Ultrasonic / magnetic resonance dual-mode nanoprobe as well as preparation method and application thereof

Abstract

The invention discloses an ultrasonic / magnetic resonance dual-mode nanoprobe as well as a preparation method and application thereof, the preparation method comprises the following steps: S1, preparing an iron sulfide nanosheet FeS by taking ammonium ferrous sulfate, trisodium citrate and the like as raw materials, and resuspending FeS ethanol to obtain a FeS ethanol solution; s2, adding perfluorohexane into the FeS absolute ethyl alcohol solution, and preparing a FeS / PFH suspension by adopting an ultrasonic crusher under an ice bath; s3, mixing the FeS / PFH suspension with a 1 mg / mL dopamine hydrochloride absolute ethyl alcohol solution, stirring at normal temperature in a dark place, and washing with pure water to obtain a final product FeS / PFH / PDA nano material. According to the ultrasonic / magnetic resonance dual-mode nanoprobe as well as the preparation method and the application thereof, the synthesis method for preparing the ultrasonic / magnetic resonance dual-mode nanoprobe is simple, convenient and high in operability; the synthesized product is stable, has repeatability, and is beneficial to 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, modern medical imaging technology plays a vital role in the early diagnosis of diseases and the evaluation of treatment effects. At present, commonly used imaging technologies include ultrasound imaging (US), magnetic resonance imaging (MRI), photoacoustic imaging (PAI), CT imaging, PET imaging, etc. However, single-modality imaging technology often has certain limitations and cannot obtain comprehensive and detailed diagnostic information. Therefore, the integration and combined use of the above two or three imaging technologies to achieve multimodal imaging can overcome the shortcomings of a single imaging technology, provide clearer and more accurate images for clinical diagnosis, and provide more molecular, functional and anatomical information for the diagnosis and treatment of diseases.

[0003] Among many imaging technologies, ultrasound imaging (US) has the advantages of low cost, real-time imaging, safety and no radiation, but its tissue resolution and image clarity are low; magnetic resonance imaging (MRI) has high soft tissue resolution and image clarity, and can perform multi-directional, multi-parameter, and multi-sequence imaging, but it cannot achieve real-time imaging. Combining US and MR imaging technologies to achieve US / MR dual-modality imaging can obtain richer, more complete, and more accurate imaging information and improve the detection rate of diseases. Therefore, it is very important to synthesize a US / MR dual-modality nanoprobe with good stability and good biocompatibility in the field of biomedicine. 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 has strong operability. 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, using ammonium ferrous sulfate, trisodium citrate, etc. as raw materials to prepare iron sulfide nanosheets FeS, and resuspending FeS in ethanol to obtain a FeS ethanol solution;

[0007] S2, adding perfluorohexane to the FeS anhydrous ethanol solution, and using an ultrasonic crusher under an ice bath to prepare a FeS / PFH suspension;

[0008] S3. Mix the FeS / PFH suspension with 1 mg / mL dopamine hydrochloride anhydrous ethanol solution, stir at room temperature in the dark, and wash 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, stirring at room temperature to obtain solution A;

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

[0012] S13, subjecting solution C to a solvothermal reaction, centrifuging after the reaction is completed, washing with ethanol, collecting the precipitate, and resuspending it in 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 method for preparing the ultrasound / magnetic resonance dual-mode nanoprobe as described above, and the particle size of FeS / PFH / PDA is 140-160nm.

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

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

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

[0023] (2) The synthesis method of 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 by 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 The transmission electron microscope image of the FeS nanomaterial prepared in the embodiment;

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

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

[0030] Figure 4 The results of the cell-blood compatibility 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 This is the ultrasound imaging effect 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 mice; (b) is the relative signal-to-noise ratio statistics. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings and embodiments. 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 reacted for 120 min at room temperature with magnetic stirring at 800 rpm to obtain solution A.

[0040] 56.3475 mg of thioacetamide was dissolved in 15 mL of ethylene glycol to obtain solution B. Solution B was added to solution A, and the mixture was reacted for 5 min at room temperature and under magnetic stirring at 800 rpm; 2 mL of triethanolamine was added dropwise, and the mixture was reacted for 5 min at room temperature and under magnetic stirring at 800 rpm.

[0041] After the reaction, the liquid was transferred to a stainless steel autoclave lined with Teflon and heated at 200°C for 24 hours. Finally, the mixture was collected by centrifugation at 14,000 rpm for 10 minutes and washed with anhydrous ethanol until the supernatant was clear to obtain the product 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 crusher to prepare a FeS / PFH suspension under ice bath. Then, under ice bath ultrasonic conditions, mix the FeS / PFH suspension with a volume ratio of 1:1 and a 1 mg / mL dopamine hydrochloride anhydrous ethanol solution, continue stirring at room temperature and away from light for 6 hours, and 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, resuspend in 100 μL 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 to prepare a transmission electron microscopy sample, and observe with a transmission electron microscope.

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

[0047] (2) ICP-OES quantitative Fe 2+ The same concentration of FeS and FFP 200 μL, using UV spectrophotometer to detect the absorption curve.

[0048] like Figure 2 As shown, UV-visible spectroscopy confirmed that the absorbance of FFP at 808 nm was higher than that of FeS, thus confirming 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) (strength: 1×10 4 cells / well) and cultured for 24 hours at 37°C and 5% CO2 humidity. Subsequently, the original culture medium was replaced with 100 μL of fresh culture medium containing different concentrations of FFP nanomaterials (0, 20, 40, 80, 160 μg / mL) and incubated for 24 hours. Finally, the cell viability was determined by MTT method.

[0050] The results are as follows Figure 3 As shown in the figure, 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 that FFP has good biocompatibility 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 concentrations 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 mixture was incubated at 37°C for 3 hours, centrifuged and photographed. The absorbance of the sample at 542 nm was then detected using an ELISA reader to calculate the hemolysis rate.

[0052] The results are as follows Figure 4 As shown, 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. During the injection process, care should be taken to avoid the generation of bubbles. The concentration of FFP was 8 mg / mL. A GE LOGIQ E9 ultrasound diagnostic instrument was used, the contrast mode was selected, and the L9 probe (MI 0.6) was used to record the imaging using the ultrasound diagnostic instrument's video recording function.

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

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

[0056] (7) Press Fe 2+ The experimental group MC38 mice were injected with FFP (100 μL) nanomaterials via the tail vein at a concentration of 8 mg / kg to inject Fe 2+ The two groups of FP (100 μL) with a concentration of 8 mg / kg and 0.9% NaCl (100 μL) were used as control groups. After injection, the tumor area of ​​the tumor-bearing mice was immediately scanned using a GE LOGIQ E9 ultrasound diagnostic instrument (9L linear array probe, contrast mode selected, MI0.6), and dynamic images were saved. Figure 7 As shown, 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 solution of the present invention rather than to limit it. 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 solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe, characterized in that: The steps include: S1, using ammonium ferrous sulfate, trisodium citrate, etc. as raw materials to prepare iron sulfide nanosheets FeS, and resuspending FeS in ethanol to obtain a 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. Mix the FeS / PFH suspension with 1 mg / mL dopamine hydrochloride anhydrous ethanol solution, stir at room temperature in the dark, and wash 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 S1, the specific steps of preparing FeS include: S11, dissolving ammonium ferrous sulfate and trisodium citrate in ethylene glycol, adding polyethyleneimine after complete dissolution, stirring at room temperature to obtain solution A; S12, adding 0.05M thioacetamide ethylene glycol solution to solution A to obtain solution B; adding triethanolamine dropwise to solution B, stirring at room temperature to obtain solution C; S13, subjecting solution C to a solvothermal reaction, centrifuging after the reaction is completed, washing with ethanol, collecting the precipitate, and resuspending it in ethanol to obtain an ethanol solution of FeS.

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

4. The method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to claim 2, characterized in that: In step S12, the volume ratio of the ethylene glycol solution of thioacetamide to solution A is 15:

20.

5. The method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to claim 2, characterized in that: In step S12, the volume ratio of solution B to triethanolamine is 35:

2.

6. The method for preparing an ultrasound / magnetic resonance dual-mode nanoprobe according to claim 2, characterized in that: In step S13, the temperature of the solvent thermal reaction is 200° C. and the reaction time is 24 h.

7. 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.

8. 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.

9. 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 8, characterized in that: The particle size of FeS / PFH / PDA is 140-160 nm.

10. Use of the ultrasound / magnetic resonance dual-mode nanoprobe FeS / PFH / PDA as claimed in claim 9 in ultrasound imaging, magnetic resonance imaging or ultrasound / magnetic resonance dual-mode imaging for non-medical purposes.

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