Application of EPN3 modified perovskite nanocrystalline probe in diagnosis of lymph node metastasis of breast cancer

Abstract

The invention provides an application of a dendrimer modified perovskite nanocrystal probe in prediction of mammary gland lymph node metastasis. The probe is formed by coupling a biological material of a targeted EPN3 gene and a water-soluble perovskite nanocrystal through electrostatic interaction, the water-soluble perovskite nanocrystal is formed by packaging perovskite quantum dots and a dendritic macromolecular packaging material through the coordination bonding effect of chemical bonds. The EPN3 modified water-soluble perovskite nanocrystalline probe provided by the invention can be used for high-sensitivity rapid detection of tumor tissues or cells with breast cancer lymph node metastasis.

Description

Technical Field

[0001] The present invention relates to the field of rapid diagnosis of cancer pathology, and in particular to the application of an EPN3-modified perovskite nanocrystal probe in the diagnosis of breast cancer lymph node metastasis. Background Art

[0002] The Epsin family is a family of proteins involved in clathrin-dependent endocytosis and is primarily involved in regulating multiple signaling pathways. The Epsin family includes Epsin1 (EPN1), Epsin2 (EPN2), and Epsin3 (EPN3). These members play important roles in maintaining normal cardiovascular development and nervous system homeostasis. The Epsin family also exhibits important biological functions in a variety of diseases.

[0003] The extent of lymph node metastasis in breast cancer determines the scope of surgery, especially in early breast cancer surgery. Rapid pathological diagnosis can help doctors determine in real time whether axillary lymph node dissection is necessary. Through rapid pathological diagnosis, surgeons can determine whether to expand the scope of surgery or take other measures based on whether there is cancer cell metastasis in the lymph nodes during surgery. Therefore, the development of a highly sensitive targeted probe for breast cancer lymph node metastasis is of great significance to clinical medicine. Highly specific and high-contrast fluorescent probes can greatly assist pathologists in determining the degree of canceration of tumors in fluorescent immunohistochemistry pathology. Existing fluorescent dyes have low fluorescence luminescence efficiency, weak resistance to photobleaching, and insufficient specificity. It is particularly important to prepare a fluorescent probe with high specificity and high luminescence efficiency. Summary of the Invention

[0004] In response to the deficiencies in the prior art, the present invention provides an EPN3-modified perovskite nanocrystal probe for use in the diagnosis of breast cancer lymph node metastasis.

[0005] The present invention provides a perovskite nanocrystal probe, which is formed by electrostatic interaction or covalent coupling between a biomaterial targeting an EPN3 gene and a perovskite nanocrystal modified with a dendrimer.

[0006] According to an embodiment of the present invention, the biomaterial targeting the EPN3 gene includes biomaterials such as antibodies, polypeptides, aptamers, enzymes, etc. targeting the EPN3 gene; preferably, the antibody targeting the EPN3 gene is Anti-EPN3.

[0007] According to an embodiment of the present invention, the mass ratio (mg:mg) of the dendrimer-modified perovskite nanocrystals to the biomaterial targeting the EPN3 gene is 10:0.01-0.5, preferably 10:0.02-0.4, for example, 10:0.03, 10:0.05, 10:0.08, 10:0.1, 10:0.13, 10:0.15, 10:0.18, 10:0.2, 10:0.25, and 10:0.3.

[0008] According to an embodiment of the present invention, the dendrimer-modified perovskite nanocrystals include perovskite quantum dots and dendrimer materials.

[0009] According to an embodiment of the present invention, the dendrimer-modified perovskite nanocrystals are formed by chemically coordinating the bonding of perovskite quantum dots and dendrimer materials.

[0010] According to an embodiment of the present invention, the dendrimer material is selected from any one, two or more of the following: polyamidoamine (PAMAM), polylysine (PLL), polyethyleneimine (PEI), and polypropyleneimine (PPI).

[0011] According to an embodiment of the present invention, the terminal functional groups of the dendrimer-modified perovskite nanocrystals may be carboxyl, amino, thiol, and the like.

[0012] According to an embodiment of the present invention, the perovskite quantum dots may be selected from CsPbBr3.

[0013] According to an embodiment of the present invention, the dendrimer-modified perovskite nanocrystals are water-soluble.

[0014] According to an embodiment of the present invention, the spectral range of the dendrimer-modified perovskite nanocrystal is 520nm±20nm, for example, 500nm, 505nm, 510nm, 515nm, 520nm, 525nm, 530nm, 535nm, or 540nm; preferably, the spectral position of the dendrimer-modified perovskite nanocrystal is 520nm.

[0015] According to an embodiment of the present invention, the particle size of the dendrimer-modified perovskite nanocrystal is 20 nm ± 3 nm, for example, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm or 23 nm; preferably, the particle size of the dendrimer-modified perovskite nanocrystal is 20 nm.

[0016] According to an embodiment of the present invention, the average particle size of the perovskite quantum dots is 5 nm to 15 nm, for example, 8 nm to 12 nm; illustratively, the average particle size of the perovskite quantum dots is 8 nm, 9 nm, 10 nm, 11 nm or 12 nm.

[0017] According to an embodiment of the present invention, the average particle size of the dendrimer-modified perovskite nanocrystals is greater than the average particle size of the perovskite quantum dots.

[0018] According to an embodiment of the present invention, the mass ratio (mg:mg) of the perovskite quantum dots to the dendrimers is 116:(20-90), 116:(30-100); for example, it can be 116:20, 116:25, 116:30, 116:40, 116:50, 116:60, 116:70, 116:80, 116:100.

[0019] According to an embodiment of the present invention, dendrimers are coated on the surface of perovskite quantum dots; preferably, the coating is complete. In one embodiment, complete coating can be achieved when the mass ratio (mg:mg) of perovskite quantum dots to dendrimers is at least 116:30.

[0020] Among them, the dendrimer-modified perovskite nanocrystals appear yellow-green under visible light and green under ultraviolet light (e.g., 365nm excitation).

[0021] According to an embodiment of the present invention, the electrical property of the dendrimer-modified perovskite nanocrystal is positive charge.

[0022] The present invention provides a method for preparing the above-mentioned perovskite nanocrystal probe, which comprises: subjecting dendrimer-modified perovskite nanocrystals to electrostatic interaction or covalent coupling with a biomaterial targeting the EPN3 gene, thereby obtaining the perovskite nanocrystal probe.

[0023] According to an embodiment of the present invention, the preparation method is specifically as follows: dissolving the dendrimer-modified perovskite nanocrystals in water, adding a biomaterial targeting the EPN3 gene, mixing, and adding bovine serum albumin for blocking treatment to obtain the perovskite nanocrystal probe.

[0024] According to an embodiment of the present invention, the mass ratio (mg:mg) of the dendrimer-modified perovskite nanocrystals to the biomaterial targeting the EPN3 gene is 10:0.01-0.5, preferably 10:0.02-0.4, for example, 10:0.03, 10:0.05, 10:0.08, 10:0.1, 10:0.13, 10:0.15, 10:0.18, 10:0.2, 10:0.25, and 10:0.3.

[0025] According to an embodiment of the present invention, the method for preparing the dendrimer-modified perovskite nanocrystals comprises the following steps: heating the dendrimers and perovskite quantum dots to react, thereby obtaining the dendrimer-modified perovskite nanocrystals.

[0026] According to an embodiment of the present invention, the dendrimer-modified perovskite nanocrystals are prepared by the following method:

[0027] (1) mixing cesium bromide, lead bromide, dendrimers and an organic solvent to form a stable solution, adding oleylamine and oleic acid to react;

[0028] Preferably, in step (1), the reaction temperature is 50°C-70°C, for example 60°C;

[0029] Preferably, in step (1), the organic solvent is selected from any one of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), γ-butyrolactone (GBL), and N,N-dimethylacetamide (DMA);

[0030] (2) adding the solution after the reaction in step (1) to an anti-solvent to react and precipitate dendrimer-modified perovskite nanocrystals;

[0031] Preferably, in step (2), the anti-solvent is selected from at least one of toluene, chlorobenzene, and n-hexane;

[0032] Preferably, in step (2), the reaction temperature is 35°C-50°C, for example 45°C.

[0033] According to an embodiment of the present invention, the method for preparing the dendrimer-modified perovskite nanocrystals specifically comprises the following steps:

[0034] (A1) 42.5 mg of cesium bromide (CsBr), 73.4 mg of lead bromide (PbBr2), and 30 mg of PAMAM were added to 5 mL of N,N-dimethylformamide (DMF) to form a stable solution. 0.25 mL of oleylamine and 0.5 mL of oleic acid were added as a ligand stabilizing solution, and the reaction was carried out at 60°C.

[0035] (A2) adding 0.75 mL of the reaction solution from step (A1) to 15 mL of toluene, heating the mixture at 45° C. to precipitate the dendrimer-modified perovskite nanocrystals (using an antisolvent supersaturation method) to prepare water-soluble dendrimer-modified perovskite nanocrystals;

[0036] (A3) centrifuging the water-soluble dendrimer-modified perovskite nanocrystals described in step (A2), and drying the precipitate to obtain solid water-soluble dendrimer-modified perovskite nanocrystals;

[0037] (A4) dispersing the solid water-soluble dendrimer-modified perovskite nanocrystals obtained in step (A3) in water to obtain a water-soluble dendrimer-modified perovskite nanocrystal solution.

[0038] The present invention also provides a perovskite nanocrystal probe prepared by the above preparation method.

[0039] The present invention also provides a kit, which includes the above-mentioned perovskite nanocrystal probe.

[0040] In one embodiment of the present invention, the kit further comprises a handheld ultraviolet lamp, a flushing solution, a pipette, a standard colorimetric card and a storage medium.

[0041] Preferably, the storage medium is an RFID tag, an IC chip, a magnetic code or a bar code.

[0042] Preferably, the flushing liquid is water, or a buffer solution.

[0043] The present invention provides use of the perovskite nanocrystal probe or the kit in detecting the EPN3 gene or in preparing a product for detecting the EPN3 gene.

[0044] The present invention provides use of the above-mentioned perovskite nanocrystal probe or the above-mentioned kit in preparing a product for detecting and / or diagnosing breast cancer lymph node metastasis.

[0045] According to an embodiment of the present invention, the product is a detection reagent.

[0046] Beneficial effects

[0047] Based on the significant expression differences of the EPN3 gene in normal breast tissue, breast carcinoma in situ and breast cancer lymph node metastasis, perovskite quantum dots are combined with the EPN3 gene target to form a pathological diagnostic probe for predicting breast cancer lymph node metastasis, which can be used for high-sensitivity and rapid detection of cancer tissues or cells that have metastasized to breast cancer or breast cancer lymph nodes.

[0048] The probe provided by the present invention has the advantages of good labeling stability (for example, electrostatic binding of biomolecules to perovskite nanocrystals), rapid and high sensitivity (for example, the fluorescence intensity of perovskite nanocrystals is high, and high-contrast staining effect is achieved after forming a targeted probe with EPN3), simple and fast operation, short detection time, easy result interpretation, and both qualitative and quantitative analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a transmission electron microscopy image of the perovskite nanocrystal modified with dendrimer (PAMAM) in Example 1;

[0050] Figure 2The PL spectrum (orange curve) and absorption spectrum (green curve) of the dendrimer (PAMAM) modified perovskite nanocrystals in Example 1;

[0051] Figure 3 This is the Confocal image of fluorescence imaging of the EPN3 modified perovskite nanocrystal probe kit in Example 3 on normal breast tissue, breast carcinoma in situ and breast lymph node metastasis tissue sections. The Confocal laser excitation wavelength is 405nm and 488nm. DETAILED DESCRIPTION

[0052] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative 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 encompassed within the scope of protection that the present invention is intended to protect.

[0053] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0054] CsBr: cesium bromide. PbBr2: lead bromide.

[0055] PAMAM: polyamidoamine dendrimer.

[0056] PNCs: perovskite nanocrystals.

[0057] Anti-EPN3: anti-EPN3 polyclonal antibody (purchased from Aibixin (Shanghai) Biotechnology Co., Ltd., product number: abs147854).

[0058] BSA: bovine serum albumin.

[0059] Example 1 Preparation of water-soluble PAMAM-modified perovskite nanocrystals

[0060] 1. Dissolve 42.5 mg of CsBr, 73.4 mg of PbBr2, and 30 mg of PAMAM in 5 mL of N,N-dimethylformamide (DMF) solvent. After complete dissolution, add 0.5 mL of oleic acid and 0.25 mL of oleylamine to form a stable precursor solution.

[0061] 2. Take 750 μL of the above precursor solution and slowly add it dropwise to 15 mL of vigorously stirred toluene solution to obtain a perovskite quantum dot solution; stir the reaction for 4 hours to ensure complete coating to obtain a PAMAM-modified perovskite nanocrystal solution.

[0062] 3. Separation and purification of perovskite nanocrystals: The PAMAM-modified perovskite nanocrystal solution in step (2) was centrifuged at 8000 r for 10 min, and the obtained precipitate was dried in a fume hood for 1 h to obtain a light yellow powder, which turned green under ultraviolet light.

[0063] 4. The powder was dispersed in water by ultrasonication for 1 min and the water-soluble PAMAM-modified perovskite nanocrystals (i.e., PAMAM@PNCs) were stored at room temperature.

[0064] Transmission electron microscopy images of perovskite nanocrystals modified with dendrimers (PAMAM) Figure 1 As shown. The PL spectrum of perovskite nanocrystals modified with dendrimers (PAMAM) and the absorption spectrum are shown as follows. Figure 2 shown.

[0065] The results show that the particle size of PAMAM-modified perovskite nanocrystals is about 20 nm, and the PL emission peak of PAMAM-modified perovskite nanocrystals is 520 nm.

[0066] Example 2 Preparation of ENP3-modified perovskite nanocrystal probes

[0067] 10 mg of the PAMAM-modified perovskite nanocrystal powder prepared in step (3) of Example 1 was dissolved in ultrapure water, mixed with Anti-EPN3 (100 μL, 1 mg / mL) and vortexed for 10 s, and 0.02% BSA solution was added to block the unbound sites to obtain ENP3-modified perovskite nanocrystal probes.

[0068] Example 3: An EPN3-modified perovskite nanocrystal kit for rapid breast cancer lymph node metastasis cancer tissue imaging

[0069] The kit includes a handheld UV lamp, detection solution, rinse solution, pipette, and standard colorimetric cards.

[0070] Detection solution: ENP3-modified perovskite nanocrystal probe prepared in Example 2. Rinse solution: ultrapure water.

[0071] Instructions for use: Take 200-300 μL of the test solution and add it to the slice to be tested. Let it react at room temperature for 5-8 minutes. After the reaction, rinse it three times with a flushing solution (such as ultrapure water). Use a handheld ultraviolet lamp to observe the image and compare it with the standard colorimetric card to determine whether breast cancer has lymph node metastasis.

[0072] Evaluation criteria for distinguishing between tumor and normal tissue: Use a 365nm ordinary handheld UV lamp to illuminate the section to be tested. Based on the principle of antibody-antigen specific recognition, when the relevant antigen of the tumor area in the tissue section to be tested is detected, the detection solution is added to the tissue area, reacted for 5-8 minutes, and then rinsed three times with the rinse solution. Under the excitation of the handheld UV lamp, if the tumor area fluoresces, and the color is compared with the standard colorimetric card, the test result is strong fluorescence, which indicates breast cancer lymph node metastasis. Under the excitation of the handheld UV lamp, if the tumor area fluoresces, and the color is compared with the standard colorimetric card, the test result is moderate intensity fluorescence, which indicates breast cancer in situ. Under the excitation of the handheld UV lamp, if the tumor area fluoresces, and the color is compared with the standard colorimetric card, the test result is weak fluorescence or almost no fluorescence, which indicates normal tissue area, i.e., non-tumor area. The higher the fluorescence intensity, the higher the probability of breast cancer lymph node metastasis in the tissue section to be tested; conversely, the lower the fluorescence intensity, the lower the probability of metastasis.

[0073] Confocal images of fluorescence imaging of EPN3 modified perovskite nanocrystal probe kit on normal breast tissue, breast carcinoma in situ and breast lymph node metastasis tissue sections are shown in Figure 3 DAPI was used to stain cell nuclei, which colocalized with the perovskite probe. As can be seen in the image, tumor sections from breast lymph node metastases exhibit strong green fluorescence, exceeding the fluorescence intensity of breast carcinoma in situ, while normal breast tissue exhibited almost no fluorescence. This is consistent with the results of bioinformatics analysis of EPN3 gene expression.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. 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 solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A perovskite nanocrystal probe, wherein the perovskite nanocrystal probe is formed by electrostatic interaction or covalent coupling between a biomaterial targeting the EPN3 gene and a dendrimer-modified perovskite nanocrystal.

2. The perovskite nanocrystal probe according to claim 1, characterized in that The biological materials targeting the EPN3 gene include antibodies, polypeptides, aptamers, enzymes and other biological materials targeting the EPN3 gene; preferably, the antibody targeting the EPN3 gene is Anti-EPN3; Preferably, the mass ratio of the dendrimer-modified perovskite nanocrystal to the biomaterial targeting the EPN3 gene is 10:0.01-0.5, preferably 10:0.02-0.4; Preferably, the terminal functional groups of the dendrimer-modified perovskite nanocrystals are carboxyl, amino, or thiol.

3. The perovskite nanocrystal probe according to claim 1 or 2, characterized in that: The dendrimer-modified perovskite nanocrystals include perovskite quantum dots and dendrimer materials; Preferably, the dendrimer-modified perovskite nanocrystals are formed by chemically coordinating perovskite quantum dots and dendrimer materials; Preferably, the dendrimer material is selected from any one, two or more of the following: polyamidoamine, polylysine, polyethyleneimine, polypropyleneimine; Preferably, the perovskite quantum dots are selected from CsPbBr3; Preferably, the mass ratio of the perovskite quantum dots to the dendrimers is 116:(30-100); Preferably, the spectral range of the dendrimer-modified perovskite nanocrystals is 520 nm ± 20 nm; Preferably, the particle size of the dendrimer-modified perovskite nanocrystals is 20 nm ± 3 nm; Preferably, the average particle size of the perovskite quantum dots is 5 nm to 15 nm.

4. The method for preparing the perovskite nanocrystal probe according to any one of claims 1 to 3, comprising: The dendrimer-modified perovskite nanocrystals are subjected to electrostatic interaction or covalent coupling with biomaterials targeting the EPN3 gene, thereby obtaining a perovskite nanocrystal probe; Preferably, the mass ratio of the dendrimer-modified perovskite nanocrystal to the biomaterial targeting the EPN3 gene is 10:0.01-0.5, preferably 10:0.02-0.

4.

5. The preparation method according to claim 4, characterized in that The preparation method of the dendrimer-modified perovskite nanocrystals comprises the following steps: heating the dendrimers and perovskite quantum dots to react, thereby obtaining the dendrimer-modified perovskite nanocrystals; Preferably, the dendrimer-modified perovskite nanocrystals are prepared by the following method: (1) mixing cesium bromide, lead bromide, dendrimers and an organic solvent to form a stable solution, adding oleylamine and oleic acid to react; (2) adding the solution after the reaction in step (1) into an anti-solvent to react and precipitate dendrimer-modified perovskite nanocrystals.

6. The perovskite nanocrystal probe prepared by the preparation method according to claim 4 or 5.

7. A kit comprising the perovskite nanocrystal probe according to any one of claims 1-3 and 6.

8. The kit according to claim 7, characterized in that The kit also includes a handheld ultraviolet lamp, a flushing solution, a pipette, a standard colorimetric card, and a storage medium.

9. Use of the perovskite nanocrystal probe according to any one of claims 1 to 3 and 6, or the kit according to claim 7 or 8, in detecting the EPN3 gene or in preparing a product for detecting the EPN3 gene.

10. Use of the perovskite nanocrystal probe according to any one of claims 1 to 3 and 6, or the kit according to claim 7 or 8, in the preparation of a product for detecting and / or diagnosing breast cancer lymph node metastasis.

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