Application of perovskite nanocrystalline probe in preparation of tumor imaging reagent

Abstract

The invention provides an application of a perovskite nanocrystalline probe in preparation of a tumor imaging reagent. The perovskite nanocrystalline probe is formed by electrostatic interaction or covalent coupling of water-soluble perovskite nanocrystalline and a cell nucleus targeting reagent. The water-soluble perovskite nanocrystal is formed by in-situ coating and packaging of perovskite quantum dots and a polymer packaging material. The polymer packaging material is selected from polyvinylpyrrolidone; and the perovskite quantum dots are selected from CsPbBr3. The method can be used for rapid imaging of tumor cells and pathology, can meet the requirement of visual detection, does not need large-scale fluorescence detection equipment, and is suitable for POCT detection scene application.

Description

Technical Field

[0001] The present invention relates to the technical field of luminescent materials, and in particular to an application of a perovskite nanocrystal probe in the preparation of a tumor imaging agent. Background Art

[0002] The nucleus is the largest and most important cell structure in eukaryotic cells. Nuclear fluorescent probes can directly stain cells or tissues, thereby locating the nucleus, determining the distribution of cells in tissues, and tracking changes in the nucleus throughout the process from mitosis to apoptosis. Particles with a size below 100nm can penetrate the cell membrane and enter the cell, while particles larger than 100nm cannot enter the cell membrane. Therefore, the size of fluorescent probes targeting the nucleus must be less than 100 nanometers.

[0003] Perovskite quantum dot materials have excellent fluorescence properties and are a new generation of display materials. However, they are not resistant to water and oxygen, which limits their application in the biological field. How to use perovskite quantum dot materials to achieve rapid positioning of the cell nucleus for rapid imaging is an urgent problem to be solved. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an application of a perovskite nanocrystal probe in tumor cell and pathology imaging, which solves the problem in the prior art that the cell nucleus cannot be quickly located for imaging.

[0005] In a first aspect, the present invention provides a perovskite nanocrystal probe, which is formed by electrostatic interaction or covalent coupling of water-soluble perovskite nanocrystals and a cell nucleus targeting agent.

[0006] According to an embodiment of the present invention, the water-soluble perovskite nanocrystals include perovskite quantum dots and polymer encapsulation materials;

[0007] Preferably, the water-soluble perovskite nanocrystals are formed by in-situ coating and encapsulation of perovskite quantum dots and polymer encapsulation materials.

[0008] Preferably, the polymer encapsulation material is selected from polyvinyl pyrrolidone (PVP).

[0009] Preferably, the perovskite quantum dots are selected from CsPbBr 3 .

[0010] According to an embodiment of the present invention, the particle size of the water-soluble perovskite nanocrystal is 20 to 100 nm, for example, 20 nm, 30 nm, 40 nm, 50 nm or 80 nm. Preferably, the particle size of the water-soluble perovskite nanocrystal is 30 nm.

[0011] According to an embodiment of the present invention, the mass ratio (mg:mg) of the perovskite quantum dots to polyvinyl pyrrolidone is 116:(10-100); for example, it can be 116:10, 116:30, 116:60, 116:80, or 116:100.

[0012] According to an embodiment of the present invention, the molecular weight of the polyvinyl pyrrolidone is not less than 10,000, preferably 10,000 to 60,000, for example, 10,000, 24,000, 29,000, 40,000, 55,000 or 58,000.

[0013] According to an embodiment of the present invention, the coating is complete coating. In one embodiment, when the mass ratio (mg:mg) of the perovskite quantum dots to polyvinyl pyrrolidone is 116:10, complete coating can be achieved.

[0014] Among them, CsPbBr 3 The perovskite quantum dot solution appears yellow under visible light and green under ultraviolet light (e.g., 365nm excitation).

[0015] According to an embodiment of the present invention, the polymer encapsulation material of the water-soluble perovskite nanocrystal forms a stable chemical bond by bonding with Pb ions in the perovskite quantum dots through C=O, and is inserted into the perovskite structure to form a polymer network-encapsulated structure.

[0016] According to an embodiment of the present invention, the water-soluble perovskite nanocrystals are prepared by the following method: polyvinyl pyrrolidone and perovskite quantum dots are heated to react to obtain the water-soluble perovskite nanocrystals.

[0017] According to an embodiment of the present invention, the water-soluble perovskite nanocrystal is prepared by the following method:

[0018] (1) mixing cesium bromide, lead bromide, polyvinyl pyrrolidone and an organic solvent to form a stable solution, adding oleylamine and oleic acid, and reacting;

[0019] Preferably, the reaction temperature is 50-70°C, for example 60°C;

[0020] Preferably, the organic solvent includes any one of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), γ-butyrolactone (GBL), and N,N-dimethylacetamide (DMA);

[0021] (2) adding the solution of step (1) into an anti-solvent to react and precipitate water-soluble perovskite nanocrystals;

[0022] Preferably, the anti-solvent is selected from at least one of toluene, chlorobenzene and n-hexane.

[0023] Preferably, the reaction temperature is 35-50°C, such as 45°C.

[0024] According to an embodiment of the present invention, the water-soluble perovskite nanocrystal is prepared by the following method:

[0025] (1) 0.2 mmol of cesium bromide (CsBr) and 0.2 mmol of lead bromide (PbBr) were added. 2 ) and polyvinyl pyrrolidone were added with 5 mL of N,N-dimethylformamide (DMF) to form a stable solution, and 0.25 mL of oleylamine and 0.5 mL of oleic acid were added as a ligand stabilizing solution. The entire reaction was carried out at 60 °C.

[0026] (2) adding 0.75 mL of the stabilizing solution in step (A1) into 15 mL of toluene as an anti-solvent, heating the mixture at 45° C. to react, and precipitating water-soluble perovskite nanocrystals by an anti-solvent supersaturation method to obtain water-soluble perovskite nanocrystals.

[0027] According to an embodiment of the present invention, the more the amount of polyvinyl pyrrolidone is added, the larger the nanoparticle size it forms. According to an embodiment of the present invention, the larger the molecular weight of the polyvinyl pyrrolidone used, the larger the nanoparticle size it forms. According to an embodiment of the present invention, the amount of polyvinyl pyrrolidone limited is 100mg, and the molecular weight is 58000, and the nanoparticle size formed is below 100 nanometers.

[0028] According to an embodiment of the present invention, the cell nucleus targeting agent is a biological material targeting the cell nucleus, for example, it may include antibodies, peptides, aptamers, enzymes and other biological materials. Preferably, the antibody includes Anti-BIRC 5 .

[0029] In a second aspect, the present invention provides a method for preparing a perovskite nanocrystal probe, comprising covalently coupling a perovskite nanocrystal with a cell nucleus targeting agent to obtain a perovskite nanocrystal probe;

[0030] Preferably, the cell nucleus targeting perovskite nanocrystal probe is the probe mentioned above.

[0031] According to an embodiment of the present invention, the covalent coupling includes activating the perovskite nanocrystal and then performing a coupling reaction with a cell nucleus targeting agent.

[0032] Preferably, the activators used are EDC and NHS.

[0033] According to one embodiment of the present invention, the method for preparing the cell nucleus-targeting perovskite nanocrystal probe comprises the following steps:

[0034] (A1) 0.2 mmol of cesium bromide (CsBr), 0.2 mmol of lead bromide (PbBr) 2 ) and polyvinyl pyrrolidone were added with 5 mL of N,N-dimethylformamide (DMF) to form a stable solution, and 0.25 mL of oleylamine and 0.5 mL of oleic acid were added as a ligand stabilizing solution. The entire reaction was carried out at 60 °C.

[0035] (A2) adding 0.75 mL of the stabilizing solution described in step (A1) to 15 mL of toluene as an anti-solvent, heating the mixture at 45° C. to precipitate water-soluble perovskite nanocrystals using an anti-solvent supersaturation method, thereby preparing water-soluble perovskite nanocrystals;

[0036] (A3) dispersing the solid water-soluble perovskite nanocrystals obtained in step (A2) in water to obtain a water-soluble perovskite nanocrystal solution;

[0037] (A4) activating the water-soluble perovskite nanocrystal solution obtained in step (A3) and performing a coupling reaction with a cell nucleus targeting agent.

[0038] Among them, the size of the water-soluble perovskite nanocrystals formed after coating can be regulated by adding the amount and molecular weight of polyvinyl pyrrolidone.

[0039] In a third aspect, the present invention provides a kit comprising the above-mentioned perovskite nanocrystal probe.

[0040] In a fourth aspect, the present invention provides use of the above-mentioned perovskite nanocrystal probe and the above-mentioned kit in the preparation of tumor cell imaging reagents.

[0041] The cell nucleus targeting agent is Anti-BIRC5. The target is BIRC5 (Survivin gene). The cancer gene atlas TCGA database contains the expression of relevant BIRC5 in all tumors and their corresponding normal tissue cells, showing that BIRC5 is highly expressed in tumor cells and lowly expressed or not expressed in normal cells.

[0042] The operation process of perovskite nanocrystal probe staining cells is as follows:

[0043] Tumor cells and normal cells were mixed and fixed with 4% paraformaldehyde for 10 minutes, blocked with goat serum for 30 minutes at room temperature, and stained with an appropriate amount of perovskite nanocrystal probe targeting the cell nucleus. After washing with ultrapure water, they were irradiated with a handheld ultraviolet lamp. The green fluorescent area was the tumor cell, and the non-fluorescent area was the normal cell.

[0044] A fifth aspect of the present invention provides the use of the above-mentioned perovskite nanocrystal probe in the preparation of tumor pathology imaging agents.

[0045] The operation process of the perovskite nanocrystal probe staining pathology is as follows:

[0046] Add an appropriate amount of perovskite nanocrystal probe to the processed paraffin section or the sample area frozen during surgery for staining, wash with ultrapure water, and then use a handheld ultraviolet lamp to illuminate. The green fluorescent area is the tumor area; the non-fluorescent area is the normal area.

[0047] The present invention has the following beneficial effects:

[0048] The perovskite nanocrystal probe provided by the present invention can quickly target the cell nucleus and can be used for rapid imaging of tumor cells and pathology. The perovskite quantum dot probe can be illuminated by a portable handheld ultraviolet lamp, which can meet the needs of visual detection without the need for large-scale fluorescence detection equipment, and is suitable for POCT detection scene applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the expression of BIRC5 in all tumors in the Cancer Gene Atlas TCGA database.

[0050] Figure 2 TEM images of water-soluble perovskite nanocrystals (30 nm and 200 nm) in Example 1;

[0051] Figure 3 Confocal images of fluorescence imaging of perovskite nanocrystal probes (30nm and 200nm) targeting the cell nucleus on gastric cancer MGC803. The Confocal laser excitation wavelengths are 405nm and 488nm.

[0052] Figure 4 Confocal images of fluorescence imaging of perovskite nanocrystal probes targeting cell nuclei on colorectal cancer and its corresponding normal tissue sections were taken. The Confocal laser excitation wavelengths were 405nm and 488nm. DETAILED DESCRIPTION

[0053] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0054] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0055] Example 1 Preparation of water-soluble perovskite nanocrystals

[0056] (1) 42.5 mg CsBr and 73.4 mg PbBr2 Dissolve 10 mg PVP with a molecular weight of 10,000 and 100 mg PVP with a molecular weight of 60,000 in 5 mL of N,N-dimethylformamide (DMF) solvent respectively. After complete dissolution, add 0.5 mL of oleic acid and 0.25 mL of oleylamine to form a stable precursor solution.

[0057] (2) Take 750L of the above solution and slowly add it dropwise into 15mL of vigorously stirred toluene solution to obtain a perovskite quantum dot solution; stir and react for 4h to complete the coating to obtain water-soluble perovskite nanocrystals (PVP@PNCs). PVP@PNCs is a structure in which PVP forms a stable chemical bond with Pb ions through C=O, and is inserted into the perovskite structure to form a polymer network encapsulation structure.

[0058] Transmission electron microscopy images of water-soluble perovskite nanocrystals Figure 2 As shown in the figure, it can be seen that the particle size of the water-soluble perovskite nanocrystals with a molecular weight of 10,000 and a molecular weight of 10 mg PVP is about 30 nm; the particle size of the water-soluble perovskite nanocrystals with a molecular weight of 60,000 and a molecular weight of 200 nm.

[0059] Example 2 Preparation of perovskite nanocrystal probes targeting cell nuclei

[0060] 10 mg of perovskite nanocrystal powder was dissolved in ultrapure water, reacted with Anti-BIRC5 (100 uL, 1 mg / mL) through EDC (40 mg / mL) and NHS (40 mg / mL) activators at room temperature for 20 min, and centrifuged through an ultrafiltration centrifuge tube to obtain perovskite nanocrystals targeting the cell nucleus (PVP@PNCs@Anti-BIRC5).

[0061] The Cancer Gene Atlas TCGA database contains the expression of BIRC5 in all tumors and their corresponding normal tissue cells. Figure 1 As shown, it is obvious that BIRC5 is highly expressed in almost all tumor cells, but not expressed or low expressed in normal cells.

[0062] Example 3: Nucleus-targeted perovskite nanocrystal probe for rapid imaging of tumor cells

[0063] (1) Gastric cancer cell MGC803 cells were cultured in Corning cell culture dishes and placed in a carbon dioxide incubator at 37°C and 5% CO 2 The culture medium was Gibco's DMEM mixed with 10% FBS and 1% double antibody.

[0064] (2) Fix the cells with 4% paraformaldehyde mixed with Triton X-100 for 10 min and rinse with PBS buffer.

[0065] (3) Block the cells with 1 mL of goat serum blocking solution for 20 min and rinse with PBS buffer.

[0066] (4) 1 mL of 30 nm and 200 nm targeted nucleus perovskite nanocrystal probes were added to the cell culture dish for staining for 10 min, and then rinsed with PBS buffer.

[0067] (5) Use DAPI to stain the cell nucleus for 10 minutes, protect from light, and rinse with PBS buffer. Figure 3 shown.

[0068] The results show that the 30nm perovskite nanocrystal probe targeting the cell nucleus can co-localize with the DAPI-stained cell nucleus staining position, while the 200nm perovskite nanocrystal probe targeting the cell nucleus appears scattered and star-shaped, making it difficult to enter the cell-targeted cell nucleus.

[0069] Example 4: Targeting the nucleus with perovskite nanocrystal probes for rapid imaging of tumor pathology sections

[0070] Take 200-300 μL of the detection solution and drop it onto the slice to be detected. Let it react at room temperature for 15 minutes. After the reaction, rinse it with a rinse solution (such as ultrapure water) for 5-6 times and observe the image with a handheld ultraviolet lamp.

[0071] Evaluation criteria for distinguishing tumors from normal tissues: Use an ordinary handheld ultraviolet lamp to irradiate the slice area to be tested. According to the principle of antibody-antigen specific recognition, when the relevant antigens in the tumor area in the tissue slice to be tested are detected, the test solution is added to the tissue area, reacts for 15 minutes, and then rinses the test solution 5-6 times with a rinse solution.

[0072] When the tumor area is excited by the handheld ultraviolet lamp, fluorescence appears, and the test result is the tumor area; conversely, if the tissue section to be tested is normal tissue, that is, it does not contain relevant antigens of the tumor area, there is no fluorescence in the tumor area under the handheld ultraviolet lamp, and the test result is the normal tissue area, that is, the non-tumor area.

[0073] The higher the fluorescence intensity, the higher the degree of tumor canceration in the tissue section to be tested. Conversely, the lower the fluorescence intensity, the lower the degree of canceration. Figure 4 shown.

[0074] From the results, we can see that Figure 4Confocal images of fluorescence imaging of perovskite nanocrystal probes targeting the cell nucleus on colorectal cancer and corresponding normal intestinal tissue sections. The Confocal laser excitation wavelengths are 405nm and 488nm. Normal intestinal tissue sections show no green fluorescence, while colorectal cancer tissue sections show strong green fluorescence. This is consistent with the theory that BIRC5 is highly expressed in tumors and lowly or not expressed in normal tissues.

[0075] 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 the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A perovskite nanocrystal probe, characterized in that: The perovskite nanocrystal probe is formed by electrostatic interaction or covalent coupling of water-soluble perovskite nanocrystals and a cell nucleus targeting agent.

2. A perovskite nanocrystal probe as claimed in claim 1, characterized in that: The water-soluble perovskite nanocrystals include perovskite quantum dots and polymer encapsulation materials; Preferably, the water-soluble perovskite nanocrystals are formed by in-situ encapsulation of perovskite quantum dots and polymer encapsulation materials; Preferably, the polymer encapsulation material is selected from polyvinyl pyrrolidone; Preferably, the perovskite quantum dots are selected from CsPbBr3; Preferably, the mass ratio of the perovskite quantum dots to polyvinyl pyrrolidone is 116:(10-100); Preferably, the molecular weight of the polyvinyl pyrrolidone is not less than 10,000, more preferably 10,000-60,000.

3. A perovskite nanocrystal probe as claimed in claim 1, characterized in that: The particle size of the water-soluble perovskite nanocrystal is 20 to 100 nm.

4. A perovskite nanocrystal probe as claimed in claim 1, characterized in that: The preparation method of the water-soluble perovskite nanocrystal comprises heating polyvinyl pyrrolidone and perovskite quantum dots to obtain.

5. A perovskite nanocrystal probe as claimed in claim 4, characterized in that: The following steps are involved: (1) mixing cesium bromide, lead bromide, polyvinyl pyrrolidone and an organic solvent to form a stable solution, adding oleylamine and oleic acid, and reacting; Preferably, the reaction temperature is 50-70°C; Preferably, the organic solvent includes any one of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), γ-butyrolactone (GBL), and N,N-dimethylacetamide (DMA); (2) adding the solution of step (1) into an anti-solvent to react and precipitate water-soluble perovskite nanocrystals; Preferably, the anti-solvent is selected from at least one of toluene, chlorobenzene and n-hexane. Preferably, the reaction temperature is 35-50°C.

6. A perovskite nanocrystal probe as claimed in claim 1, characterized in that: The cell nucleus targeting agent includes a biological material that targets the cell nucleus; Preferably, the biomaterial comprises an antibody, a polypeptide, an aptamer, an enzyme; more preferably, the biomaterial is Anti-BIRC5.

7. A method for preparing a perovskite nanocrystal probe according to any one of claims 1 to 6, characterized in that: The method comprises covalently coupling the perovskite nanocrystal with a cell nucleus targeting agent to obtain a perovskite nanocrystal probe; Preferably, the covalent coupling comprises the perovskite nanocrystals being activated and then undergoing a coupling reaction with a cell nucleus targeting agent; Preferably, the activators used are EDC and NHS.

8. A kit, characterized in that: A perovskite nanocrystal probe comprising any one of claims 1-6.

9. Use of a perovskite nanocrystal probe according to any one of claims 1 to 6 and a kit according to claim 8 in the preparation of a tumor cell imaging agent.

10. Use of a perovskite nanocrystal probe according to any one of claims 1 to 6 and a kit according to claim 8 in the preparation of a tumor pathology imaging agent.

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