Preparation method and application of magnetic nano-capture system based on molybdenum selenide

Abstract

The present invention discloses a magnetic nano-capture system based on molybdenum selenide and its preparation method and application. The preparation method comprises: (1) ultrasonically stripping block-shaped molybdenum selenide into flaky molybdenum selenide nanosheets; (2) through hydrothermal reaction, at a certain temperature, reacting ferric chloride, molybdenum selenide nanosheets and L-ascorbic acid under high pressure in a polytetrafluoroethylene liner to generate molybdenum selenide nanosheets loaded with ultra-small ferroferric oxide; (3) adding dopamine hydrochloride to a suspension of molybdenum selenide nanosheets loaded with ferroferric oxide, and after washing, adding anti-EpCAM antibodies to obtain a magnetic nano-capture system of molybdenum selenide. The present invention utilizes EpCAM, which is a receptor protein highly expressed by most tumor cells, and ferroferric oxide has good magnetic attraction ability, as well as the excellent photothermal performance of molybdenum selenide, so that the magnetic nano-system can not only target tumor cells, but also capture cells for detection and analysis under the action of a magnetic field, and can also achieve in situ killing of tumor cells in vivo.

Description

Technical Field

[0001] The present invention belongs to the field of cancer treatment, and in particular relates to tumor recurrence, and is used for the prognosis and treatment of cancer. Background Art

[0002] Cancer is becoming a major global threat to human health, and metastasis from primary tumors accounts for nearly 90% of cancer mortality. Despite the availability of a variety of clinical cancer treatments, such as chemotherapy, radiotherapy, and surgical resection, many malignancies remain remarkably susceptible to recurrence and metastasis. During cancer progression, some tumor cells can detach from the extracellular matrix of the primary tumor and enter the circulatory system (such as the bloodstream or lymphatic system). These cells, known as circulating tumor cells (CTCs), may then colonize and proliferate at secondary sites, potentially establishing metastatic cancer. Therefore, early detection and clearance of these CTCs from the patient's bloodstream is crucial. Furthermore, CTCs share similar genomic information and protein expression profiles with cells at the primary tumor site, making them crucial for diagnosis and monitoring, particularly given the heterogeneity and significant inter-individual variability of tumors. Therefore, CTCs offer promising biomarkers in liquid biopsies for early diagnosis of primary tumor progression and real-time monitoring of treatment efficacy.

[0003] However, regardless of the tumor type and the malignant stage of the tumor, the CTCs content in the bloodstream of cancer patients is usually extremely rare compared with the large number of blood cells. 2 Therefore, before removing the dispersed CTCs from the circulation system or obtaining downstream diagnostic information from them, we urgently need an efficient strategy to enrich CTCs in the bloodstream. Current methods for enriching CTCs are mainly based on their physical properties (such as size-based capture), immune affinity-mediated technology involving tumor-specific antibodies, or a combination of the two. However, the capture efficiency of the capture systems reported so far is relatively low, or they can only capture CTCs under static conditions, but cannot achieve this goal under the condition of blood flow in the human body. Moreover, they cannot kill CTCs remaining in the body in situ and need to be supplemented with other therapeutic drugs, which increases the burden on patients and greatly limits their application in clinical medicine. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the primary purpose of the present invention is to provide a method for preparing a magnetic nano-capture system based on MoSe.

[0005] Another object of the present invention is to provide a magnetic nano-capture system based on molybdenum selenide.

[0006] Another object of the present invention is to provide an application of molybdenum selenide in preparing a magnetic nano-capture system.

[0007] Another object of the present invention is to provide an application of a molybdenum selenide magnetic nano-capture system in the preparation of cancer prognosis or treatment drugs or adjuvants.

[0008] Molybdenum selenide (MoSe2) nanomaterials, including 2D nanosheets and 3D nanoflowers, are widely recognized for their applications in radiofrequency / photothermal tumor therapy. Due to their high photothermal efficiency, MoSe2 nanosheets are expected to be used for tumor ablation under near-infrared lasers. By leveraging the advantages of nanocarriers to load ferroferric oxide and modifying EpCAM antibodies to construct a magnetic nanocapture system, after capturing CTCs in vivo for monitoring and analysis, tumor cells can be ablated in situ using photothermal methods. Therefore, such a magnetic nanosystem has promising medical applications and is a worthy new area of research.

[0009] The present invention provides a method for preparing a molybdenum selenide magnetic nano-capture system, comprising the following steps:

[0010] (1) Preparation of a stock solution: Molybdenum selenide was dissolved in N-methyl-2-pyrrolidone (NMP) to prepare a molybdenum selenide stock solution with a concentration of 8-12 mg / mL; dopamine hydrochloride was dissolved in Tris buffer to prepare a dopamine stock solution with a pH of 8.3-8.8 and a concentration of 9-11 mM;

[0011] (2) preparing a mixed solution: treating the molybdenum selenide stock solution with a probe ultrasonic treatment at a power of 240-280 W for 7-9 hours, and washing with ultrapure water by centrifugation to form the mixed solution;

[0012] (3) adding L-ascorbic acid, NaHCO3 and FeCl3·6H2O to the mixed solution, then transferring the mixture to a polytetrafluoroethylene-lined autoclave, hydrothermally reacting at 145-165°C for 5-7 hours, and then centrifuging and washing to obtain a magnetic nanosystem loaded with ferrosoferric oxide;

[0013] (4) Adding dopamine stock solution to the magnetic nanosystem, washing with water by centrifugation, and then adding 14-20 μg / mL streptavidin and 4-20 μg / mL EpCAM antibody in sequence to obtain the final molybdenum selenide magnetic nanocapture system. The molybdenum selenide magnetic nanocapture system is evenly dispersed in water and stored at 0-4°C for future use.

[0014] Furthermore, step (3) comprises: adding FeCl3·6H2O, NaHCO3 and L-ascorbic acid to a mixed solution with a concentration of 0.2-0.3 mmol / L, mixing uniformly, transferring the solution to a stainless steel autoclave lined with polytetrafluoroethylene, performing a hydrothermal reaction at 140-160°C in a muffle furnace for 5-7 hours, transferring the solution to a centrifuge tube for centrifugal washing, and adsorbing the product with a magnet close to the centrifuge tube. The collected black product is the magnetic nanosystem loaded with ferrosoferric oxide.

[0015] Furthermore, the centrifugal washing in step (2) is performed at a centrifugal speed of 12000-15000 rpm and a centrifugal time of 10 min-20 min.

[0016] Furthermore, the centrifugal washing in step (4) is performed by placing a magnet close to the centrifuge tube for 5-10 minutes to adsorb the product.

[0017] The above-mentioned solution of the present application brings the following beneficial effects: the magnetic nano-capture system of molybdenum selenide of the present application can effectively target and enrich tumor cells, and can also achieve in situ photothermal killing of tumor cells. In addition, the preparation method provided by the present application is simple, rapid, safe, reliable, and easy to mass-produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a transmission electron microscope image of a single molybdenum selenide nanosheet in an embodiment of the present application;

[0019] Figure 2 This is a transmission electron micrograph of a molybdenum selenide nanosheet (MoSe2-Fe3O4) loaded with ultra-small ferrosoferric oxide in an embodiment of the present application;

[0020] Figure 3 This is a transmission electron micrograph of the molybdenum selenide magnetic nanocapture system (MoSe2-Fe3O4-pDA-EpCAM, NMs) in the embodiment of the present application;

[0021] Figure 4 This is a fluorescence image of the molybdenum selenide magnetic nanocapture system (NMs) targeting HepG2 cells in the example of this application;

[0022] Figure 5 This is a fluorescence image of HepG2 cells captured by a molybdenum selenide magnetic nanocapture system (NMs) and then adsorbed by a magnet in an embodiment of the present application;

[0023] Figure 6 The capture efficiency of HepG2 cells by the molybdenum selenide magnetic nanocapture system (NMs) in the embodiment of the present application in the state without liquid flow and in the state of liquid flow;

[0024] Figure 7This is a statistical graph of the cell viability test after HepG2 cells were enriched using the molybdenum selenide magnetic nanocapture system (NMs) in the embodiment of this application;

[0025] Figure 8 This is a fluorescence image of the MoSe magnetic nanocapture system (NMs) capturing HepG2 cells and killing them in situ in the examples of this application. DETAILED DESCRIPTION

[0026] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0027] Example 1, the preparation method of the molybdenum selenide magnetic nanosystem provided by the present invention comprises the following steps:

[0028] (1) Prepare a stock solution by dissolving 1 g of molybdenum selenide in 100 mL of N-methyl-2-pyrrolidone (NMP) to prepare a molybdenum selenide stock solution; dissolve dopamine hydrochloride in Tris buffer to prepare a dopamine stock solution with a pH of 8.5 and a concentration of 10 mM;

[0029] (2) preparing a mixed solution by ultrasonically treating 100 mL of molybdenum selenide stock solution at 250 W power for 8 h, and washing the solution with ultrapure water by centrifugation at a speed of 12,000 rpm for 20 min to form a mixed solution;

[0030] (3) 0.022 g of ascorbic acid, 0.1654 g of NaHCO3, and 0.1773 g of FeCl3·6H2O were added to a mixed solution with a concentration of 0.2 mmol / L, and then transferred to a polytetrafluoroethylene-lined autoclave. After hydrothermal reaction at 150 °C for 6 h, the solution was centrifuged and washed to obtain a magnetic nanosystem loaded with ferrosoferric oxide.

[0031] (4) Dopamine stock solution and 20 μg / mL coumarin 6 were added to the magnetic nanosystem. After centrifugation and washing with water, 15 μg / mL streptavidin and 5 μg / mL EpCAM antibody were added in sequence to obtain a molybdenum selenide magnetic nanocapture system labeled with coumarin 6. The magnetic nanocapture system was evenly dispersed in water and stored at 4°C for future use.

[0032] (5) Detection: The molybdenum selenide magnetic nanocapture system MoSe2-Fe3O4-pDA-EpCAM (NMs) was digested according to the national standard method (GB5009.93-2017), and the iron content was determined by atomic fluorescence spectrometry, and the EpCAM content was determined by BCA protein concentration determination kit (Biyuntian Biotechnology, P0010S).

[0033] (6) MoSe magnetic nanocapture system (NMs) can target and capture tumor cells: The transmission electron microscopy image of the prepared MoSe magnetic nanocapture system is shown in Figure 2. Figure 3 As shown, Figure 1 This is a transmission electron microscope image of a single MoSe nanosheet. Figure 2 This is a transmission electron microscope image of molybdenum selenide nanosheets (MoSe2-Fe3O4) loaded with ultra-small ferrosoferric oxide. Figure 1-3 It can be seen that the MoSe magnetic nano-capture system is based on Figure 1 Figure 2 The nanostructure shown has been successfully constructed, and the prepared molybdenum selenide magnetic nanocapture system (NMs) is a sheet-like nanostructure with a particle size between 100-300nm. Tumor cells labeled with Dil were incubated with molybdenum selenide magnetic nanocapture system (NMs-Ce6) labeled with coumarin 6 for 30 minutes. Figure 4 It can be seen that the molybdenum selenide magnetic nanocapture system (NMs-Ce6) labeled with coumarin 6 (green) is wrapped around the tumor cells labeled with Dil (red). The results prove that the molybdenum selenide magnetic nanocapture system (NMs) can efficiently capture tumor cells.

[0034] Example 2, the preparation method of the molybdenum selenide magnetic nanosystem provided by the present invention comprises the following steps:

[0035] (1) Prepare a stock solution by dissolving 1.2 g of molybdenum selenide in 100 mL of N-methyl-2-pyrrolidone (NMP) to prepare a molybdenum selenide stock solution; dissolve dopamine hydrochloride in Tris buffer to prepare a dopamine stock solution with a pH of 8.5 and a concentration of 10 mM;

[0036] (2) preparing a mixed solution by ultrasonically treating 100 mL of molybdenum selenide stock solution at 250 W power for 9 h, and washing the solution by centrifugation with ultrapure water at a centrifugal speed of 14,000 rpm for 15 min to form a mixed solution;

[0037] (3) 0.022 g of ascorbic acid, 0.1654 g of NaHCO3, and 0.1773 g of FeCl3·6H2O were added to a mixed solution with a concentration of 0.25 mmol / L, mixed evenly, and then transferred to a stainless steel autoclave lined with polytetrafluoroethylene. After hydrothermal reaction at 150°C in a muffle furnace for 7 h, the mixture was transferred to a centrifuge tube for centrifugal washing, and a magnet was placed against the centrifuge tube to adsorb the product. The collected black product was the magnetic nanosystem loaded with ferroferric oxide.

[0038] (4) Add dopamine stock solution to the magnetic nanosystem, wash with water by centrifugation, and adsorb the product by pressing the centrifuge tube against a magnet for 5-10 minutes. Then, add 18 μg / mL streptavidin and 12 μg / mL EpCAM antibody in sequence to obtain a molybdenum selenide magnetic nanocapture system. The molybdenum selenide magnetic nanocapture system is evenly dispersed in water and stored at 4°C for future use.

[0039] Example 3, the preparation method of the molybdenum selenide magnetic nanosystem provided by the present invention comprises the following steps:

[0040] (1) Prepare a stock solution by dissolving 0.8 g of molybdenum selenide in 100 mL of N-methyl-2-pyrrolidone (NMP) to prepare a molybdenum selenide stock solution; dissolve dopamine hydrochloride in Tris buffer to prepare a dopamine stock solution with a pH of 8.8 and a concentration of 12 mM;

[0041] (2) preparing a mixed solution by ultrasonically treating 100 mL of molybdenum selenide stock solution at 280 W power for 8 h with a probe, and washing the solution with ultrapure water by centrifugation at a speed of 15,000 rpm for 10 min to form a mixed solution;

[0042] (3) 0.022 g of ascorbic acid, 0.1654 g of NaHCO3, and 0.1773 g of FeCl3·6H2O were added to a mixed solution with a concentration of 0.3 mmol / L, mixed evenly, and then transferred to a stainless steel autoclave lined with polytetrafluoroethylene. After hydrothermal reaction at 150°C in a muffle furnace for 7 h, the solution was centrifuged and washed, and the product was adsorbed by a magnet against the centrifuge tube. The collected black product was the magnetic nanosystem loaded with ferroferric oxide.

[0043] (4) Add dopamine stock solution to the magnetic nanosystem, wash with water by centrifugation, and adsorb the product by pressing the centrifuge tube against a magnet for 5-10 minutes. Then, add 20 μg / mL streptavidin and 20 μg / mL EpCAM antibody in sequence to obtain a molybdenum selenide magnetic nanocapture system. The molybdenum selenide magnetic nanocapture system is evenly dispersed in water and stored at 4°C for future use.

[0044] The preparation method of the magnetic nano-capture system of molybdenum selenide in the present application adopts a hydrothermal synthesis method combined with modern nano-synthesis technology, takes advantage of molybdenum selenide as a carrier to load ultra-small ferrosoferric oxide, and wraps a polydopamine (pDA) layer on the surface of the nanosheet to link EpCAM antibodies, so that the capture system can well target and enrich tumor cells, and at the same time can achieve in situ photothermal killing of tumor cells, thereby achieving prognosis of cancer treatment.

[0045] The preparation method provided in this application is simple, rapid, safe, reliable, and easy to produce on a large scale.

[0046] In addition, the magnetic nano-capture system of the present invention is a safe and stable nano-material, which makes the magnetic nano-capture system of molybdenum selenide more potential for clinical transformation and application.

[0047] Compared to other nanomaterial carriers, 2D MoSe nanosheets possess superior biosafety and exceptional photothermal efficiency. Modified with EpCAM antibodies, they can effectively target tumor cells. Under the influence of a magnetic field, they attract and mobilize Fe3O4, thereby enriching tumor cells and achieving photothermal ablation of tumors. This holds great significance and application value for further research and treatment of tumors.

[0048] By using a nano-delivery system that uses in situ growth of ultra-small, long iron oxide on molybdenum selenide nanosheets, the capture system can avoid destroying the morphology and genetic material of tumor cells after targeting them, and can target a large number of tumor cells over time. When extracting tumor cells for analysis and monitoring, the tumor cells can be rapidly migrated to the end of the magnetic field under the action of an additional magnetic field, thereby achieving the enrichment of circulating tumor cells. To prevent circulating tumor cells from metastasizing to the distal end of the primary tumor with the flow of blood, the molybdenum selenide magnetic nano-capture system can ablate the tumor through photothermal therapy after targeting circulating tumor cells, thereby avoiding the metastasis of tumor cells. The above implementation cases show that the magnetic nano-capture system based on molybdenum selenide has good clinical translation prospects and application value.

[0049] The magnetic nanocapture system with molybdenum selenide nanosheets as carriers can be applied to tumor monitoring, especially for tumors with high expression of EpCAM.

[0050] The photothermal effect of molybdenum selenide as a drug carrier, the targeting effect of EpCAM antibodies and the magnetic attraction of ferroferric oxide can also be used to enable the circulating tumor cell capture system to target tumor cells and at the same time photothermally ablate tumor cells in situ, thereby achieving a better prognosis in cancer treatment.

[0051] Example 4: This example examines the enrichment process of a molybdenum selenide magnetic nanocapture system (NMs) after capturing tumor cells. The method is as follows:

[0052] After incubating the suspended tumor cells with a magnetic nanocapture system of molybdenum selenide labeled with coumarin 6 (NMs-Ce6) for 30 minutes, the cells were placed in a capillary tube and the initial position of the cells was obtained using a fluorescence microscope. A magnet was then placed on one side of the capillary tube and the migration changes of the tumor cells were recorded using a fluorescence microscope. Figure 5 It can be seen that cells targeted by the MoSe magnetic nanocapture system (NMs) can be efficiently enriched under the action of the magnetic field.

[0053] Example 5: In this embodiment, the enrichment efficiency of a molybdenum selenide magnetic nanocapture system (NMs) after capturing tumor cells was investigated. The method is as follows:

[0054] After the suspended tumor cells were counted using a flow cytometer, the suspended cells were incubated with a molybdenum selenide magnetic nanocapture system (NMs) for 30 minutes. After proper mixing, they were transferred to a 2 mL ep tube, and a magnet was placed on one side of the ep tube. The adsorbed tumor cells and non-adsorbed tumor cells were separated, and the number of captured cells was counted using a flow cytometer. At the same time, the suspended cells that had been counted and mixed with the molybdenum selenide magnetic nanocapture system (NMs) were injected into the catheter at a uniform speed through a syringe to simulate the environment of human blood flow, and a magnet was placed on one side of the catheter to collect the discharged tumor cells. The capture efficiency of the molybdenum selenide magnetic nanocapture system (NMs) for capturing tumor cells under this flow state was calculated by deducting the tumor cells discharged from the catheter. Figure 6 It can be seen that the capture efficiency of the MoSe magnetic nanocapture system (NMs) will greatly increase with the change of incubation time. Moreover, the capture efficiency of the MoSe magnetic nanocapture system (NMs) in the above two states can exceed 70%, and even under static conditions, the capture efficiency can reach a maximum of 90%, indicating that the MoSe magnetic nanocapture system (NMs) has excellent tumor cell capture and enrichment capabilities.

[0055] Example 6: This embodiment examines the viability of enriched tumor cells using a molybdenum selenide magnetic nanocapture system (NMs). The method is as follows:

[0056] After the suspended tumor cells were counted by flow cytometry, they were incubated with a MoSe magnetic nanocapture system (NMs) for 30 minutes. After proper mixing, they were transferred to a 2 mL eppendorf tube and a magnet was placed on one side of the eppendorf tube for 30 minutes. The adsorbed tumor cells were separated from the non-adsorbed tumor cells and the enriched tumor cells were transferred to a culture flask. The cell viability was monitored on days 1, 3, and 5. Figure 7It can be seen that after five days of culture, there was no significant difference in the absorbance values of untreated cells and cells captured by the capture system, indicating that the molybdenum selenide magnetic nanocapture system (NMs) has less toxicity to tumor cells, does not affect tumor proliferation, and greatly maintains the integrity of the cells, making them useful for analysis and monitoring.

[0057] Example 7: This example investigates the photothermal killing of in situ tumor cells using a molybdenum selenide magnetic nanocapture system (NMs). The method is as follows:

[0058] After incubating the suspended cells, calcein / propidium iodide (AM / PI) and molybdenum selenide magnetic nanocapture system (NMs) for 30 minutes, the cells were placed into a capillary tube and fluorescence images of the cells without 808nm laser irradiation were taken using a fluorescence microscope. Then, fluorescence changes of the cells under 808nm laser irradiation were captured using a fluorescence microscope. Figure 8 It can be seen that before and after 808nm laser irradiation, the cell fluorescence changes from green to red (generally, live cells are labeled green by AM, and dead cells are labeled red by PI). This shows that the MoSe magnetic nanocapture system (NMs) has excellent photothermal killing ability and can achieve in situ photothermal ablation of tumors.

[0059] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing a molybdenum selenide magnetic nano-capture system, characterized in that: The following steps are involved: (1) Preparation of a stock solution: Molybdenum selenide was dissolved in N-methyl-2-pyrrolidone to prepare a molybdenum selenide stock solution with a concentration of 8-12 mg / mL; dopamine hydrochloride was dissolved in Tris buffer to prepare a dopamine stock solution with a pH of 8.3-8.8 and a concentration of 9-11 mM; (2) preparing a mixed solution: treating the molybdenum selenide stock solution with a probe ultrasonic treatment for 7-9 hours, and washing the solution with ultrapure water by centrifugation to form the mixed solution; (3) adding L-ascorbic acid, NaHCO3 and FeCl3·6H2O to the mixed solution, then transferring the mixture to a polytetrafluoroethylene-lined autoclave, hydrothermally reacting at 145-165°C for 5-7 hours, and then centrifuging and washing to obtain a magnetic nanosystem loaded with ferrosoferric oxide; (4) Adding dopamine stock solution to the magnetic nanosystem, washing with water by centrifugation, and then adding 14-20 μg / mL streptavidin and 4-20 μg / mL EpCAM antibody in sequence to obtain the final molybdenum selenide magnetic nanocapture system. The molybdenum selenide magnetic nanocapture system is evenly dispersed in water and stored at 0-4°C for future use.

2. The preparation method according to claim 1, characterized in that Step (3) comprises: adding FeCl3·6H2O, NaHCO3 and L-ascorbic acid to a mixed solution with a concentration of 0.2-0.3 mmol / L, mixing uniformly, transferring the solution to a stainless steel autoclave lined with polytetrafluoroethylene, performing a hydrothermal reaction at 140-160°C in a muffle furnace for 5-7 hours, transferring the solution to a centrifuge tube for centrifugal washing, and adsorbing the product with a magnet close to the centrifuge tube. The collected black product is a magnetic nanosystem loaded with ferroferric oxide.

3. The preparation method according to claim 1, characterized in that The centrifugal washing in step (2) is performed at a centrifugal speed of 12,000-15,000 rpm and a centrifugal time of 10-20 min.

4. The preparation method according to claim 1, characterized in that The centrifugal washing described in step (4) is to centrifuge and wash and adsorb the product by keeping the centrifuge tube close to the magnet for 5-10 minutes.

5. A molybdenum selenide magnetic nano-capture system prepared by the preparation method according to any one of claims 1 to 4.

6. The molybdenum selenide magnetic nano-capture system according to claim 5 is a magnetic nano-capture system using molybdenum selenide as a carrier and carrying ultra-small ferrosoferric oxide.

7. Use of the molybdenum selenide magnetic nanocapture system prepared by the preparation method according to any one of claims 1 to 4 in the preparation of cancer therapeutic drugs.

8. The application according to claim 7, characterized in that: Specifically, it is used in the preparation of drugs targeting circulating tumor cells.

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