Ultrasonic / magnetic resonance dual-mode nanoprobe and its preparation method and application

Abstract

The present invention discloses an ultrasound / magnetic resonance dual-mode nanoprobe and its preparation method and application. The preparation method comprises the following steps: S1, preparing iron sulfide nanosheets (FeS) using ammonium ferrous sulfate, trisodium citrate, etc. as raw materials, and resuspending the FeS in ethanol to obtain an FeS ethanol solution; S2, adding perfluorohexane to the FeS anhydrous ethanol solution, and using an ultrasonic crusher under an ice bath to prepare a FeS / PFH suspension; S3, mixing the FeS / PFH suspension with a 1 mg / mL dopamine hydrochloride anhydrous ethanol solution, stirring at room temperature in the dark, and washing with pure water to obtain the final product, FeS / PFH / PDA nanomaterial. The present invention discloses an ultrasound / magnetic resonance dual-mode nanoprobe and its preparation method and application. The synthesis method for preparing the ultrasound / magnetic resonance dual-mode nanoprobe is simple and highly operable; the synthesized product is stable and reproducible, which is beneficial for biomedical research and clinical disease diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of dual-mode nanoprobes, and in particular to an ultrasound / magnetic resonance dual-mode nanoprobe and a preparation method and application thereof. Background Art

[0002] With the development of imaging science, modern medical imaging technology plays a vital role in the early diagnosis of diseases and the evaluation of treatment effects. Currently, commonly used imaging technologies include ultrasound imaging (US), magnetic resonance imaging (MRI), photoacoustic imaging (PAI), CT imaging, and PET imaging. However, single-modality imaging technologies often have certain limitations and cannot obtain comprehensive and detailed diagnostic information. Therefore, the integration and combined use of two or three of these imaging technologies to achieve multimodal imaging can overcome the shortcomings of single imaging technologies, provide clearer and more accurate images for clinical diagnosis, and provide more molecular, functional, and anatomical information for disease diagnosis and treatment.

[0003] Among numerous imaging technologies, ultrasound imaging (US) offers advantages such as low cost, real-time imaging, safety, and the absence of radiation. However, its tissue resolution and image clarity are relatively low. Magnetic resonance imaging (MRI) offers higher soft tissue resolution and image clarity and is capable of multi-directional, multi-parameter, and multi-sequence imaging, but lacks real-time imaging. Combining US and MR imaging techniques to achieve US / MR dual-modality imaging can yield richer, more complete, and more accurate imaging information, improving disease detection rates. Therefore, the synthesis of stable and biocompatible US / MR dual-modality nanoprobes is crucial in the biomedical field. Summary of the Invention

[0004] To solve the above problems, the present invention provides an ultrasound / magnetic resonance dual-mode nanoprobe and its preparation method and application. The synthesis method of the prepared ultrasound / magnetic resonance dual-mode nanoprobe is simple and highly operable. The synthesized product is stable and repeatable, which is beneficial to biomedical research and clinical disease diagnosis.

[0005] To achieve the above object, the present invention provides a method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe, comprising the following steps:

[0006] S1, preparing iron sulfide nanosheets FeS using ammonium ferrous sulfate, trisodium citrate, etc. as raw materials, and resuspending FeS in ethanol to obtain an FeS ethanol solution;

[0007] S2. Add perfluorohexane to the FeS anhydrous ethanol solution and use an ultrasonic disruptor under ice bath to prepare a FeS / PFH suspension;

[0008] S3. The FeS / PFH suspension was mixed with a 1 mg / mL dopamine hydrochloride anhydrous ethanol solution, stirred at room temperature in the dark, and washed with pure water to obtain the final product, FeS / PFH / PDA nanomaterial.

[0009] Preferably, in step S1, the specific steps of preparing FeS include:

[0010] S11, dissolving ammonium ferrous sulfate and trisodium citrate in ethylene glycol, adding polyethyleneimine after complete dissolution, and stirring at room temperature to obtain solution A;

[0011] S12, adding a 0.05 M thioacetamide solution in ethylene glycol to solution A to obtain solution B; adding triethanolamine dropwise to solution B and stirring at room temperature to obtain solution C;

[0012] S13. Solution C is subjected to a solvothermal reaction. After the reaction is completed, the solution is centrifuged and washed with ethanol. The precipitate is collected and resuspended with ethanol to obtain an ethanol solution of FeS.

[0013] Preferably, in step S11, the mass volume ratio of ammonium ferrous sulfate: trisodium citrate: ethylene glycol: polyethyleneimine is 235.284 mg: 58.82 mg: 20 mL: 500 mg.

[0014] Preferably, in step S12, the volume ratio of the ethylene glycol solution of thioacetamide to solution A is 15:20.

[0015] Preferably, in step S12, the volume ratio of solution B to triethanolamine is 35:2.

[0016] Preferably, in step S13, the temperature of the solvent thermal reaction is 200° C., and the reaction time is 24 h.

[0017] Preferably, in step S2, the mass volume ratio of perfluorohexane to FeS in the suspension is 40 μL:1 mg.

[0018] Preferably, in step S3, the volume ratio of the FeS / PFH suspension to the 1 mg / mL dopamine hydrochloride anhydrous ethanol solution is 1:1.

[0019] An ultrasound / magnetic resonance dual-mode nanoprobe FeS / PFH / PDA is prepared by the above-mentioned preparation method of the ultrasound / magnetic resonance dual-mode nanoprobe, and the particle size of FeS / PFH / PDA is 140-160 nm.

[0020] An application of the ultrasound / magnetic resonance dual-mode nanoprobe FeS / PFH / PDA as described above in non-medical ultrasound imaging, magnetic resonance imaging or ultrasound / magnetic resonance dual-mode imaging.

[0021] The ultrasound / magnetic resonance dual-mode nanoprobe of the present invention and its preparation method and application have the following beneficial effects:

[0022] (1) The present invention successfully constructed an innovative dual-modal nanoprobe, in which FeS gives the nanoprobe T2 magnetic resonance imaging (MRI) capability, and PFH gives the nanoprobe ultrasound imaging capability. The two are integrated into a nanoplatform to achieve dual-modal imaging with complementary advantages, providing a new tool for precision medicine;

[0023] (2) The synthetic method for preparing the ultrasound / magnetic resonance dual-mode nanoprobe of the present invention is simple and has strong operability; the synthesized product is stable and reproducible;

[0024] (3) The ultrasound / magnetic resonance dual-mode nanoprobe synthesized in the present invention has good biocompatibility;

[0025] (4) The ultrasound / magnetic resonance dual-mode nanoprobe synthesized by the present invention is beneficial to biomedical research and clinical disease diagnosis.

[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a transmission electron microscope image of the FeS nanomaterial prepared in Example;

[0028] Figure 2 UV-visible spectra of FeS nanomaterials and FFP nanomaterials;

[0029] Figure 3 The results of cell biocompatibility tests of FFP nanomaterials;

[0030] Figure 4 The results of the cell-hemocompatibility test of FFP nanomaterials;

[0031] Figure 5 In vitro ultrasound imaging of FFP nanomaterials;

[0032] Figure 6 In vitro magnetic resonance imaging of FFP nanomaterials;

[0033] Figure 7 Ultrasound imaging of mice after tail vein injection of FFP nanomaterials;

[0034] Figure 8 This is the 9.4T magnetic resonance imaging effect of mice after tail vein injection of FFP nanomaterials, where (a) is the magnetic resonance image of the mouse; (b) is the relative signal-to-noise ratio statistics. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages disclosed in the embodiments of the present invention more clear, the embodiments of the present invention are further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0036] Example

[0037] A method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe comprises the following steps:

[0038] Preparation of S1 and FeS nanomaterials

[0039] 235.284 mg of ammonium ferrous sulfate and 58.82 mg of trisodium citrate were dissolved in 20 mL of ethylene glycol. After complete dissolution, 500 mg of polyethyleneimine was added and the mixture was reacted at room temperature with magnetic stirring at 800 rpm for 120 min to obtain solution A.

[0040] Dissolve 56.3475 mg of thioacetamide in 15 mL of ethylene glycol to obtain solution B. Add solution B to solution A and react at room temperature with magnetic stirring at 800 rpm for 5 minutes. Add 2 mL of triethanolamine dropwise and react at room temperature with magnetic stirring at 800 rpm for 5 minutes.

[0041] After the reaction, the entire liquid was transferred to a Teflon-lined stainless steel autoclave and heated at 200°C for 24 hours. Finally, the solution was centrifuged at 14,000 rpm for 10 minutes and washed with anhydrous ethanol until the supernatant was clear, yielding the FeS nanosheets. The product was dispersed in ethanol and stored at 4°C or -20°C.

[0042] S2. Preparation of FFP nanomaterials

[0043] Take 1 mL of the ethanol solution of FeS nanosheets prepared in step S1 (containing 1 mg of FeS nanosheets), add 40 μL of perfluorohexane, and use an ultrasonic disruptor in an ice bath to prepare a FeS / PFH suspension. Then, under ice bath sonication, mix the FeS / PFH suspension at a 1:1 volume ratio with a 1 mg / mL solution of dopamine hydrochloride in anhydrous ethanol. Continue stirring at room temperature in the dark for 6 hours, then wash with pure water to obtain the final product, FeS / PFH / PDA (FFP) nanomaterial.

[0044] Experimental analysis

[0045] (1) Take 100 μg of FeS nanosheets in ethanol, centrifuge to remove the supernatant, and resuspend in 100 μL of pure water to prepare a 1 mg / mL solution. Use a 10 μL pipette to take 10 μL of the solution and drop it onto a copper grid, a total of 30-40 μL, dry it to prepare a transmission electron microscopy sample, and observe it with a transmission electron microscope.

[0046] like Figure 1 As shown, the FeS nanomaterial is in the shape of flakes and has a particle size of about 80-110 nm.

[0047] (2) ICP-OES quantitative Fe 2+ 200 μL of FeS and FFP with the same concentration were added, and their absorption curves were detected using a UV spectrophotometer.

[0048] like Figure 2 As shown, UV-visible spectroscopy confirmed that the absorbance of FFP at 808 nm was higher than that of FeS, which further confirmed the successful encapsulation of PDA.

[0049] (3) 3T3 cells were seeded in 96-well plates using DMEM medium (containing 10% FBS and 1% penicillin-streptomycin) (density: 1×10 4 cells / well) and incubated at 37°C, 5% CO₂, and humidity for 24 hours. Subsequently, the culture medium was replaced with 100 μL of fresh culture medium containing different concentrations of FFP nanomaterials (0, 20, 40, 80, and 160 μg / mL) and incubated for 24 hours. Finally, cell viability was determined using the MTT assay.

[0050] The results are as follows Figure 3 As shown in the results, when the FFP concentration reached 160 μg / mL, the viability of 3T3 cells did not decrease significantly compared with the control group. This result fully demonstrates the good biocompatibility of FFP and lays a solid foundation for subsequent in-depth research and application.

[0051] (4) Whole blood was collected after removing the mouse eyeball, and red blood cells were separated by low-temperature centrifugation and washed twice with cold sterile saline. Different concentration gradients of FFP were added to the red blood cell suspension as the experimental group, the red blood cell suspension diluted with PBS was used as the negative control, and the red blood cell suspension diluted with ultrapure water was used as the positive control. The blood was incubated at 37°C for 3 hours, centrifuged, and photographed. The absorbance of the sample at 542 nm was then measured using a microplate reader to calculate the hemolysis rate.

[0052] The results are as follows Figure 4 As shown in Figure 3, the hemolysis rate of FFP was observed to be less than 5%, confirming the good blood biocompatibility of FFP.

[0053] (5) The sample solutions of the experimental group and the negative control group in (4) were appropriately injected into the agarose gel sample pool prepared in advance. Care should be taken to avoid the generation of bubbles during the injection process. The concentration of FFP was 8 mg / mL. A GE LOGIQ E9 ultrasound diagnostic instrument was used, with the contrast mode selected and the L9 probe (MI 0.6). The imaging was recorded using the ultrasound diagnostic instrument's video function.

[0054] The results are as follows Figure 5 As shown, the load of PFH and the good acoustic phase change response of FFP are directly verified.

[0055] (6) MC38 cells treated with different concentrations of FFP were subjected to MR scanning to detect T2 relaxation rate. Figure 6 As shown, a typical concentration-dependent darkening effect was exhibited.

[0056] (7) Press Fe 2+ The experimental group MC38 mice were injected with FFP (100 μL) nanomaterials via tail vein at a concentration of 8 mg / kg to inject Fe 2+ Two groups were injected with 8 mg / kg FP (100 μL) and 100 μL 0.9% NaCl as control groups. Immediately after injection, the tumor area of ​​the mice was scanned using a GE LOGIQ E9 ultrasound diagnostic apparatus (9L linear array probe, contrast mode selected, MI 0.6), and dynamic images were saved. Figure 7 As shown in the figure, compared with the control group, the echo signal of the tumor site treated with FFP nanomaterials increased, indicating that FFP nanomaterials have good ultrasound imaging capabilities.

[0057] (8) After the MC38 mouse model was injected with FFP nanomaterials via the tail vein at a dose of 8 mg / kg, T2 magnetic resonance imaging was performed immediately after the injection of contrast agent.

[0058] The results are as follows Figure 8 As shown, part (a) is the T2 magnetic resonance imaging image of the mouse at different times before and after the injection of contrast agent, including coronal (COR) and transverse axial (TRA) images; part (b) is the image signal-to-noise ratio of part (a).

[0059] It can be seen that after the injection of FFP nanomaterials, the image signal-to-noise ratio increased compared with before injection and reached the maximum value at 60 minutes, indicating that FFP nanomaterials have the ability of T2 magnetic resonance imaging.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe, characterized in that: The steps include: S1, preparing iron sulfide nanosheets FeS using ammonium ferrous sulfate, trisodium citrate, etc. as raw materials, and resuspending FeS in ethanol to obtain an FeS ethanol solution; In S1, the specific steps of preparing the FeS ethanol solution include: S11, dissolving ammonium ferrous sulfate and trisodium citrate in ethylene glycol, adding polyethyleneimine after complete dissolution, and stirring at room temperature to obtain solution A; S12, adding a 0.05 M thioacetamide solution in ethylene glycol to solution A to obtain solution B; adding triethanolamine dropwise to solution B and stirring at room temperature to obtain solution C; S13, subjecting solution C to a solvothermal reaction, centrifuging after the reaction, washing with ethanol, collecting the precipitate, and resuspending it in ethanol to obtain an ethanol solution of FeS; S2. Add perfluorohexane to the FeS anhydrous ethanol solution and use an ultrasonic crusher under ice bath to prepare a FeS / PFH suspension; S3. The FeS / PFH suspension was mixed with a 1 mg / mL dopamine hydrochloride anhydrous ethanol solution, stirred at room temperature in the dark, and washed with pure water to obtain the final product, FeS / PFH / PDA nanomaterial.

2. The method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to claim 1, characterized in that: In step S11, the mass volume ratio of ammonium ferrous sulfate: trisodium citrate: ethylene glycol: polyethyleneimine is 235.284 mg: 58.82 mg: 20 mL: 500 mg.

3. The method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to claim 1, characterized in that: In step S12, the volume ratio of the ethylene glycol solution of thioacetamide to solution A is 15:

20.

4. The method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to claim 1, wherein: In step S12, the volume ratio of solution B to triethanolamine is 35:

2.

5. The method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to claim 1, wherein: In step S13, the temperature of the solvent thermal reaction is 200° C., and the reaction time is 24 h.

6. The method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to claim 1, characterized in that: In step S2, the mass volume ratio of perfluorohexane to FeS in the suspension is 40 μL:1 mg.

7. The method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to claim 1, characterized in that: In step S3, the volume ratio of the FeS / PFH suspension to the 1 mg / mL dopamine hydrochloride anhydrous ethanol solution is 1:

1.

8. An ultrasound / magnetic resonance dual-mode nanoprobe FeS / PFH / PDA prepared by the method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to any one of claims 1 to 7, characterized in that: The particle size of FeS / PFH / PDA is 140-160 nm.

9. Use of the ultrasound / magnetic resonance dual-mode nanoprobe FeS / PFH / PDA according to claim 8 in the preparation of products for ultrasound imaging, magnetic resonance imaging or ultrasound / magnetic resonance dual-mode imaging.

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