Nano material, preparation method thereof and application of nano material in preparation of medicine for inhibiting nasopharyngeal carcinoma metastasis

Abstract

The invention provides a nano-material and a preparation method thereof, the nano-material is an organic-inorganic hybrid nano-material (TA-BSA-coated CuS) with copper sulfide as a core and albumin and polyphenol as a shell, which is formed by triggering oxidative coupling of BSA and polyphenol (TA) with albumin (BSA) as a template. The nasopharyngeal carcinoma cell membrane overexpressing Gas6 is wrapped to form the Gas6 cell membrane / TA-BSA-CuS nano material, and the material can effectively inhibit lung metastasis of tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a nanomaterial and a preparation method thereof, and application of the nanomaterial in preparing a drug for inhibiting metastasis of nasopharyngeal carcinoma. Background Art

[0002] As a common malignant tumor of the head and neck, the metastatic characteristics of nasopharyngeal carcinoma seriously affect the patient's quality of life and prognosis. At present, clinical treatment methods face many challenges in inhibiting the metastasis of nasopharyngeal carcinoma, such as limited treatment effects and large side effects. Therefore, it is urgent to explore new treatment strategies and materials. The rise of nanomaterials in the field of tumor treatment has brought new hope for solving this problem, but the application of existing nanomaterials in the treatment of nasopharyngeal carcinoma still has problems such as insufficient targeting, unsatisfactory efficacy, and biocompatibility needs to be improved.

[0003] Therefore, it is necessary to develop a new nanomaterial with high efficiency in inhibiting NPC metastasis. Summary of the invention

[0004] The purpose of the present invention is to provide a nanomaterial and a preparation method thereof and an application thereof in the preparation of a drug for inhibiting the metastasis of nasopharyngeal carcinoma. A new organic-inorganic hybrid nanomaterial (TA-BSA@CuS) is prepared based on copper sulfide, and the effectiveness of the nanomaterial in inhibiting tumor metastasis is detected after preparation.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a method for preparing a nanomaterial is provided, the method comprising:

[0007] BSA was dissolved in a buffer solution, and then TA was added to initiate an oxidative coupling reaction between BSA and TA under stirring. Then, a copper source solution was added dropwise and stirred to carry out a biomineralization reaction, and unreacted substances were removed to obtain TA-BSA@CuS nanomaterials.

[0008] Select appropriate nasopharyngeal carcinoma cell lines, use genetic engineering technology to make them overexpress Gas6, culture them in a medium containing serum and growth factors until the logarithmic growth phase, collect cells and extract to obtain cell membranes;

[0009] The TA-BSA@CuS nanomaterial is mixed with the cell membrane, incubated in a buffer solution to allow the cell membrane to wrap the nanomaterial, and then centrifuged and washed to remove unwrapped substances to obtain Gas6-cell membrane / TA-BSA@CuS nanomaterial.

[0010] Furthermore, the mass ratio of the BSA to the TA is in the range of 3:1-15:1.

[0011] Furthermore, the conditions of the oxidative coupling reaction include: a temperature range of 310-323K, a pH range of 11-13.5, and the conditions of the biomineralization reaction include: a temperature range of 30-50°C, and a pH range of 11-13.5.

[0012] Furthermore, the copper source solution is selected from one of copper nitrate, copper chloride and copper acetate.

[0013] Furthermore, the method of selecting a suitable nasopharyngeal carcinoma cell line and using genetic engineering technology to overexpress Gas6 comprises the following specific steps:

[0014] 293T cells were transfected with Lipo8000 transfection reagent, oe-Gas6 plasmid and packaging plasmid (pMD2G and psPAX2). After 48 hours, the supernatant containing lentivirus was collected and transfected into 5-8F cells. Puromycin (5 μg / mL) was added to select 5-8F cells that stably overexpressed Gas6.

[0015] Furthermore, the formula of the culture medium containing serum and growth factors is DMEM high-glucose culture medium sterilized by filtration through a 0.1 μm filter membrane, pH 7.0-7.4, containing 25 mM D-glucose, supplemented with 4.0 mM L-alanyl-L-glutamine and 1.0 mM sodium pyruvate, and supplemented with 10% fetal bovine serum.

[0016] Furthermore, the mass ratio of the TA-BSA@CuS nanomaterial to the cell membrane is in the range of 1.5-1.75, and the buffer solution is pure water.

[0017] In the second aspect of the present invention, a nanomaterial prepared by the method is provided.

[0018] In the third aspect of the present invention, provided is the use of the nanomaterial in preparing a drug for inhibiting the metastasis of nasopharyngeal carcinoma.

[0019] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0020] This invention discloses for the first time a new type of nanomaterial for inhibiting the metastasis of nasopharyngeal carcinoma. The nanomaterial uses albumin (BSA) as a template to trigger the oxidative coupling of BSA and polyphenol (TA), forming an organic-inorganic hybrid nanomaterial (TA-BSA@CuS) with copper sulfide as the core and albumin and polyphenol as the shell. It also wraps the nasopharyngeal carcinoma cell membrane that overexpresses Gas6 to form a Gas6-cell membrane / TA-BSA@CuS nanomaterial. It provides a new means for the targeted treatment of tumors. This material is scientifically reliable and feasible, does not cause additional harm to the human body, is easy to operate, and is worthy of promotion. Improvements and changes to certain steps of this patent still reflect the technical principles of the patent of this invention, and similar technical methods all fall within the technical protection scope of this patent. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0022] Figure 1 Transmission electron microscopy of cell membrane-encapsulated material.

[0023] Figure 2 In vivo imaging of the material inhibiting nasopharyngeal carcinoma metastasis in nude mice. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0025] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0026] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0027] The general idea of ​​the present invention is as follows:

[0028] According to a typical embodiment of the present invention, a method for preparing a nanomaterial is provided, the method comprising:

[0029] Step S1, dissolving BSA in deionized water, then adding TA, inducing an oxidative coupling reaction between BSA and TA under stirring, then dripping a copper source solution and stirring to carry out a biomineralization reaction, removing unreacted substances, and obtaining TA-BSA@CuS nanomaterials;

[0030] In the step S1,

[0031] The mass ratio of the BSA to the TA is in the range of 3:1-15:1. If the mass ratio is too small, it is difficult to form a stable BSA and TA complex, while if it is too large, TA cannot be incorporated into the material.

[0032] The conditions of the oxidative coupling reaction include: a temperature range of 310-323K and a pH range of 11-13.5.

[0033] The copper source solution is selected from one of copper nitrate, copper chloride and copper acetate.

[0034] The conditions of the biomineralization reaction include: a temperature range of 30-50° C. and a pH range of 11-13.5.

[0035] Step S2, selecting a suitable nasopharyngeal carcinoma cell line, using genetic engineering technology to make it overexpress Gas6, culturing it in a medium containing serum and growth factors until the logarithmic growth phase, collecting the cells and extracting to obtain cell membranes;

[0036] In the step S2,

[0037] The method of selecting a suitable nasopharyngeal carcinoma cell line and using genetic engineering technology to overexpress Gas6 comprises the following specific steps:

[0038] 293T cells were transfected with Lipo8000 transfection reagent, oe-Gas6 plasmid and packaging plasmid (pMD2G and psPAX2). After 48 hours, the supernatant containing lentivirus was collected and transfected into 5-8F cells. Puromycin (5 μg / mL) was added to select 5-8F cells that stably overexpressed Gas6.

[0039] The formula of the culture medium containing serum and growth factors is DMEM high-glucose culture medium sterilized by filtration through a 0.1 μm filter membrane, pH 7.0-7.4, containing 25 mM D-glucose, supplemented with 4.0 mM L-alanyl-L-glutamine and 1.0 mM sodium pyruvate, and 10% fetal bovine serum.

[0040] Step S3, mixing the TA-BSA@CuS nanomaterial with the cell membrane, incubating in a buffer solution to allow the cell membrane to wrap the nanomaterial, centrifuging and washing to remove unwrapped substances, and obtaining Gas6-cell membrane / TA-BSA@CuS nanomaterial.

[0041] In the step S3,

[0042] The mass ratio of the TA-BSA@CuS nanomaterial to the cell membrane is in the range of 1.5-1.75:1. If the mass ratio is too small, the cell membrane may adhere and aggregate, resulting in excessive precipitation in the material solution, affecting the stability of the system. If the mass ratio is too large, the amount of nanomaterial will be relatively excessive, and the cell membrane cannot wrap it all, which will not only cause waste of materials, but may also cause other adverse reactions, thereby adversely affecting the experimental results or application effects.

[0043] The buffer solution is purified water.

[0044] The present application will be described in detail below with reference to embodiments and experimental data.

[0045] Example 1. Gas6-cell membrane / TA-BSA@CuS nanomaterial and preparation method thereof

[0046] The embodiment of the present invention provides a method for preparing Gas6-cell membrane / TA-BSA@CuS nanomaterial, comprising the following steps:

[0047] 1. Preparation and characterization of the new nanomaterial TA-BSA@CuS

[0048] Dissolve BSA in deionized water and adjust to 50-100 mg / ml (preferably 80 mg / ml in this embodiment), add 5-20 mg TA (preferably 15 mg in this embodiment), stir and react at 37°C and pH≈13 to initiate the oxidative coupling reaction of BSA and TA. Slowly drop 10 mg / ml copper nitrate solution, continue stirring, and perform biomineralization reaction to form TA-BSA@CuS nanomaterials. Use dialysis and other techniques to remove unreacted substances to obtain pure TA-BSA@CuS nanomaterials.

[0049] 2. Preparation and verification of cell membrane-wrapped nanomaterials Gas6-cell membrane / TA-BSA@CuS

[0050] Culture of nasopharyngeal carcinoma cells overexpressing Gas6: 293T was transfected with a mixture of Lipo8000 transfection reagent (purchased from Beyotime, catalog number C0533-1.5ml), oe-Gas6 plasmid (sequence: 5′-GCCCTGTTAGACATCATTCAAGAGATGATGTCTAACAGGGC-3′, SEQ ID NO.1, purchased from GeneCare) and packaging plasmids (pMD2G and psPAX2, purchased from Fenghui Bio, catalog numbers BR037 and BR036). After 48 hours, the supernatant containing lentivirus was collected and transfected into 5-8F cells. Puromycin (5 μg / mL, purchased from Beyotime, catalog number ST551-10 mg) was added to select 5-8F cells that stably overexpressed Gas6.

[0051] Cell membrane extraction: 5-8F cells overexpressing Gas6 were incubated in Tris buffer (pH 7.4, containing 1x EDTA-free protease inhibitor) at 4°C for 1 h, then collected with a cell scraper and centrifuged at 500g for 10 min. After centrifugation, the supernatant was ultrasonicated for 10 min, and then centrifuged at 10000g for 10 min to collect the supernatant. Next, the precipitate was collected by high-speed centrifugation at 100000g for 1 hour, dissolved in pure water at a concentration of 1 mg / mL, and finally ultrasonicated for 30s to obtain a uniform cell membrane.

[0052] Cell membrane-encapsulated nanomaterials: TA-BSA@CuS nanomaterials were mixed with extracted cell membranes at a mass ratio of 1.5:1 and incubated in a suitable buffer for a specific time to allow the cell membrane to encapsulate the nanomaterials. Unencapsulated materials were removed by centrifugation, washing, and other operations to obtain Gas6-cell membrane / TA-BSA@CuS nanomaterials.

[0053] Transmission electron microscopy (TEM) was used to analyze the cell membrane wrapping of nanomaterials. Figure 1 As shown, it is shown that the cell membrane encloses the material.

[0054] Example 2: Application experiment of inhibiting nasopharyngeal carcinoma metastasis

[0055] 1. Methods

[0056] 5-8F cells were transfected with luciferase lentivirus solution (purchased from Bio-Tech, catalog number C4001-1 ml) and virus infection reagent (purchased from GeneCare, catalog number REVG005). Puromycin (5 μg / mL) was added to select 5-8F cells that stably expressed luciferase.

[0057] After 1 week of adaptive feeding, BALB / c nude mice (≈16 g, female, 5 weeks old, 25 mice) were injected intrathoracically with 5-8F cells transfected with luciferase gene (5×106 / mouse) to prepare a nasopharyngeal carcinoma lung metastasis model. After 7 days, luciferin was injected intraperitoneally, and then the nude mice were imaged whole-body to detect the tumor formation in the chest cavity. After confirming the tumor formation, they were randomly divided into 5 groups (5 mice in each group): (a) PBS, (b) BSA@Cus, (c) TA-BSA@Cus, (d) cell membrane / TA-BSA@Cus, (e) Gas6-cell membrane

[0058] / TA-BSA@Cus.

[0059] On the first day, the nude mice in group ae were injected with different drugs through the tail vein. 24 hours after the drug administration, the nude mice in the five groups were anesthetized with gas at 0.8W / cm 2 The tumor was irradiated with 808nm laser with a power density of 100 nm for 10 minutes, and then repeated once after an interval of 1 day. After 14 days of treatment, fluorescein was injected intraperitoneally, and then the whole body imaging of the nude mice was performed and the fluorescence intensity was statistically analyzed. After the treatment, the nude mice were euthanized, and the tumors and major organs (heart, liver, spleen, lung, and kidney) of the nude mice were immediately collected, and the number of nodules on the lung surface was recorded to evaluate tumor metastasis.

[0060] 2. Results

[0061] The results are as follows Figure 2 As shown, it indicates that:

[0062] After completing the 14-day group treatment regimen, all nude mice were imaged and statistically analyzed based on the fluorescence intensity. The results showed that the tumor fluorescence intensity in the PBS group was the highest, while the intensity in the Gas6-cell membrane / TA-BSA@CuS group was the lowest. In addition, the lung tissues of nude mice were taken out after sacrifice for photography and HE analysis. Multiple obvious metastatic foci were observed in the lungs of group a, fewer lung metastatic foci in groups b, c, and d, and no obvious metastatic foci were observed in the lungs of group e. This shows that Gas6-cell membrane / TA-BSA@CuS combined with phototherapy has a significant inhibitory effect on nasopharyngeal carcinoma lung metastasis.

[0063] Compared with the control, the Gas6-cell membrane / TA-BSA@CuS of the embodiment of the present invention can more effectively inhibit the lung metastasis of the tumor.

[0064] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0065] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0066] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a nanomaterial, characterized in that: The method comprises: TA was added to BSA, and an oxidative coupling reaction between BSA and TA was initiated under stirring. Then, a copper source solution was added dropwise and stirred to carry out a biomineralization reaction, and unreacted substances were removed to obtain TA-BSA@CuS nanomaterials. Select appropriate nasopharyngeal carcinoma cell lines, use genetic engineering technology to make them overexpress Gas6, culture them in a medium containing serum and growth factors until the logarithmic growth phase, collect cells and extract to obtain cell membranes; The TA-BSA@CuS nanomaterial is mixed with the cell membrane, incubated in a buffer solution to allow the cell membrane to wrap the nanomaterial, and then centrifuged and washed to remove unwrapped substances to obtain Gas6-cell membrane / TA-BSA@CuS nanomaterial.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the BSA to the TA is in the range of 3:1-15:

1.

3. The preparation method according to claim 1, characterized in that: The conditions of the oxidative coupling reaction include: a temperature range of 310-323K and a pH range of 11-13.

5.

4. The preparation method according to claim 1, characterized in that: The conditions of the biomineralization reaction include: a temperature range of 30-50° C. and a pH range of 11-13.

5.

5. The preparation method according to claim 1, characterized in that: The copper source solution is selected from one of copper nitrate, copper chloride and copper acetate.

6. The preparation method according to claim 1, characterized in that: The method of selecting a suitable nasopharyngeal carcinoma cell line and using genetic engineering technology to overexpress Gas6 comprises the following specific steps: After transfecting 293T cells with a mixture of liposome transfection reagent, oe-Gas6 plasmid and packaging plasmid, the supernatant containing lentivirus was collected and transfected into cells, and puromycin was added to select cells that stably overexpressed Gas6.

7. The preparation method according to claim 1, characterized in that: The formula of the culture medium containing serum and growth factors is DMEM high-glucose culture medium filtered and sterilized, pH 7.0-7.4, containing 25±1mM D-glucose, supplemented with 4.0±1mM L-alanyl-L-glutamine and 1.0±1mM sodium pyruvate, and supplemented with 10±2% fetal bovine serum.

8. The preparation method according to claim 1, characterized in that: The mass ratio of the TA-BSA@CuS nanomaterial to the cell membrane is in the range of 1.5-1.75, and the buffer solution is pure water.

9. A Gas6-cell membrane / TA-BSA@CuS nanomaterial prepared by the method according to any one of claims 1 to 8.

10. Use of the nanomaterial according to claim 9 in the preparation of a drug for inhibiting metastasis of nasopharyngeal carcinoma.

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