applied to various imaging modalities, including optical imaging, positron emission tomography and magnetic resonance imaging. Magnetic particle imaging (MPI) was first introduced in 2005 as a new non-invasive and tomographic medical imaging modality, in which the signal only comes from the non-linear dynamic magnetization response of superparamagnetic nanoparticles, aiming to provide real-time, functional and quantitative non-invasive imaging. By directly detecting magnetic nanoparticles (MNPs), it provides high sensitivity, avoids ionizing radiation and minimizes tissue background interference, and can overcome the limitations of current imaging modalities. However, since MPI is a new molecular imaging technology and its tracers are still in the development stage, developing MNPs with high MPI imaging performance is an urgent need for the current development of MPI technology.

[0003] As the core tracer of MPI technology, the magnetic properties of MNPs directly determine the imaging resolution and sensitivity. Research shows that when the size of MNPs is <30 nm, their superparamagnetic properties can significantly enhance the magnetization reversal rate, thereby increasing the MPI signal intensity. However, existing synthesis techniques generally face the paradox of "size - performance - biocompatibility": Although the traditional coprecipitation method has a simple process, the product particle size distribution is wide (20 - 200 nm) and the crystallinity is low, resulting in poor magnetic response consistency; Although the thermal decomposition method can prepare highly uniform MNPs, it relies on organometallic precursors and high-temperature conditions (>300 °C), which not only generates a biocompatible hydrophobic surface but also increases the preparation cost due to the complex phase transfer process; The hydrothermal method can improve the crystallinity, but its high-pressure and airtight environment poses safety hazards, and there is a lack of precision in particle size control. More notably, existing methods generally exhibit performance antinomies. Reducing the annealing temperature to pursue small size will lead to an increase in crystal defects and a decrease in magnetic moment; while the high-temperature treatment required to improve crystallinity will cause particle agglomeration and increase the size. This contradiction directly results in the difficulty of improving the sensitivity of the MPI performance of current commercial MNPs (such as Resovist). In addition, the extensive use of organic solvents, high-energy-consuming reaction conditions, and complex post-treatment steps in traditional processes seriously violate the principles of green chemistry and restrict the clinical transformation process of MNPs.

[0004] Therefore, developing a green preparation technology for MNPs with small size (<10 nm), highly crystalline magnetic cores, good biocompatibility, and environmental friendliness has become a key issue for breaking through the bottleneck of MPI clinical applications. This can not only improve the sensitivity of lesion detection to the level of hundreds of cells but also promote the development of diagnostic and therapeutic integrated probes. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a magnetic nanoprobe with small size and high magnetic particle imaging performance, as well as a preparation method and application thereof. The magnetic nanoprobe prepared by the method of the present invention has a small size, high-sensitive imaging performance, and excellent hydrophilic performance, and is expected to be used for the diagnosis of micro-lesions.

[0006] In the first aspect, the present invention provides a preparation method for a magnetic nanoprobe with small size and high magnetic particle imaging performance, which is realized by adopting the following technical solutions.

[0007] A preparation method for a magnetic nanoprobe with small size and high magnetic particle imaging performance includes the following steps: S1. Dissolve ferric chloride in an ethylene glycol solution and stir evenly; S2. Add manganese chloride to the ethylene glycol solution containing ferric chloride and stir evenly; wherein, the molar ratio of ferric chloride to manganese chloride is 1:(0 - 0.6); S3. Dissolve sodium acetate in the solution obtained in step S2 and stir evenly; wherein, the molar ratio of sodium acetate to ferric chloride is (1-7):1; S4. Dissolve sodium citrate in the solution obtained in step S3 and stir evenly; the molar ratio of sodium citrate to ferric chloride is (1-2):1; S5. React the mixed solution obtained in step S4 by solvothermal method, and cool the reaction product to room temperature; S6. Wash and centrifuge the product obtained in step S5, and collect the supernatant; concentrate the collected supernatant, then perform dialysis, and concentrate the product after dialysis again to obtain a magnetic nanoprobe with small size and high magnetic particle imaging performance.

[0008] Further, in step S5, the reaction conditions of the solvothermal method are to react at 160-200 °C for 6-12 h.

[0009] Further, in step S6, the centrifugal washing conditions are: wash with ethanol and water, centrifuge at 8000-11000 rpm for 10-20 min, and wash 3-5 times.

[0010] Further, in step S6, the supernatant collected by centrifugation is concentrated at 40-60 °C for 12-48 h.

[0011] Further, in step S6, the cut-off molecular weight of the dialysis bag is 1000-3500 kDa, and the dialysis time is 1-2 days.

[0012] Further, in step S6, the product after dialysis is concentrated again, and the concentration multiple is 10-20 times.

[0013] In the second aspect, the present invention provides a magnetic nanoprobe with small size and high magnetic particle imaging performance, which is realized by the following technical solutions.

[0014] A magnetic nanoprobe with small size and high magnetic particle imaging performance prepared by the above preparation method.

[0015] In the third aspect, the present invention provides a use of a magnetic nanoprobe with small size and high magnetic particle imaging performance, which is realized by the following technical solutions.

[0016] An application of the above magnetic nanoprobe with small size and high magnetic particle imaging performance in the preparation of products for diagnosing micro-lesions.

[0017] This application has the following beneficial effects.

[0018] (1) The manganese-doped MNPs prepared by the present invention simultaneously have small size, high MPI performance and good hydrophilicity; (2) The MNPs prepared by the present invention are prepared with magnetic particles smaller than 10 nm through the selection and purification of raw materials. (3) Through the doping of manganese element, the present invention effectively improves the MPI performance. The MPI signal of the prepared MNPs is stronger than that of the commercial gold standard Vivotrax under the condition of the same concentration. (4) The operation of preparing manganese-doped MNPs by the present invention is simple, with high biosafety and strong hydrophilicity. Description of the Drawings

[0019] Figure 1 is the schematic diagram of the preparation method of the MNPs of the present invention; Figure 2 are the photos of the MNPs doped with different elements in Example 1 and Example 2 of the present invention dispersed in water (in the figure, from left to right are the MNPs doped with cobalt element, the MNPs doped with manganese element, the MNPs, the MNPs doped with sulfur element, and the MNPs doped with nickel element); Figure 3 are the MPI images of the MNPs doped with different elements in Example 1 and Example 2 of the present invention; Figure 4 is the result graph of the MPI signal values of the MNPs doped with different elements in Example 1 and Example 2 of the present invention under the same concentration; Figure 5 is the saturation magnetization curve graph of the MNPs doped with different elements in Example 1 and Example 2 of the present invention; Figure 6 is the spectral signal spectrum of the cobalt element-doped MNPs (Co 0.4 Fe 2.6 O4) of Example 2 of the present invention; Figure 7 is the spectral signal spectrum of the manganese element-doped MNPs (Mn 0.4 Fe 2.6 O4) of Example 2 of the present invention; Figure 8 is the spectral signal spectrum of the MNPs (Fe3O4) of Example 1 of the present invention; Figure 9 is the spectral signal spectrum of the sulfur element-doped MNPs (FeS) of Example 2 of the present invention; Figure 10 is the spectral signal spectrum of the nickel element-doped MNPs (Ni 0.4 Fe 2.6 O4) of Example 2 of the present invention; Figure 11 is the MNPs (Mn x Fe 3-x(O4) Photos of dispersion in water (in the figure, the dosages of manganese chloride from left to right are 1 mmol, 0.8 mmol, 0.6 mmol, 0.5 mmol, 0.4 mmol, and 0.2 mmol respectively); Figure 12 MNPs (Mn x Fe 3-x O4) with different ratios of manganese element doping prepared in Example 3 of the present invention; Figure 13 MNPs (Mn x Fe 3-x O4) with different ratios of manganese element doping prepared in Example 3 of the present invention; Statistical result graphs of hydrodynamic diameter, potential, and PDI; Figure 14 MNPs (Mn x Fe 3-x O4) with different ratios of manganese element doping prepared in Example 3 of the present invention; MPI image (in the figure, 1: Mn 0.4 Fe 2.6 O4; 2: Mn 0.8 Fe 2.2 O4; 3: MnFe2O4; 4: Mn 1.2 Fe 1.8 O4; 5: Mn 1.6 Fe 1.4 O4; 6: Mn2FeO4); Figure 15 MNPs (Mn x Fe 3-x O4) with different ratios of manganese element doping prepared in Example 3 of the present invention; MPI signal value result graph; Figure 16 MNPs (Mn x Fe 3-x O4) with different ratios of manganese element doping prepared in Example 3 of the present invention; Saturation magnetization curve graph; Figure 17 MNPs (Mn 0.8 Fe 2.2 O4) with manganese element doping (0.4 mmol) prepared in Example 3 of the present invention; Spectrum signal graph; Figure 18 MNPs (MnFe2O4) with manganese element doping (0.5 mmol) prepared in Example 3 of the present invention; Spectrum signal graph; Figure 19 MNPs (Mn 1.2 Fe 1.8 O4) with manganese element doping (0.6 mmol) prepared in Example 3 of the present invention; Spectrum signal graph; Figure 20For the spectral signal map of MNPs (Mn 1.6 Fe 1.4 O4) doped with 0.8 mmol of manganese element in Example 3 of the present invention; Figure 21 For the spectral signal map of MNPs (Mn2FeO4) doped with 1 mmol of manganese element in Example 3 of the present invention; Figure 22 For the transmission electron microscope image of MNPs (Mn 0.4 Fe 2.6 O4) prepared by doping with the optimal amount of manganese element in the present invention; Figure 23 For the TEM size statistical chart of MNPs (Mn 0.4 Fe 2.6 O4) prepared by doping with the optimal amount of manganese element in the present invention. Detailed implementation manners

[0020] The present invention provides a method for preparing MNPs with small size, high MPI performance and good hydrophilicity. The initial raw materials for preparing MNPs include ferric chloride, sodium acetate, sodium citrate, the reaction solvent is ethylene glycol, and raw materials containing different doping elements: manganese chloride, reduced glutathione, cobalt chloride, nickel chloride. The method of the present invention provides a simple and green preparation and purification route for preparing highly sensitive and biocompatible MPI tracers MNPs.

[0021] The following further describes this patent application with reference to the drawings and embodiments.

[0022] Example 1 A method for preparing a magnetic nanoprobe with small size and high magnetic particle imaging performance, comprising the following steps: Step 1: Dissolve ferric chloride in an ethylene glycol solution and stir magnetically to form a uniform yellow transparent solution; add 1 millimole of ferric chloride to every 10 mL of the ethylene glycol solution.

[0023] Step 2: Dissolve sodium acetate in the ethylene glycol solution containing ferric chloride and stir magnetically to form a uniform brown transparent solution; add 7 millimoles of sodium acetate to every 10 mL of the ethylene glycol solution.

[0024] Step 3: Dissolve sodium citrate in the above ethylene glycol solution containing ferric chloride and sodium acetate and stir magnetically to form a uniform solution; add 2 millimoles of sodium citrate to every 10 mL of the ethylene glycol solution.

[0025] Step 4: Transfer the mixed solution in Step 3 into a high-pressure reactor with a polytetrafluoroethylene lining, place it in an oil bath, and react for 12 h at 180 °C by solvothermal method. Cool the reaction product to room temperature.

[0026] Step 5: Wash the product in Step 4 with ethanol and water (10,000 rpm, centrifuge for 10 min), wash five times, and collect the supernatant each time; concentrate the collected supernatant (60 o °C, 24 h), dialyze in deionized water for 1 day (cut-off molecular weight of the dialysis bag: 3500 kDa), and concentrate the dialyzed product to 5 mL again; finally, store the concentrated MNPs sample in a refrigerator at 4 o °C.

[0027] Example 2 Preparation of MNPs doped with different elements Step 1: Dissolve ferric chloride in ethylene glycol solution and stir magnetically to form a uniform yellow transparent solution; add 1 millimole of ferric chloride to every 10 mL of ethylene glycol solution.

[0028] Step 2: Add reagents containing different elements (cobalt chloride, nickel chloride, manganese chloride, reduced glutathione) to the ethylene glycol solution containing ferric chloride respectively, and stir magnetically to form a uniform transparent solution; add 0.2 millimole of the reagent containing different elements to every 10 mL of ethylene glycol solution.

[0029] Step 3: Dissolve sodium acetate in the ethylene glycol solution obtained in the second step and stir magnetically to form a uniform brown transparent solution; add 7 millimoles of sodium acetate to every 10 mL of ethylene glycol solution.

[0030] Step 4: Dissolve sodium citrate in the ethylene glycol solution obtained in the third step and stir magnetically to form a uniform solution; add 2 millimoles of sodium citrate to every 10 mL of ethylene glycol solution.

[0031] Step 5: Transfer the mixed solution in Step 4 into a high-pressure reactor with a polytetrafluoroethylene lining, place it in an oil bath, and react for 12 h at 180 °C by solvothermal method. Cool the reaction product to room temperature.

[0032] Step 6: Wash the product in Step 5 with ethanol and water (10,000 rpm, centrifuge for 10 min), wash five times, and collect the supernatant each time; concentrate the collected supernatant (60 o °C, 24 h), dialyze in deionized water for 1 day (cut-off molecular weight of the dialysis bag: 3500 kDa), and concentrate the dialyzed product to 5 mL again; finally, store the MNPs doped with different elements in a refrigerator at 4o Refrigerator of C.

[0033] The following tests the product performance of the products prepared in Example 1 and Example 2: Refer to Figure 2 , Figure 2 is the white light photograph of the dispersion of MNPs doped with different elements in water prepared by the above solvothermal method and purification. By observation, the MNPs prepared by this method have strong hydrophilicity.

[0034] Refer to Figure 3 , different MNPs (the concentration of iron is 100 microliters) are placed in an ELISA strip and imaged by an MPI scanner, as Figure 3 shown. The MPI imaging signal shows that highly sensitive MNPs can be obtained by doping manganese during the preparation process. This result demonstrates that the doping of manganese elements can improve the sensitivity of MPI.

[0035] Refer to Figure 4 , Figure 4 is Figure 3 the MPI signal value after imaging. Through Figure 3 the result, it can be more intuitively observed that a feeding ratio of 0.2 mmol of manganese chloride can prepare MNPs with high MPI performance (Mn 0.4 Fe 2.6 O4).

[0036] Refer to Figure 5 , Figure 5 are the saturation magnetization curves of MNPs prepared by doping different elements in Example 1 and Example 2, which are Co 0.4 Fe 2.6 O4, Mn 0.4 Fe 2.6 O4, FeS, Fe3O4, Ni 0.4 Fe 2.6 O4 respectively. Figure 5 It shows that under the same test conditions, the doping of manganese can obtain a higher saturation magnetization intensity (Mn 0.4 Fe 2.6 O4).

[0037] Refer to Figures 6 - 10 , Figures 6 - 10 are the spectral signal maps of Co 0.4 Fe 2.6 O4, Mn 0.4 Fe 2.6 O4, Fe3O4, FeS, Ni 0.4 Fe 2.6 O4 respectively. The spectral signals of the materials are tested by a magnetic particle spectrometer, as Figure 7As shown, after doping with manganese element, the 9th harmonic number appears, proving that the doping of manganese element can not only improve the MPI signal of MNPs, but also improve the resolution of magnetic particles.

[0038] The above data prove that the doping of manganese element can effectively improve the MPI imaging performance of MNPs. Next, by changing the feeding ratio of manganese element, the optimal feeding ratio is explored.

[0039] Example 3 The difference between this example and Example 2 is that: the feeding ratio of manganese element (0.2 - 1 mmol) is changed to prepare Mn x Fe 3-x O4 (where x is the feeding amount of manganese element). The specific feeding ratios are: fixing the feeding of ferric chloride at 1 mmol, and adding different ratios of manganese chloride (0.2, 0.4, 0.5, 0.6, 0.8, and 1 mmol) respectively. The white light photos of the MNPs dispersed in water collected by centrifugation, dialysis, and concentration for the products prepared by the solvothermal method are as Figure 11 shown, and this figure proves that the prepared MNPs have good dispersibility in water.

[0040] Refer to Figure 12 , Figure 12 is the hydrated particle size distribution diagram of MNPs doped with different ratios of manganese element (Mn x Fe 3-x O4). As shown in the figure, as the doping ratio of manganese increases, the hydrated particle size of the prepared Mn x Fe 3-x O4 gradually increases. This shows that the doping of manganese element makes the magnetic particle core gradually increase.

[0041] Refer to Figure 13 , Figure 13 is the statistics of the hydrated particle size, potential, and PDI of Mn x Fe 3-x O4 prepared with different feeding ratios of manganese element (0.2 - 1 mmol). Through data display, it is found that as the feeding ratio of manganese element gradually increases, the hydrated particle size gradually increases. At the same time, the prepared MNPs show negative potential and good dispersibility.

[0042] Refer to Figures 14 - 15 , Figure 14 is the MPI image of 100 μL of Mn x Fe 3-x O4 doped with different ratios of manganese element. Through quantitative analysis of the imaging signals, as Figure 15 shown, it shows that the doping of 0.2 mmol of manganese chloride can significantly improve the MPI imaging performance.

[0043] Refer toFigures 16 - 21 , the imaging performance of MNPs was verified by testing the saturation magnetization curve ( Figure 16 ) and detecting the signals ( Figures 17 - 21 ). It was shown by Figure 17 that as the doping amount of manganese element gradually increased, the saturation magnetization intensity of the magnetic particles gradually decreased, and at the same time, the harmonic order also gradually decreased. The above data demonstrated that the doping of 0.2 mmol of manganese chloride could significantly improve the saturation magnetization intensity and imaging resolution.

[0044] Referring to Figures 22 - 23 , Figure 22 was the transmission electron microscope image of Mn 0.4 Fe 2.6 O4 prepared with the optimal manganese element doping. It was found that the prepared MNPs had monodisperse and spherical nanoparticles. Through statistical analysis of their sizes, it was shown that the average size of the prepared magnetic nanoparticles was 6.29 ± 0.19 nm.

[0045] Based on the above data, it was demonstrated that the magnetic nanoparticles prepared by this invention had small sizes and high MPI imaging performance. At the same time, they had higher imaging signals than commercial Vivotrax. Therefore, this invention provided a simple and green preparation method for preparing magnetic nanoparticles with high MPI performance.

[0046] The examples of this specific embodiment are all preferred embodiments of the present invention, and do not limit the protection scope of the present invention accordingly. Therefore, all equivalent changes made according to the structure, shape, and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A preparation method of a magnetic nanoprobe with small size and high magnetic particle imaging performance, characterized in that: It includes the following steps: S1. Dissolve ferric chloride in ethylene glycol solution and stir evenly; S2. Add manganese chloride to the ethylene glycol solution containing ferric chloride and stir evenly; wherein, the molar ratio of ferric chloride to manganese chloride is 1:(0 - 0.6); S3. Dissolve sodium acetate in the solution obtained in step S2 and stir evenly; wherein, the molar ratio of sodium acetate to ferric chloride is (1 - 7):1; S4. Dissolve sodium citrate in the solution obtained in step S3 and stir evenly; the molar ratio of sodium citrate to ferric chloride is (1 - 2):1; S5. React the mixed solution obtained in step S4 by solvothermal method, and cool the reaction product to room temperature; S6. Wash and centrifuge the product obtained in step S5, and collect the supernatant; concentrate the collected supernatant, then perform dialysis, and concentrate the product after dialysis again to obtain a magnetic nanoprobe with small size and high magnetic particle imaging performance.

2. The preparation method of a magnetic nanoprobe with small size and high magnetic particle imaging performance according to claim 1, characterized in that: In step S5, the reaction conditions of the solvothermal method are to react at 160 - 200 °C for 6 - 12 h.

3. The preparation method of a magnetic nanoprobe with small size and high magnetic particle imaging performance according to claim 1, characterized in that: In step S6, the centrifugation and washing conditions are: wash with ethanol and water, centrifuge at 8000 - 11000 r / min for 10 - 20 min, and wash 3 - 5 times.

4. The preparation method of a magnetic nanoprobe with small size and high magnetic particle imaging performance according to claim 1, characterized in that: In step S6, the supernatant collected by centrifugation is concentrated at 40 - 60 °C for 12 - 48 h.

5. The preparation method of a magnetic nanoprobe with small size and high magnetic particle imaging performance according to claim 1, characterized in that: In step S6, the cut-off molecular weight of the dialysis bag is 1000 - 3500 kDa, and the dialysis time is 1 - 2 days.

6. The preparation method of a magnetic nanoprobe with small size and high magnetic particle imaging performance according to claim 1, characterized in that: In step S6, the product after dialysis is concentrated again, and the concentration multiple is 10 - 20 times.

7. A magnetic nanoprobe with small size and high magnetic particle imaging performance prepared by the preparation method according to any one of claims 1 - 6.

8. Use of the magnetic nanoprobe with small size and high magnetic particle imaging performance according to claim 7 in the preparation of products for diagnosing micro-lesions.

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