A specific targeted nanoprobe for integrated diagnosis and treatment of breast cancer, its preparation method and application

Abstract

This invention belongs to the field of biotechnology and discloses a nanoprobe for the integrated diagnosis and treatment of breast cancer, its preparation method, and its application. Specifically, the nanoprobe for the integrated diagnosis and treatment of breast cancer is an aminated bimodal nanoprobe modified with CA153 antibody. This invention's nanoprobe is obtained by surface functionalization modification of the aminated bimodal nanoprobe with CA153 antibody. This design effectively improves the affinity and specificity of the nanoprobe, enabling it to bind to breast cancer cells with high selectivity, rapidness, and sensitivity, achieving fluorescence imaging of breast cancer cells. It also exhibits targeted inhibitory activity against the growth of breast cancer cells, inducing tumor cell apoptosis, thereby effectively inhibiting tumor cell proliferation and spread, achieving effective treatment of breast cancer, and realizing the integration of in vitro and in vivo treatment and diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a nanoprobe that integrates specific targeting for the diagnosis and treatment of breast cancer, its preparation method, and its application. Background Technology

[0002] Breast cancer is a condition in which breast epithelial cells proliferate uncontrollably under the influence of various carcinogenic factors. Early symptoms often include breast lumps, nipple discharge, and enlarged axillary lymph nodes. In later stages, distant metastasis of cancer cells can lead to multi-organ complications, directly threatening the patient's life. Breast cancer is one of the most common malignant tumors in women, accounting for 7-10% of all malignant tumors according to statistics.

[0003] Early diagnosis and effective treatment of breast cancer remain a challenge in current medical research and clinical practice. Traditional cancer diagnosis and treatment are two separate processes. Traditional diagnostic methods often rely on the detection of biomarkers. CA153 protein, as a biomarker for breast cancer, shows significantly higher expression levels on the surface of breast cancer cells than in normal cells, thus providing an effective target. However, existing diagnostic methods often lack sufficient sensitivity and specificity. Drug treatment methods, based on different mechanisms of action, can be divided into chemotherapy, endocrine therapy (hormone therapy), and targeted therapy. Chemotherapy drugs for breast cancer generally interfere with cancer cell proliferation by altering or inhibiting the biochemical metabolic processes of cancer cells, primarily using anthracyclines and taxanes. Endocrine therapy (hormone therapy) inhibits cancer cell growth by removing or blocking the effects of hormones. Compared to chemotherapy, endocrine therapy has advantages such as definite efficacy, low toxicity, ease of use, no need for hospitalization, and better patient tolerance. Although its onset of action is slower, the remission period is longer, making it particularly suitable for patients with hormone receptor (ER / PR) positive breast cancer at all stages. Targeted therapy is a treatment method that specifically interferes with and thus blocks tumor growth. Compared with chemotherapy, it has less impact on normal cells, and patients tolerate it better during treatment. It is suitable for HER-2 positive breast cancer patients. However, there is currently no integrated diagnostic and therapeutic technology that can precisely target breast cancer cells and achieve excellent therapeutic effects, combining the diagnosis and treatment processes into one. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a nanoprobe that integrates specific targeting for the diagnosis and treatment of breast cancer.

[0005] The nanoprobes of this invention are obtained by surface functionalization modification of amino-modified bimodal nanoprobes with CA153 antibody. This design effectively improves the affinity and specificity of the nanoprobes, enabling them to bind to breast cancer cells with high selectivity, speed and sensitivity, achieving fluorescence imaging of breast cancer cells, and effectively inhibiting the proliferation and spread of tumor cells, thus achieving effective treatment of breast cancer and realizing the integration of in vivo and in vitro treatment and diagnosis.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned nanoprobe.

[0007] This invention modifies the surface of an amino-modified bimodal nanoprobe by attaching a CA153 antibody to it via ester bonds. This stable immobilization of the CA153 antibody on the nanoprobe surface endows the nanoprobe with the ability to specifically recognize and bind to breast cancer cells expressing the CA153 protein. This specific binding is achieved through a specific interaction between the antibody and the antigen, ensuring high selectivity of the nanoprobe for the target cells. Simultaneously, the surface modification endows the nanoprobe with specific functions, exhibiting targeted inhibitory activity against breast cancer cells, inducing tumor cell apoptosis, and thus effectively inhibiting tumor cell proliferation and spread.

[0008] Another object of the present invention is to provide the use of the above-mentioned nanoprobes in the preparation of drugs for the prevention and / or treatment and / or diagnosis of breast cancer.

[0009] The nanoprobes of this invention exhibit excellent fluorescence imaging performance on breast cancer cells, responding rapidly and sensitively to and binding to them, making the tumor cells clearly visible under a fluorescence microscope. This characteristic is crucial for the early diagnosis and study of the biological characteristics of breast cancer. Furthermore, the nanoprobes of this invention also demonstrate targeted inhibition of breast cancer cell growth, inducing tumor cell apoptosis and effectively inhibiting tumor cell proliferation and spread. This characteristic makes them a powerful tool in the field of breast cancer treatment and holds promise for playing an important role in future cancer therapy. The bimodal nanoprobes of this invention show great potential in the integrated diagnosis and treatment of breast cancer, and their unique design and function make them a promising tool for biomedical research and clinical applications.

[0010] The objective of this invention is achieved through the following solution:

[0011] A specific targeted nanoprobe for the diagnosis and treatment of breast cancer (DMNPs-AbCA153) is specifically an amino-modified bimodal nanoprobe modified with CA153 antibody.

[0012] Furthermore, the CA153 antibody is modified onto the surface of the aminated bimodal nanoprobe via ester bonds.

[0013] Furthermore, the aminated bimode nanoprobe is obtained by surface hydroxylation modification of the bimode nanoprobe with 3-aminopropyltriethoxysilane (APTES).

[0014] Furthermore, the aminated bimode nanoprobe is obtained by mixing and reacting 3-aminopropyltriethoxysilane (APTES) with the bimode nanoprobe.

[0015] Furthermore, the mixing reaction is carried out in an aqueous solution.

[0016] Furthermore, the mixing reaction can be carried out using ultrasound. The mixing reaction time can be 1-30 minutes.

[0017] Furthermore, after the mixing reaction is completed, the aminated bimodal nanoprobe can be obtained by centrifugation.

[0018] Furthermore, the dual-mode nanoprobe has a four-layer core-shell structure, consisting of a noble metal nanoparticle core connected with Raman signal molecules, a first modification layer encapsulating the core, a conjugated polymer fluorescent molecule layer covering the first modification layer, and a second modification layer encapsulating the conjugated polymer fluorescent molecule layer, from the inside out.

[0019] Furthermore, the precious metal nanoparticles are gold nanoparticles or silver nanoparticles.

[0020] Furthermore, the modified layer is silicon dioxide.

[0021] Furthermore, the conjugated polymer is MEH-PPV or BDMO-PPV.

[0022] The dual-mode nanoprobe described in this invention is obtained by coating a first modification layer after mixing Raman signal molecules with noble metal nanoparticles, then compounding it with conjugated polymer fluorescent molecules, and finally coating the outermost layer with a second modification layer. The preparation method is a conventional method in the art.

[0023] Furthermore, the specific preparation method of the dual-modal nanoprobe can be found in CN107880872A.

[0024] The specific targeted breast cancer diagnosis and treatment nanoprobe of the present invention can be obtained by mixing and reacting CA153 antibody with an amino-modulated bimodal nanoprobe in an aqueous solution.

[0025] Furthermore, the reaction can be carried out under the action of a crosslinking agent; the crosslinking agent can be glutaraldehyde.

[0026] Furthermore, the reaction is preferably carried out in an ice-water bath. The reaction time can be 10-100 min.

[0027] This invention synthesizes a bimodal nanoprobe using existing methods, obtains an aminoated bimodal nanoprobe through APTES modification, and further modifies the surface of the probe with CA153 antibody to obtain a targeted nanoprobe, DMNPs-AbCA153. This nanoprobe can accurately locate breast cancer cells (such as 4T1 breast cancer cells) and simultaneously kill cells, targeting and inhibiting the growth of breast cancer cells, inducing tumor cell apoptosis, and effectively inhibiting tumor cell proliferation and spread. Experimental results show that the cell-killing mechanism of DMNPs-AbCA153 is that the nanoprobe activates the ROS and JNK pathways, stimulating cell apoptosis, with minimal damage to the organism.

[0028] This invention also provides the application of the above-mentioned specific targeted breast cancer diagnosis and treatment nanoprobe in the preparation of breast cancer prevention drugs.

[0029] This invention also provides the application of the above-mentioned specific targeted breast cancer diagnosis and treatment nanoprobe in the preparation of drugs for treating breast cancer.

[0030] The nanoprobes of this invention exhibit excellent fluorescence imaging of breast cancer cells, responding rapidly and sensitively to and binding to breast cancer cells, enabling sensitive fluorescence imaging that makes tumor cells clearly visible under a fluorescence microscope. This characteristic is crucial for the early diagnosis and study of the biological characteristics of breast cancer.

[0031] The present invention also provides the application of the above-mentioned specific targeted breast cancer diagnosis and treatment nanoprobe in the preparation of reagents for detecting, screening and / or diagnosing breast cancer.

[0032] This invention also provides the application of the above-mentioned specific targeted breast cancer diagnosis and treatment integrated nanoprobe in the preparation of breast cancer cell imaging agents.

[0033] The present invention also provides a kit for detecting breast cancer, the kit containing the above-mentioned nanoprobe.

[0034] The nanoprobes of this invention can effectively accumulate in tumor tissue, exhibiting targeted inhibitory growth ability on breast cancer cells, inducing tumor cell apoptosis, and effectively inhibiting tumor cell proliferation and spread. This characteristic makes it a powerful tool in the field of breast cancer treatment and is expected to play an important role in future cancer treatment. The bimodal nanoprobes of this invention show great potential in the integrated diagnosis and treatment of breast cancer, not only improving the early diagnosis rate of breast cancer but also providing patients with more precise treatment options. Its unique design and function make it a promising tool for biomedical research and clinical applications. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The fluorescence spectra of two probes, DNMPs and DMNPs-AbCA153, are shown.

[0037] Figure 2 Laser confocal image showing the co-localization of DNMPs and DMNPs-AbCA153 in 4T1 cells.

[0038] Figure 3 This is a graph showing the toxicity analysis of different concentrations of the DMNPs-AbCA153 probe of this invention on 4T1 cells.

[0039] Figure 4 This is a laser confocal image showing the effect of the DMNPs-AbCA153 probe of this invention on ROS enzymes in 4T1 cells.

[0040] Figure 5 This is a flow cytometry diagram showing the effect of the DMNPs-AbCA153 probe of the present invention on ROS enzymes in 4T1 cells. In the diagram, a represents the blank control, b represents the antibody CA153, c represents DMNPs, d represents DMNPs-AbCA153, and e represents DMNPs-AbCA153+NAC.

[0041] Figure 6 This is a Western blotting image of 4T1 cells after treatment with the DMNPs-AbCA153 probe of this invention.

[0042] Figure 7 The expression levels of P65, P-P65, p-JNK, JNK, Blc2, and Bax proteins are statistically represented.

[0043] Figure 8 This is a statistical graph showing the effect of the DMNPs-AbCA153 probe of this invention on tumor size.

[0044] Figure 9 This is an HE staining image showing the effect of the DMNPs-AbCA153 probe of this invention on tumor tissue.

[0045] Figure 10 This is a statistical analysis of the effect of the DMNPs-AbCA153 probe of this invention on the body weight of mice.

[0046] Figure 11This is a statistical graph showing the effects of the DMNPs-AbCA153 probe of this invention on liver and kidney function in mice. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all materials involved in the following embodiments are commercially available. Unless otherwise specified, all methods are conventional methods. The proportions of each component are expressed in parts by mass and parts by volume, in mg and mL.

[0048] One embodiment is a specific targeted breast cancer diagnosis and treatment integrated nanoprobe (DMNPs-AbCA153), specifically an amino-modified bimodal nanoprobe modified with CA153 antibody.

[0049] In one embodiment, the CA153 antibody is modified onto the surface of the aminated bimodal nanoprobe via ester bonds.

[0050] In one embodiment, the aminated bimode nanoprobe is obtained by surface hydroxylation modification of the bimode nanoprobe with 3-aminopropyltriethoxysilane (APTES).

[0051] In one embodiment, the aminated bimodal nanoprobe is obtained by reacting 3-aminopropyltriethoxysilane (APTES) and the bimodal nanoprobe. The 3-aminopropyltriethoxysilane (APTES) serves for surface hydroxylation modification, and its amount is conventional in the art. In one embodiment, the ratio of 3-aminopropyltriethoxysilane (APTES) to the bimodal nanoprobe can be greater than or equal to 10 parts per part by mass of the bimodal nanoprobe. -8 Molar amounts of 3-aminopropyltriethoxysilane, g, mol.

[0052] In one embodiment, the ratio of 3-aminopropyltriethoxysilane (APTES) to the dual-mode nanoprobe is 10 mg / mL for every part by mass of the dual-mode nanoprobe. -8 In another embodiment, the molar ratio of 3-aminopropyltriethoxysilane (APTES) to the dual-mode nanoprobe is 10 molars per part by mass of the dual-mode nanoprobe. -7 Molar amounts of 3-aminopropyltriethoxysilane; in another embodiment, the ratio of 3-aminopropyltriethoxysilane (APTES) to the dual-mode nanoprobe is 10 molar amounts per part by mass of dual-mode nanoprobe. -6 Molar amounts of 3-aminopropyltriethoxysilane.

[0053] In one embodiment, the mixing reaction is carried out in an aqueous solution.

[0054] In one embodiment, the mixing reaction can be carried out in ultrasound.

[0055] In one embodiment, the mixing reaction time is 1-30 min. In one embodiment, the mixing reaction time is 5 min; in another embodiment, the mixing reaction time is 10 min; and in yet another embodiment, the mixing reaction time is 20 min.

[0056] In one embodiment, after the mixing reaction is completed, the aminated bimodal nanoprobe can be obtained by centrifugation.

[0057] In one embodiment, the aminated bimode nanoprobe can be prepared by a method including the following steps: resuspending the bimode nanoprobe in water, adding 3-aminopropyltriethoxysilane (APTES) under ultrasonic conditions, and reacting for 1-30 mins to obtain the aminated bimode nanoprobe.

[0058] In one embodiment, the reaction is followed by standing for 10-60 minutes to obtain a fully reacted aminated bimodal nanoprobe.

[0059] In one embodiment, the aminated bimodal nanoprobe can be obtained by centrifugation after the reaction; the obtained aminated bimodal nanoprobe can be resuspended in water to obtain an aqueous solution of the aminated bimodal nanoprobe.

[0060] In one embodiment, the dual-mode nanoprobe has a four-layer core-shell structure, consisting of a noble metal nanoparticle core connected with Raman signal molecules, a first modification layer encapsulating the core, a conjugated polymer fluorescent molecule layer covering the first modification layer, and a second modification layer encapsulating the conjugated polymer fluorescent molecule layer, from the inside out.

[0061] In one embodiment, the noble metal nanoparticles are gold nanoparticles or silver nanoparticles. In another embodiment, the noble metal nanoparticles are gold nanoparticles; in yet another embodiment, the noble metal nanoparticles are silver nanoparticles.

[0062] In one embodiment, the modification layer is silicon dioxide.

[0063] In one embodiment, the conjugated polymer is MEH-PPV or BDMO-PPV. In another embodiment, the conjugated polymer is MEH-PPV; in the third embodiment, the conjugated polymer is BDMO-PPV.

[0064] In one embodiment, the dual-mode nanoprobe is obtained by coating a first modification layer with a mixture of Raman signal molecules and noble metal nanoparticles, followed by compounding with a conjugated polymer fluorescent molecule, and finally coating the outermost layer with a second modification layer. The preparation method is a conventional method in the art.

[0065] Furthermore, the specific preparation method of the dual-mode nanoprobe can be found in CN107880872A.

[0066] In one embodiment, the dual-mode nanoprobe can be prepared by a method comprising the following steps:

[0067] S1. A Raman signal molecule solution was added to an aqueous dispersion of noble metal nanoparticles, and the mixture was ultrasonicated to obtain a mixture;

[0068] S2. Under ice-water bath conditions, transfer the mixture of S1 to anhydrous ethanol, stir and mix well, then add ammonia water, continue stirring and mixing well, then add tetraethyl silicate / ethanol solution dropwise, stir and react for 1-5 hours; then raise the temperature to room temperature and continue the reaction for 12-24 hours, centrifuge and wash to obtain SERS active particles.

[0069] S3. Prepare a tetrahydrofuran solution of the conjugated polymer and inject it into water under ultrasonic conditions to prepare conjugated polymer nanoparticles (CPNs).

[0070] S4. The SERS active particles of S2 and CPNs of S3 were modified with 3-aminopropyltriethoxysilane respectively. After ultrasonication to homogenize them so that active functional groups were attached to their surfaces, the two were mixed and ultrasonicated again to homogenize them so that the CPNs were adsorbed onto the surface of the SERS active particles.

[0071] S5. Add NH4OH and TEOS / EtOH solutions sequentially to the mixed solution in S4, and coat a second layer of silica using the Stöber method to obtain a dual-mode nanoprobe.

[0072] In one embodiment, the dual-mode nanoprobe can be obtained by centrifugation after coating.

[0073] In one embodiment, the specific targeted breast cancer diagnostic and therapeutic nanoprobe can be obtained by reacting a CA153 antibody with an aminated bimodal nanoprobe in an aqueous solution. The amount of CA153 antibody used can be based on conventional methods in the art. For example, in one embodiment, the ratio of CA153 antibody to aminated bimodal nanoprobe can be greater than or equal to 10 mg / mL of the aminated bimodal nanoprobe per part by mass. -7 One mass of CA153 antibody is sufficient.

[0074] In one embodiment, the ratio of the CA153 antibody to the aminated bimodal nanoprobe can be 10 mg / mL for every part by weight of the aminated bimodal nanoprobe. -7 In another embodiment, the ratio of the CA153 antibody to the aminated bimodal nanoprobe can be 10 mg / mL of the aminated bimodal nanoprobe per part by weight. -5 In another embodiment, the ratio of the CA153 antibody to the aminated bimodal nanoprobe can be 10 mg / mL of the aminated bimodal nanoprobe per part by weight. -6Mass fraction of CA153 antibody.

[0075] In one embodiment, the reaction can be carried out under the action of a crosslinking agent; the crosslinking agent can be glutaraldehyde. The amount of the crosslinking agent used is conventional; for example, in one embodiment, the ratio of the crosslinking agent used can be greater than or equal to 10 per part by mass of the aminated bimodal nanoprobe. -3 A mass fraction of crosslinking agent is sufficient.

[0076] In one embodiment, the crosslinking agent can be added at a ratio of 10 mg / part aminated bimodal nanoprobe. -3 In another embodiment, the crosslinking agent is added in proportions of 10 parts by weight per part by weight of the aminated bimodal nanoprobe. -2 Parts by weight of crosslinking agent; in one embodiment, the proportion of crosslinking agent may be 0.1 parts by weight of crosslinking agent added per part by weight of aminated bimodal nanoprobe.

[0077] In one embodiment, the reaction is preferably carried out in an ice-water bath. In one embodiment, the reaction time is 0.5-4 hours. In one embodiment, the reaction time is 0.5 hours; in another embodiment, the reaction time is 1 hour; and in yet another embodiment, the reaction time is 4 hours.

[0078] In one embodiment, the specific targeting nanoprobe for breast cancer diagnosis and treatment can be prepared by the following steps: mixing CA153 antibody with an aminated bimodal nanoprobe in an aqueous solution and reacting under ice-water bath conditions for 0.5-4 hours to obtain an aminated bimodal nanoprobe modified with CA153 antibody.

[0079] In one embodiment, the reaction can be carried out under conditions of slow shaking.

[0080] One embodiment is the application of the above-mentioned specific targeted breast cancer diagnosis and treatment nanoprobe in the preparation of breast cancer treatment drugs.

[0081] One embodiment is the application of the above-mentioned specific targeted breast cancer diagnosis and treatment nanoprobe in the preparation of reagents for detecting, screening and / or diagnosing breast cancer.

[0082] One embodiment is the application of the above-mentioned specific targeting nanoprobe for integrated diagnosis and treatment of breast cancer in the preparation of breast cancer cell imaging agents.

[0083] One embodiment is a kit for detecting breast cancer, the kit containing the above-mentioned nanoprobe.

[0084] Example 1: Preparation of a specific targeted nanoprobe for breast cancer diagnosis and treatment (DMNPs-AbCA153)

[0085] (1) Preparation of dual-mode nanoprobes:

[0086] S1. Gold nanoparticles were prepared using conventional methods, such as adding 1 volume part of 0.97 g / mL HAuCl4 aqueous solution to 380 volume parts of boiling water, continuing to boil for 3 min, and then adding 2 volume parts of 1% trisodium citrate aqueous solution to obtain gold nanoparticles with a diameter of about 35 nm.

[0087] S2. Dilute 3 volumes of the gold nanoparticle dispersion prepared in S1 with water to 5 volumes, add 0.02 volumes of 10 mM 4-MBA / ethanol solution, and sonicate or stir for 30 min to allow 4-MBA to react fully with the gold nanoparticles.

[0088] S3. Add the mixture from S2 to 200 parts by volume of anhydrous ethanol. Under ice-water bath conditions, add 7.5 parts by volume of ammonia (28%), stir continuously, and then slowly add 1.5 parts by volume of 10mM TEOS / EtOH solution in two drops. Stir the reaction for 2 hours, raise the temperature to room temperature and continue the reaction for 18 hours. Centrifuge and wash to obtain SERS active particles, and redisperse them in 3 parts by volume of anhydrous ethanol for later use.

[0089] S4. BDMO-PPV CPNs were prepared by injecting a 0.01 mg / mL BDMO-PPV / THF solution into water at a volume ratio of 1:4 under ultrasonic conditions. The solution was then left to stand in a vacuum container to allow the residual tetrahydrofuran to evaporate completely before use.

[0090] S5. Add 5 volumes of 10 to 3 volumes of the SERS active particles obtained in S3 and 1 volume of the CPNs dispersion obtained in S4, respectively. -7 M's APTES / ethanol solution was mixed evenly by sonication and then added to 100 volumes of anhydrous ethanol. The mixture was sonicated for another 30 minutes to allow CPNs to be adsorbed and fixed on the surface of SERS active particles.

[0091] S6. Repeat step S3, coat a second layer of silica using the Stöber method, centrifuge and wash to obtain SERS-MEF dual-mode probes (DMNPs) with a total size of approximately 75 nm.

[0092] (2) Preparation of nanoprobes (DMNPs-AbCA153): 1 volume fraction of SERS-MEF dual-mode nanoprobes with a concentration of 3.6 mg / mL was added to water for resuspending, and 2 × 10⁻⁶ ppm was added under ultrasonic conditions. -3 Volume concentration of 10 -4The mixture was reacted with 3-aminopropyltriethoxysilane (APTES) at a concentration of 1 mol / L for 1-30 min, then allowed to stand for 10-60 min. After centrifugation and washing, the aminated bimodal nanoprobe was obtained. The aminated bimodal nanoprobe was resuspended in water, and 1 volume fraction of a 10 mol / L solution was added. -3 μg / mL CA153 antibody, 3 volumes of 0.5×10 -3 % glutaraldehyde was added, and the reaction was carried out with gentle shaking in an ice-water bath for 0.5–4 h. The nanoprobes (DMNPs-AbCA153) were obtained by centrifugation. The CA153 antibody was purchased from Abcam (Abcam, Inc., MA, USA).

[0093] The nanoprobes prepared in this invention were characterized. Figure 1 The figures show the fluorescence spectra of two probes, DNMPs and DMNPs-AbCA153. As can be seen from the figures, the surface modification of amino-modified DNMPs with CA153 antibody significantly affects the fluorescence of the original nanomaterials.

[0094] Example 2: Aggregation of nanoprobes (DMNPs-AbCA153) in 4T1 breast cancer cells

[0095] 4T1 mouse breast cancer cells were purchased from Guangzhou Jennio Biotechnology Co., Ltd. Anti-CA153 antibody for immunofluorescence was purchased from CST (Cell Signaling Technology, Inc. USA). Cell counting kit-8 (CCK8) was purchased from Suyan Biotech Co., Ltd.

[0096] (1) 4T1 mouse breast cancer cells were cultured in RPMI 1640, 5% carbon dioxide, at 37°C. For fluorescence imaging experiments using laser confocal microscopy, the concentration was 1×10⁻⁶ cells / mL. 4 4T1 cells were seeded into laser confocal dishes. After culturing for 24 hours, the cells were washed twice with phosphate-buffered saline (PBS) at pH 7.4 before use.

[0097] (2) Immunofluorescence imaging: Cells in confocal culture dishes were fixed with 4% cell fixative, washed three times with PBST, and blocked for 20 min. Then, DMNPs and DMNPs-AbCA153 probes were added to the culture dish and incubated with the cells for 1 h. After adding anti-CA153 primary antibody, the cells were incubated for 2 h, then eluted with PBST, and fluorescent secondary antibody was added. The cells were incubated for another 1 h, and then washed three times with PBST. Finally, the cells were observed under a confocal microscope using DAPI nuclear staining.

[0098] Figure 2This is a laser confocal image showing the co-localization of DNMPs and DMNPs-AbCA153 in 4T1 cells. As can be seen from the image, the modified DMNPs-AbCA153 probe of this invention can target breast cancer cells and accurately locate them.

[0099] Example 3: Cytotoxicity assay of nanoprobes (DMNPs-AbCA153)

[0100] The potential cytotoxicity of the nanoprobe (DMNPs-AbCA153) to 4T1 cells was evaluated using the CCK8 assay. 4T1 cells were seeded into 96-well plates containing 200 μL of cell suspension. After incubation at 37°C in 5% CO2 for 24 h, 50 μL of probe dispersion (0.36 μg / mL–0.00281 μg / mL) was added to each well, and the plates were incubated for another 24 h. The plates were then washed with PBS to remove residual probe; 10 μL of CCK8 solution and 90 μL of RPMI 1640 were added to each well, and the plates were incubated for 1 h. Absorbance was measured at 450 nm using an ELISA instrument. Cell viability was expressed as a percentage of control cells to measure cytotoxicity.

[0101] Figure 3 This figure shows the toxicity analysis of different concentrations of the DMNPs-AbCA153 probe of this invention on 4T1 cells. As can be seen from the figure, the nanoprobe (DMNPs-AbCA153) of this invention has a significant inhibitory effect on 4T1 cells. When the probe concentration is 0.36 μg / mL, it can kill 80% of the 4T1 cells.

[0102] Example 4: Effect of nanoprobes (DMNPs-AbCA153) on intracellular ROS levels

[0103] Mean ROS levels in 4T1 cells were detected using laser confocal microscopy and the fluorescent probe DCFH-DA. 4T1 cells were treated with antibody CA153 (1 μg / mL), DMNPs (0.36 μg / mL), DMNPs-AbCA153 (0.36 μg / mL), and DMNPs-AbCA153 + NAC (5 mM) for 24 h, respectively. Then, 1 mL of 5 × 10⁶ cells was collected. 5After centrifugation (CFU / mL) and washing with PBS, the sample was resuspended in 500 µL of mixed binding buffer and incubated with 10 µM DCFH-DA in the dark at 37 °C for 20 min. Once inside PMVECs, DCFH-DA was hydrolyzed to non-fluorescent 2',7'-dichlorofluorescein (DCFH). Subsequently, DCFH was oxidized to highly fluorescent DCF under ROS. The fluorescence signal generated by DCF was detected using a confocal laser scanning system equipped with a Nikon Ti2-EECLIPSETie-EIB microscope. An argon laser with an excitation wavelength of 488 nm and an emission wavelength of 530 nm was used as the light source. The fluorescence intensity of DCF represents the level of reactive oxygen species. Detection was performed using a flow cytometer (Accuri C6, BD Biosciences, San Jose, USA). 1–5 × 10⁶ samples were collected from each experiment. 5 cell.

[0104] Figure 4 This is a laser confocal image showing the effect of the DMNPs-AbCA153 probe of this invention on ROS enzymes in 4T1 cells. Figure 5 This is a flow cytometry diagram showing the effect of the DMNPs-AbCA153 probe of this invention on ROS enzymes in 4T1 cells. In the diagram, a represents the blank control, b represents the antibody CA153, c represents DMNPs, d represents DMNPs-AbCA153, and e represents DMNPs-AbCA153 + NAC. Cells treated with DMNPs-AbCA153 showed a significantly high level of intracellular ROS enzymes. In the presence of the ROS enzyme inhibitor NAC, no ROS enzymes were observed in the DMNPs-AbCA153 + NAC group. Combined with the flow cytometry results, approximately 70% of the 4T1 cells treated with DMNPs-AbCA153 contained ROS enzymes. This indicates that DMNPs-AbCA153 can induce ROS enzyme production in 4T1 cells, which is a crucial factor in inducing 4T1 cell apoptosis.

[0105] Example 5: Western blotting detection of the effect of DMNPs-AbCA153 probe on 4T1 cells

[0106] 4T1 cells were cultured in a medium containing the following components: CA153 (1 μg / mL), DMNPs (0.36 μg / mL), DMNPs-AbCA153 (0.36 μg / mL), NAC (5 mM), and DMNPs-AbCA153 (0.36 μg / mL) + NAC (5 mM) for 24 h. Cells were then collected using the trypsin method and lysed with RIPA lysis buffer (Beyotime, Haimen, China) at 4 °C. The lysis buffer was centrifuged at 10,000 rpm for 10 min. Total protein content was determined using a BCA protein assay kit (ThermoFisher, USA). The lysis buffer was mixed with loading buffer and boiled at 100 °C for 10 min. Protein extracts (30 µg) were separated by 12.5% ​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were then incubated sequentially in a blocking solution (5% skim milk) for 1 h, followed by incubation for 2 h with primary antibody solutions containing P65, p-P65, JNK, p-JNK, Bcl-2, Bax, and β-actin, and then incubated for 1 h with gentle shaking in HRP-conjugated secondary antibody solution at room temperature. Before each incubation, the membranes were washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBST). Subsequently, the membranes were incubated with enhanced chemiluminescence for 1 min, and the blots were imaged using an X-ray imaging system (Bio-Rad, Harkles, CA, USA). Each experiment was repeated three times. The relative ratios of P65 to p-P65, JNK to p-JNK, Bcl-2, and Bax to β-actin were evaluated using ImageJ software.

[0107] Figure 6 This is a Western blotting image of 4T1 cells after treatment with the DMNPs-AbCA153 probe of this invention. Figure 7 The expression levels of P65, P-P65, p-JNK, JNK, Blc2, and Bax proteins are statistically represented.

[0108] As shown above, in 4T1 cells treated with DMNPs-AbCA153, the two genes that guide apoptosis, p-JNK and Bax, were significantly upregulated, while the four oncogenes, P65, P-P65, JNK, and Blc2, were downregulated. This indicates that DMNPs-AbCA153 stimulated the 4T1 apoptosis pathway and inhibited cancer cell development.

[0109] Example 6: Activity of nanoprobes (DMNPs-AbCA153) in the treatment of breast cancer

[0110] (1) Establishment of animal models

[0111] Thirty-three 5-6 week old female BALB / c mice, specifically pathogen-free (SPF), were obtained from Ruige Biotechnology Co., Ltd. (Guangzhou, China). After 7 days of rearing in a barrier facility, 18 healthy mice were randomly selected for modeling after daily observation. Mice were captured, suppressed, and injected with 4T1 cells via the right axilla, at a dose of 5 × 10⁻⁶ cells per mouse. 5 / 150μL. After the modeling process is complete, the animals' condition is continuously monitored. Once their health is confirmed to be normal, they are returned to their cages for regular feeding. The growth of the tumor at the injection site is closely observed; modeling is considered successful when the tumor volume reaches approximately 100 mm³.

[0112] (2) Grouping: Group 1 - normal mice (5 mice); Group 2 - tumor mice (5 mice); Group 3 - normal mice + PBS (5 mice); Group 4 - tumor mice + PBS (5 mice); Group 5 - normal mice + DMNPs-AbCA153 + PBS (5 mice); Group 6 - tumor mice + DMNPs-AbCA153 + PBS (6 mice). The dosage was 1 mL for all groups, and the DMNPs-AbCA153 content in each group was 0.32 mg / mL.

[0113] (3) PBS and PBS+DMNPs-AbCA153 were injected into the tail veins of groups 3, 4, 5, and 6, respectively. Fluorescence images were analyzed at 1, 24, 72, 120, and 168 h. After 168 h, the mice were sacrificed, their organs were collected, and fluorescence intensity was measured. These organs were then stained with hematoxylin and eosin.

[0114] (4) During the experiment, each group was weighed separately, and the tumor was measured every two days. The longest diameter (a) and the shortest diameter (b) of the tumor were measured using vernier calipers. During the measurement, the mouse was held firmly with the left hand. The precise measurement values ​​were recorded using the formula V = 1 / 2×a×b², and the tumor volume (TV) was calculated.

[0115] Statistical analysis was performed using Prism (GraphPad) software. Multiple comparisons were performed using one-way ANOVA, while pairwise comparisons were performed using Tukey's postoperative test or other specified methods. P < 0.05 (*P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.001; **** < 0.0001; *****P < 0.00001; ns, not significant (P > 0.05) were all considered statistically significant).

[0116] Figure 8This is a statistical graph showing the effect of the DMNPs-AbCA153 probe of this invention on tumor size. The PBS-DMNPs-AbCA153-Tumor sample group of this invention showed a significant difference compared to the control group PBS-Tumor. Figure 9 This is an HE staining image showing the effect of the DMNPs-AbCA153 probe of this invention on tumor tissue. Figure 10 This is a statistical analysis of the effect of the DMNPs-AbCA153 probe of this invention on the body weight of mice; Figure 11 This is a statistical graph showing the effects of the DMNPs-AbCA153 probe of this invention on liver function (AST / ALT) and kidney function (ALB / GLOB) in mice.

[0117] As shown in the figure, the DMNPs-AbCA153 nanoprobe of this invention significantly inhibits tumor growth in the body after a single administration, with a marked inhibitory effect compared to the blank control group. Furthermore, the absence of significant changes in mouse body weight and no significant impact on liver and kidney function indicates that the DMNPs-AbCA153 nanoprobe of this invention is non-toxic to the organism. This further demonstrates that the DMNPs-AbCA153 nanoprobe of this invention exhibits tumor-targeting specificity and biocompatibility.

[0118] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a specific targeted nanoprobe integrating diagnosis and treatment of breast cancer in the preparation of drugs for treating breast cancer, characterized in that: The specific targeted breast cancer diagnosis and treatment integrated nanoprobe was prepared by the following method: the bimodal nanoprobe was resuspended in water, and 3-aminopropyltriethoxysilane was added under ultrasonic conditions and reacted for 1-30 mins to obtain an aminated bimodal nanoprobe; CA153 antibody and aminated bimodal nanoprobe were mixed in an aqueous solution and reacted under ice-water bath conditions with glutaraldehyde for 0.5-4 h to obtain an aminated bimodal nanoprobe modified with CA153 antibody surface. Preparation of the dual-mode nanoprobe: S1. Gold nanoparticles were prepared by conventional method. One volume part of 0.97 g / mL HAuCl4 aqueous solution was added to 380 volume parts of boiling water. After boiling for 3 min, two volume parts of 1% trisodium citrate aqueous solution were added to obtain gold nanoparticles. S2. Dilute 3 volumes of the gold nanoparticle dispersion prepared in S1 with water to 5 volumes, add 0.02 volumes of 10 mM 4-MBA / ethanol solution, and sonicate or stir for 30 min to allow 4-MBA to react fully with the gold nanoparticles. S3. Add the mixture from S2 to 200 parts by volume of anhydrous ethanol. Under ice-water bath conditions, add 7.5 parts by volume of 28% ammonia water and stir continuously. Then slowly add 1.5 parts by volume of 10mM TEOS / EtOH solution in two drops. Stir the reaction for 2 hours, raise the temperature to room temperature and continue the reaction for 18 hours. Centrifuge and wash to obtain SERS active particles, and redisperse them in 3 parts by volume of anhydrous ethanol for later use. S4. BDMO-PPV CPNs were prepared by injecting a 0.01 mg / mL BDMO-PPV / THF solution into water at a volume ratio of 1:4 under ultrasonic conditions. S5. Add 5 volumes of 10 to 3 volumes of the SERS active particles obtained in S3 and 1 volume of the CPNs dispersion obtained in S4, respectively. - 7 M's APTES / ethanol solution was mixed evenly by sonication and then added to 100 volumes of anhydrous ethanol. The mixture was sonicated for another 30 minutes to allow CPNs to be adsorbed and fixed on the surface of SERS active particles. S6. Repeat step S3 and add NH4OH and TEOS / EtOH solutions to the mixed solution in S5 in sequence. Coat the second layer of silica using the Stöber method, centrifuge and wash to obtain the SERS-MEF dual-mode nanoprobe.

Images