Quinoline derivative for treating triple negative breast cancer

Abstract

The invention discloses a quinoline derivative for treating triple negative breast cancer, and relates to the technical field of triple negative breast cancer treatment. The AI E780-SG nano-construct disclosed by the invention is preferentially accumulated in TNBC cells which express TROP2 and are resistant to SG and organ-like tissues derived from a patient by utilizing a targeting effect mediated by an hRS7 antibody. Under near-infrared irradiation, AI E780 causes mitochondrial redox imbalance through local ROS (reactive oxygen species) generation, so that tumor selective immunogen cell death is caused, and the death is caused by membrane destruction and oxidative necrosis. Meanwhile, SG releases an SN-38-one potent topoisomerase I inhibitor and an hRS7 antibody fragment through acid response cleavage, and natural killer cell recruitment and activation are coordinated. In a mouse TNBC xenotransplantation model, the AI E780-SG nano-particles realize synergistic chemotherapy photodynamic tumor elimination, overcome SG resistance and reactivate anti-tumor immunity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of triple negative breast cancer treatment, and particularly relates to a quinoline derivative for treating triple negative breast cancer. BACKGROUND

[0002] Breast cancer continues to be the most common cancer affecting women, with approximately 420,000 new cases reported in China in 2022, thus representing a major part of the global disease burden. This rise in incidence not only reflects an increase in early detection rates due to widespread screening programs, but also the growing influence of western lifestyle factors, such as changes in diet, delayed childbearing, and reduced physical activity. Despite significant advances in treatment strategies, including surgery, chemotherapy, targeted therapy, and immunotherapy, the rising incidence of breast cancer constitutes a major public health problem that requires sustained efforts in prevention, early detection, and personalized treatment strategies.

[0003] TNBC (triple negative breast cancer) is characterized by lack of ER (estrogen receptor) and PR (progesterone receptor) expression and absence of HER2 expression, which is particularly aggressive and associated with a pessimistic prognosis. Approximately 85% to 90% of triple negative breast cancer (TNBC) specimens overexpress trophoblast cell surface antigen 2 (TROP2), which makes it an attractive therapeutic target.

[0004] Photodynamic therapy (PDT) is regaining attention as a minimally invasive approach to breast cancer management, distinguished by its good safety profile, PDT relies on 81 photosensitizers (PSs) that generate destructive reactive oxygen species (ROS) under light irradiation to cause cancer cell death, however, the clinical utility of traditional PSs is limited by aggregation-caused quenching (ACQ) and photobleaching, which hinders ROS production and therapeutic efficacy, in 2001, Tang et al. reported the opposite phenomenon--aggregation-induced emission (AIE)--in which molecular aggregation enhances fluorescence and ROS generation, meanwhile, the emergence of the concept of immunogenic cell death (ICD)--a pattern characterized by the release of damage-associated molecular patterns (DAMPs), particularly surface-exposed calreticulin (CRT)--as a cornerstone of modern cancer immunotherapy. ICD can correct the immunogenic defect inherent to malignant tumors such as metastatic TNBC, thereby enhancing tumor sensitivity to immunotherapeutic intervention, one of the key steps of ICD is the movement of CRT from the endoplasmic reticulum to the cell surface as an 'eat me' signal for dendritic cells (DCs) and promotes antigen presentation and adaptive anti-tumor immunity, previous efforts have produced AIE780 through a cationization strategy, endowing it with type I ROS generation ability and mitochondrial targeting properties, however, the repertoire of potent ICD inducers remains limited, planar PSs often face issues of poor solubility, insufficient photostability, and weak membrane affinity, leading to suboptimal ROS production and ICD induction, there is an urgent need to design PSs with optimized molecular structures that can combine strong ROS generation ability, ideal tumor antigen targeting, aqueous solubility stability, and enhanced cell permeability, for this purpose, we propose a quinoline derivative for treating triple-negative breast cancer. SUMMARY

[0005] The present application aims at: in order to solve the problems mentioned in the background art, the present application provides a quinoline derivative for treating triple-negative breast cancer.

[0006] In order to achieve the above-mentioned purpose, the present application specifically adopts the following technical solutions:

[0007] A quinoline derivative for treating triple-negative breast cancer, the preparation of the quinoline derivative comprises the following steps:

[0008] Step 1, Preparation of AIE780-SG NPs, 500 μg of AIE780COOH was placed in 9 mL of deionized water and shaken under an ultrasonic cell crusher, followed by stirring evaporation of THF at 25°C, filtration, 2 mL of NP suspension was diluted to 8 mL and reacted with 500 μg of SG, using Sulfo-NHS and EDC as coupling agents, and reacted at 25°C for 4 hours, the product was removed by centrifugation Unbound reagents, followed by two additional PBS wash-centrifugation cycles, the final NPs were resuspended in 8 mL of PBS as a stock solution;

[0009] Step 2, cell culture, breast cancer cell lines were collected and cultured in RPMI-1640 medium, and 10% fetal bovine serum was added, BT-20 cells were cultured in a special CM-0324 culture medium, and the culture conditions included growing each cell line in a 95% humidity incubator at 37°C and 5% carbon dioxide and 95% air atmosphere;

[0010] Step 3, construction of three-dimensional model, a three-dimensional patient-derived organ-like model was established by tissue-specific adult stem cells or pluripotent stem cells cultured in vitro in a 3D culture system;

[0011] Step 4, role of internalized AIE780-SG NPs in TNBC-PDOs, after the growth of TNBC-PDOs, the tissue spheres were collected, and the Matrigel was dissolved using Cell Recovery Solution, 100 μl of AIE780 and AIE780-SG nanoparticles were added to the system, suspended in phosphate buffer, and stained with fluorescent dye MitoFluorTM and DAPI for 20 minutes respectively to mitochondria and nucleus, and the endocytosis process of AIE780-SG nanoparticles was dynamically observed using a table rotating disc scanning confocal microscope and its Z-stack model;

[0012] Step 5, TNBC-PDOs viability assay, in the viability assay, TrypLE Express was used to dissociate the spheroids and suspended in Matrigel at a concentration of 2000 cells / well in each 96-well plate, 4 μL of Matrigel per well, after which the spheroids were incubated for 7 days, the PDOs were treated with SG at concentrations of 0, 0.032, 0.16, 0.8, 4, 20 and 100 μM for 6 days, the images of the 96-well plates were captured using a microscope, the PDO spheroids were mixed thoroughly and 100 μL samples were extracted from each well for quantitative analysis, using the Cell Counting-Lite 3D cell viability assay, 100 μL of PDO spheroid sample was dispensed into each well of a white opaque 96-well plate and an equal volume of assay reagent was added to each well, then the 96-well plate was placed on a shaker for 5 minutes to facilitate cell lysis, after incubation at room temperature for 30 minutes to allow stabilization, the luminescence signal was measured, which allowed the sensitivity to SG to be evaluated;

[0013] Step 6, TUNEL imaging, after dehydration of the mouse tissue, it was embedded in paraffin and cut into 4 μm sections, the sections were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 20 minutes, incubated at 37°C with an incubation containing terminal transferase and reaction mixture, the effect of the combined SG (photosensitizer) and PDT (photodynamic therapy) treatment was evaluated using the TUNEL Alexa Fluor imaging kit;

[0014] Step 7, cytotoxicity test, the cytotoxicity of the NK cells was evaluated by flow cytometry detection for 6 hours;

[0015] Step 8, cytotoxicity assay, TNBC-PDOs and cells were contacted with 1 μg / mL of SG, 1 μg / mL of AIE780 or 1216 μg / mL of AIE780-SGNPs, respectively, after which they were washed with PBS and irradiated for 30 minutes with or without white light irradiation, the survival rate of these cells relative to untreated cells was measured using the standard CCK-8 assay;

[0016] Step 9, flow cytometry assay for apoptosis, the cells were contacted with 1 μg / mL of SG, 1 μg / mL of AIE780 or 1 μg / mL of AIE780-SG nanoparticles (NPs), respectively, after which they were washed with PBS and irradiated for 30 minutes with or without 3 mW / cm 2 of white light irradiation, the apoptosis was quantitatively detected using Annexin V-FITC / PI and Annexin V-FITC / 7-AAD and the organ-like damage was evaluated;

[0017] Step 10, Protein detection and quantification, detected by Western blotting.

[0018] Further, the model of the ultrasonic cell pulverizer in the preparation of the AIE780-SG NPs is SCIENTZ-11D, the power is 80%, the vibration time is 1 minute, the pore size of the filter membrane in the filtration is 0.2 μm, the weight of Sulfo-NHS is 17.4 μg, and the weight of EDC is 15.3 μg.

[0019] Further, the size and zeta potential of the AIE780 nanoparticles and AIE780-SG nanoparticles in the preparation of the AIE780-SG NPs are evaluated at 25°C using a zeta potential analyzer, the structure of the IT-PEG-RGD nanoparticles is checked by using a FEI Tecnai transmission electron microscope (TEM) and operating at 120 kV, the fluorescence spectrum of the AIE780-SG nanoparticles is recorded by a Horiba Fluorolog-3 fluorometer using a tetrahedral colorimetric cell.

[0020] Further, the cells in the cell culture include MCF10A, MDA-MB-231, Hs578T, BT549, and BT-20.

[0021] Further, the construction of the three-dimensional model includes the following steps:

[0022] Step 31, Fresh tumor tissue obtained during surgery is immediately immersed in DMEM added with 2% penicillin / streptomycin, the tissue is cut into 1 mm thick slices, and enzyme digestion is performed in a DMEM / F12 medium containing 3 mg / ml collagenase type I and IV at a temperature of 37°C for one hour;

[0023] Step 32, After digestion, the cell suspension is filtered through a 70 μm sieve and washed twice with the medium;

[0024] Step 33, 15,000 cells and 30 μl of Matrigel are uniformly distributed in each well of a 24-well plate, then the plate is inverted and incubated in a column oven for 0.5 hours, after the Matrigel solidifies, 500 μl of medium is added, and then the medium is replaced every two days for one week.

[0025] Further, the Cell Counting-Lite 3D in the TNBC-PDOs survival ability detection needs to be thawed at room temperature.

[0026] Further, the cytotoxicity test includes the following steps:

[0027] Step 71, after the tumor cells were labeled with CellTracker CM-Dil fluorescent dye, an overnight culture was performed;

[0028] Step 72, two sets of co-culture were performed with NK cells at different effector to target ratios, after 4 hours of co-culture, Live / Dead Fixable V500-Aqua dead cell staining reagent was used to measure cell survival rate;

[0029] Step 73, data was collected by Canto II flow cytometer, and analyzed by FlowJo software version 10.

[0030] Further, the different effector to target ratios are 0.25:1, 0.5:1, 1:1 and 2:1.

[0031] Further, the protein detection and quantification comprises the following steps:

[0032] Step 101, BT-20 cells were seeded in 6-well plates at 1×104 cells per well, and placed for 12 hours, after incubation with 1 μg / mL of SG, 1 μg / mL of AIE780NPs or 1 μg / mL of AIE780-SGNPs, the cells were washed with PBS, and received 3 mW / cm2 white light irradiation for 30 minutes, after 30 minutes, the cells in each group were collected and lysed with RIPA buffer, and the total protein concentration in the lysates was determined by BCA method;

[0033] Step 102, 20 μg of protein was loaded onto a SDS-PAGE gel, and the protein was separated by gel electrophoresis;

[0034] Step 103, transferred to a PVDF membrane of Millipore, after incubation for 60 minutes in 1xTBST with 5% skim milk, the membrane was incubated at 4°C for 12 hours in 1xTBST with 5% bovine serum albumin specific primary antibody;

[0035] Step 104, after washing five times with 1xTBST, 1xTBST containing HRP-conjugated secondary antibody was added, and incubated at room temperature for 2 hours;

[0036] Step 105, after washing three times with PBS, the protein bands on the membrane were visualized by ECL on a BioRad imaging system, and the protein bands were quantified using ImageJ software.

[0037] The beneficial effects of the present application are as follows:

[0038] The AIE780-SG nanoconstruct of the present application utilizes hRS7 antibody-mediated targeting to preferentially accumulate in TROP2-overexpressing, SG-resistant TNBC cells and patient-derived organotypic tissues. Upon near-infrared irradiation, AIE780 causes mitochondrial redox imbalance through local ROS generation, leading to tumor-selective immunogenic cell death, triggered by membrane disruption and oxidative necrosis. Simultaneously, SG releases SN-38, a potent topoisomerase I inhibitor, and hRS7 antibody fragments through acid-responsive cleavage, coordinating natural killer (NK) cell recruitment and activation. In a mouse TNBC xenograft model, AIE780-SG nanoparticles enable synergistic chemo-photodynamic tumor eradication, overcoming SG resistance and reactivating antitumor immunity. A multimodal strategy is provided to enhance ADC efficacy and reprogram the suppressive TNBC microenvironment, heralding a new strategy against SG-resistant TNBC. The dual therapeutic nano-platform that combines targeted ADC delivery with photodynamic induction of ICD overcomes SG resistance in TNBC and enhances NK cell-mediated antitumor responses. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a schematic diagram of the photophysical properties of AIE780 and AIE780-SGNPs in the present application;

[0040] A, B are size distribution diagrams and TEM characterization of AIE780 and 783 AIE780-SG;

[0041] C, zeta potential distribution of AIE780 and AIE780-SG;

[0042] D, absorption spectra in PBS, respectively, at a concentration of 5x10-5M, and an excitation wavelength of 660nm;

[0043] E, photothermal effect of AIE780-SG nanoparticles (concentration of 0.5mM, power density of 380mwcm-2);

[0044] F, "on-off" temperature change of AIE780-SG within 10 minutes under 2.0Wcm-2laser irradiation, and thermal stability of AIE780-SG during five on-off cycles;

[0045] G, temperature change of PBS, AIE780 and AIE780-SG under 660nm laser irradiation, with a power density of 2.0Wcm-2

[0046] H, I are the photothermal effect of AIE780-SG nanoparticles under different molecular concentrations (power density of 380mwcm-2) and different power levels;

[0047] J, Drug release kinetics of AIE-SG in PBS;

[0048] K, L, Drug release kinetics of AIE-SG in different pH and at different temperatures in serum;

[0049] Figure 2 is a schematic diagram of TROP2 expression and SG drug sensitivity analysis of different triple-negative breast cancer cell lines in the present application, and distribution of AIE780-SG in different cell lines and its co-localization with mitochondria;

[0050] A, Representative Western blot results of TROP2 in different breast cancer cell lines;

[0051] B, Quantitative analysis of TROP2 in different breast cancer cell lines;

[0052] C, Flow cytometry analysis of TROP2 expression levels in different breast cancer cell lines;

[0053] D, IC50 curve of SG in different breast cancer cell lines;

[0054] E, Quantitative comparison of distribution of AIE780-SG in different breast cancer cell lines and its co-localization with mitochondria;

[0055] F, Distribution of AIE780 and AIE780-SG in BT-20 cells, and their co-localization with Mito at different time points after treatment with AIE780 and AIE780-SG.

[0056] Figure 3 is a schematic diagram of the effects of AIE780 / AIE780-SG nanoparticles on mitochondrial morphology, ROS generation, and apoptosis rate / protein expression in BT-20 cells after different treatments in the present application;

[0057] A, Electron microscope images of BT-20 cells after treatment with AIE780-SG under 660 nm laser irradiation or without irradiation, and their enlarged images, which show the details of mitochondrial damage;

[0058] B, Intracellular ROS generated after treatment of BT-20 cells with AIE780-SG was detected using H2DCFDA;

[0059] C, Flow cytometry analysis results of BT-20 cell apoptosis rate, AnnexinV+PI-(Q4) represents early apoptotic cells, AnnexinV+Pl+(Q2) represents late apoptotic cells, and AnnexinV-PI- represents surviving cells;

[0060] D, Apoptosis-related proteins (HSP70 / HSP90, p-elF2a / elF2

[0061] A, Representative immunoblotting images of apoptosis-related proteins (HSP70 / HSP90, p-elF2a / elF2

[0062] Figure 4 Figure 1 is a schematic diagram of the targeting of AIE780 / AIE780-SG and apoptosis of cells after different treatments in the present application;

[0063] A, Schematic diagram of organotypic culture and cell viability detection after SG treatment;

[0064] B, Microscopic images and IC50 curve of organotypic culture sensitive or resistant to SG after SG treatment;

[0065] C and D, H&E and immunohistochemical staining show high TROP2 expression and resistance to SG in TNBC organotypic culture;

[0066] E, Confocal images of TNBC tumor organotypic culture resistant to SG, stained with red AIE780 and AIE780-SG NPs, and mitotracker Green (green);

[0067] F, Apoptosis immunofluorescence staining of TNBC PODO after different treatments.

[0068] Figure 5 Figure 2 is a schematic diagram of the evaluation of tumor growth and response in vivo of BT-20 tumor-bearing mice treated with AIE780-SG NPs in the present application;

[0069] A, In vivo near-infrared fluorescence imaging of BT-20 tumor-bearing mice at different time points after intravenous injection of AIE780 and AIE780-SG NPs;

[0070] B, In vitro near-infrared fluorescence imaging of major organs and tumors removed from mice after intravenous injection of AIE780 and AIE780-SG in PBS for 12 hours and 24 hours;

[0071] C, Representative tumor images of BT-20 tumor-bearing nude mice 14 days after different treatments;

[0072] D, Tumor volume growth curve of mice after various treatments;

[0073] E. Histological analysis of tumor tissues treated with PBS, SG, AIE780, AIE780+laser, AIE780-SG and AIE780-SG+laser by hematoxylin-eosin (H&E), Ki67 and TUNEL staining.

[0074] Figure 6 is a schematic diagram of flow cytometry analysis of cytokine secretion of NK cells in the present application;

[0075] A. The surface of cells for CD80, CD86 and MHC-II was evaluated by FACS analysis after immunofluorescence staining in different groups of treatment;

[0076] B-D. Quantitative analysis of the expression level of immune markers after different treatments, taking the co-expression of CD86+, CD80+and MHC-II+as an indicator;

[0077] E. Representative flow cytometry analysis and quantification results of NK cells (CD45+CD3-CD49b+);

[0078] F. The proportion of perforin, IFN-y and GranzymeB secreting cells.

[0079] Figure 7 is a schematic diagram of immunofluorescence analysis of tumor tissues of experimental mice receiving different treatments in the present application;

[0080] A. Staining for CD49b, NKG2D, and quantifying the proportion of CD49b and NKG2D;

[0081] B. Staining for CD31, HIF-1a, DAPI, and quantifying the proportion of HIF-1a.

[0082] Figure 8 is a schematic diagram of thermal imaging observation of PBS, AIE780 and AIE780-SG under 660 nm near-infrared light for 10 minutes in the present application.

[0083] Figure 9 is a schematic diagram of measuring the intracellular ROS level of BT-20 cells after different treatments (AIE780=10 μM; AIE780-SG=10 μM), 60 nm laser irradiation power of 2 W / cm 2 , irradiation time of 5 minutes using DCFH-DA as an indicator in the present application.

[0084] Figure 10 is a schematic diagram of cell survival rate of PBS, AIE780, AIE780-SG in BT-20 cells with or without laser irradiation in the present application.

[0085] Figure 11 A, yH2AX immunofluorescence staining of BT-20 cells after different treatments;

[0086] B, Quantification of yH2AX;

[0087] C, Western blot analysis of yH2AX protein expression of BT-20 cells after different treatments.

[0088] Figure 12 is a schematic diagram of the animal imaging after intravenous injection of d-luciferin at different times in the present application, and the study of the in vivo anti-tumor effect of different treatments.

[0089] Figure 13 is a schematic diagram of the tumor weight of mice in different treatment groups in the present application.

[0090] Figure 14 A, Histopathological staining: HE staining micrographs of heart, liver, spleen, lung and kidney sections of mice in different treatment groups;

[0091] B, Albumin (U / L);

[0092] C, ALT (U / L);

[0093] D, AST (U / L);

[0094] E, Urea (mmol / L);

[0095] F, Blood urea nitrogen (mg / dL);

[0096] G, Creatinine (μmol / L).

[0097] Figure 15 A, Comparison of cytotoxicity of different treatment groups when the E:T ratio is 2:1;

[0098] B, Comparison of cytotoxicity of different treatment groups when the E:T ratio is 4:1;

[0099] C, Percentage of CD107a expression on the surface of cells in different treatment groups of mice.

[0100] Figure 16 The proportion of perforin, IFN-γ and granzyme B (GzmB) secreting cytotoxic natural killer (NK) cells is shown.

[0101] Figure 17 is a schematic diagram of the present application. DETAILED DESCRIPTION

[0102] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application.

[0103] Referring to Figure 1 Figure 17 The present application provides a quinoline derivative for treating triple-negative breast cancer, and the preparation of the quinoline derivative comprises the following steps:

[0104] Step 1, 500 μg of AIE780COOH was placed in 9 mL of deionized water and oscillated under an ultrasonic cell crusher, wherein the model of the ultrasonic cell crusher was SCIENTZ-11D, the power was 80%, the oscillation time was 1 minute, THF was evaporated under stirring at 25°C, filtration was performed, the pore size of the filter membrane was 0.2 μm, 2 mL of NP suspension was diluted to 8 mL and reacted with 500 μg of SG, and Sulfo-NHS (17.4 μg) and EDC (15.3 μg) were used as coupling agents, the reaction was performed at 25°C for 4 hours, the product was removed from the unbound reagent by centrifugation (13,000 rpm), followed by two additional PBS wash-centrifugation cycles, and finally the NPs were resuspended in 8 mL of PBS as a stock solution;

[0105] The size and zeta potential of AIE780 nanoparticles and AIE780-SG nanoparticles in the preparation of AIE780-SG NPs were evaluated at 25°C using a potential analyzer, the potential analyzer was selected from a Brookhaven Instruments Corporation Zeta Plus potential analyzer, the structure of the IT-PEG-RGD nanoparticles was checked by using a FEI Tecnai transmission electron microscope and operating at 120 kV, and the fluorescence spectrum of the AIE780-SG nanoparticles was recorded by a Horiba Fluorolog-3 fluorometer using a tetrahedral colorimetric cell.

[0106] Step 2, five human breast cancer cell lines were selected, including MCF10A, MDA-MB-231, Hs578T, BT549 and 139BT-20, the cells were cultured in a RPMI-1640 culture medium of Gibco and added with 10% ExCellBio (FSP500) fetal bovine serum, the BT-20 cells were cultured in a special CM-0324 (Wuhan P Cell Life Science and Technology Co., Ltd.) culture medium, and the standard culture conditions included growing each cell line in an incubator with a humidity of 95% at 37°C and in an atmosphere of 5% carbon dioxide and 95% air.

[0107] ​Step 3, Establishing 3D patient-derived organoid model 147 Tissue samples are 3D culture systems of tissue-specific adult stem cells or pluripotent stem cells cultured in vitro, allowing them to self-organize into structures with specific functions; to construct the TNBC-PDO model, fresh tumor tissue obtained during surgery was immediately immersed in DMEM supplemented with 2% penicillin / streptomycin, the tissue was cut into 1 mm thick slices, and enzyme digestion was performed in DMEM / F12 medium containing 3 mg / ml collagenase type I and IV at 37°C for one hour; after digestion, the cell suspension was filtered through a 70 micron sieve and washed twice with medium; 15,000 cells and 30 microliters of Matrigel were evenly distributed into each well of a 24-well plate, then the plate was inverted and incubated in a column oven for 0.5 hours, after the Matrigel solidified, 500 microliters of medium were added, and then the medium was replaced every two days for one week.

[0108] Step 4, When the TNBC-PDOs grow to the appropriate size, collect the tissue spheres and use Cell Recovery Solution (Corning, 354253) to break down the Matrigel, add 100 μl of AIE780 and AIE780-SG nanoparticles to the system, suspended in phosphate buffer, use a mitochondrial selective fluorescent dye MitoFluorTM and DAPI from Sigma-Aldrich to stain mitochondria and nuclei for 20 minutes respectively, and use a table-top spinning disk scanning confocal microscope (Oxford Instruments) and its Z-stack model to dynamically observe the endocytosis process of AIE780-SG nanoparticles.

[0109] Step 5, TNBC-PDOs viability detection, in order to evaluate the sensitivity to SG, TNBC-PDOs PDO activity assay was performed, in the activity assay, the tissue spheres were dissociated using TrypLE Express (Gibco, 12605028) and suspended in Matrigel at a concentration of 2000 cells / well in each 96-well plate, with 4 μL Matrigel per well. After 7 days of culture, PDOs were treated with SG at concentrations of 0, 0.032, 0.16, 0.8, 4, 20, and 100 μM for 6 days. The images of the 96-well plates were captured using a microscope, the PDO spheres were thoroughly mixed, and 100 μL samples were extracted from each well for quantitative analysis. The cell viability assay was performed using CellCounting-Lite3D (DD112-02, Vazyme Biotech, China). The Counting-Lite3D was thawed at room temperature. 100 μL of PDO sphere sample was dispensed into each well of a white opaque 96-well plate (BS-MP-96W, Biosharp, China). An equal volume of assay reagent was added to each well. The 96-well plate was then vigorously mixed on a shaker for 5 minutes to promote cell lysis. After incubation at room temperature for 30 minutes to allow stabilization, the luminescence signal was measured.

[0110] Step 6. To evaluate the effect of combined SG (photosensitizer) and PDT (photodynamic therapy) treatment, TUNEL imaging was performed using the TUNEL Alexa Fluor imaging kit. The mouse tissue was dehydrated, embedded in paraffin, and cut into 4 μm sections. The sections were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% TritonX-100 for 20 minutes, and incubated at 37°C with a solution containing terminal transferase and a reaction mixture.

[0111] Step 7. Cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) test The cytotoxicity of 206 NK cells was evaluated by flow cytometry for 6 hours; tumor cells were labeled with CellTracker CM-Dil fluorescent dye and cultured overnight; they were co-cultured with NK cells in duplicate at different effector-to-target ratios (0.25:1, 0.5:1, 1:1, and 2:1). After 4 hours of co-culture, cell viability was measured using Live / DeadFixable V500-Aqua dead cell staining reagent (ThermoFisher); data were collected on a Canto II flow cytometer (BD Biosciences, NJ) and analyzed using FlowJo software version 10.

[0112] Step 8, TNBC-PDOs and cells were contacted with 1 pg / mL of SG, 1 pg / mL of AIE780 or 1216 pg / mL of AIE780-SGNPs, respectively, followed by PBS wash and irradiation for 30 minutes with or without white light irradiation, and the survival rate of these cells relative to untreated cells was measured using standard CCK-8 assay.

[0113] Step 9, cells were contacted with 1 pg / mL of SG, 1 pg / mL of AIE780 or 1 pg / mL of AIE780-SG nanoparticles (NPs), respectively, followed by PBS wash and irradiation with or without 3 mW / cm2of white light irradiation (irradiation for 30 minutes), and Annexin V-FITC / PI and Annexin V-FITC / 7-AAD were used to quantitatively detect apoptosis and assess organ-like damage. 2

[0114] Step 10, protein detection and quantification, Western blotting was used for detection; BT-20 cells were seeded in 6-well plates (1 x 104cells per well), and incubated for 12 hours, after incubation with 1 pg / mL of SG, 1 pg / mL of AIE780 NPs or 1 pg / mL of AIE780-SGNPs, the cells were washed with PBS and subjected to 3 mW / cm2of white light irradiation for 30 minutes, 30 minutes later, cells in each group were collected and lysed with RIPA buffer (Thermo Scientific, Waltham, MA, USA), and the total protein concentration in the lysates was determined by BCA method (Thermo Scientific); 20 pg of protein was loaded onto SDS-PAGE gel, and the proteins were separated by gel electrophoresis; transferred to PVDF membrane of Millipore (Billerica, MA, USA), after incubation for 60 minutes in 1xTBST with 5% skim milk, the membrane was incubated with specific primary antibodies in 1xTBST with 5% bovine serum albumin for 12 hours at 4°C; after washing five times with 1xTBST, 1xTBST containing HRP-conjugated secondary antibody was added and incubated at room temperature for 2 hours; finally, after washing three times with PBS, the protein bands on the membrane were visualized by ECL (Thermo Scientific) on a BioRad imaging system (Hercules, CA, USA), and the protein bands were quantified using ImageJ software.

[0115] Experiment

[0116] BALB / c nude mice (4-5 weeks old) were injected subcutaneously with BT-20 cells (1 x 10^6) and 100 pL of PBS through the right groin, and a total of 6 injections were performed, after 7 days, when the tumor volume reached about 100 mm​3 At the same time, mice received near-infrared fluorescence imaging (NIRFLI), or were divided into six groups (5 mice per group) to receive different treatments (PBS, SG, AIE780NP, AIE780NP+Laser, Laser, AIE780-SGNP, AIE780-SGNP+Laser), laser treatment (wavelength 660 nm, power density 0.3 W / cm2, duration 10 minutes) was performed 6 hours after AIE nanoparticle injection and was repeated once a week, tumor volume and body weight were monitored every other day, calculated using the formula V = (length x width2) / 2, on the 21st day after tumor inoculation, blood tests and organ analysis were performed on mice, the main organs and tumors were fixed with 4% paraformaldehyde and HE staining and IHC staining were performed to evaluate histological changes and tumor cell proliferation. 2 , duration 10 minutes) was performed 6 hours after AIE nanoparticle injection and was repeated once a week, tumor volume and body weight were monitored every other day, calculated using the formula V = (length x width2) / 2, on the 21st day after tumor inoculation, blood tests and organ analysis were performed on mice, the main organs and tumors were fixed with 4% paraformaldehyde and HE staining and IHC staining were performed to evaluate histological changes and tumor cell proliferation.

[0117] On the 7th day after the establishment of the BT-20 breast cancer mouse model, laser treatment was performed on the six groups (PBS, SG, AIE780NP, AIE780NP+Laser, AIE780-SGNP and AIE780-SGNP+Laser; 5 mice per group), on the 21st day after BT-20 cell inoculation, all mice were sacrificed, single cell suspension was co-stained with V500-Aqua live / dead stain and labeled with antibody against CD49b-PerCP. For intracellular cytokine staining, tumor-infiltrating lymphocytes were first fixed and permeabilized according to the provided intracellular staining kit instructions, then labeled with antibodies against mouse GranzymeB-PECy7, Perforin-APC-Cy7 and IFN-y-V450 using standard methods, and immediately analyzed by flow cytometry, statistical analysis was performed using SPSS25. The sample size of each group was greater than or equal to 5, and the effect study was compared by analysis of variance (ANOVA). The two-sided LSD test was used to determine whether the difference between groups was statistically significant.

[0118] Results of the experiment

[0119] Optimizing photosensitizers to maximize ROS generation, enhance water-soluble stability and improve cellular uptake to trigger effective ICD (immune cell death) in TNBC remains a daunting challenge. To overcome these limitations, we generated the AIE780-SG construct by conjugating the humanized hRS7-targeting TROP2 to AIE780 nanoparticles (NPs). Dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis Figure 1A-B) showed that the average diameter of AIE780 NPs was 94.73 ± 1.37 nm, while that of AIE780-SG NPs was 113.24 ± 2.10 nm. These sizes are suitable for prolonged circulation and improved tumor accumulation, benefiting from the enhanced permeation and retention (EPR) effect. Drug loading resulted in a significant structural change without affecting the surface stability Figure 1 C), while the UV-Vis spectra in PBS showed the maximum absorption peaks at 605 nm (AIE780) and 635 nm (AIE780-SG) Figure 1 D). Under 660 nm near-infrared irradiation (power density of 2.0 W cm-2), AIE780-SG rapidly reached 45 °C within 2 min Figure 1 E-F). Compared with AIE780 NPs, the synthesized AIE780-SG NPs showed stable photothermal effect during the synthesis process, and there was no significant photothermal performance decline phenomenon. Systematically changing the irradiation power and NP concentration confirmed the direct proportional thermal response Figure 1 H-1). UV absorption assay determined the drug loading efficiency of AIE780-SG to be 10% Figure 1 J), pH- / temperature-dependent release studies showed that the SG release process was accelerated under acidic and high-heat conditions Figure 1 K-L). The hRS7-AIE780-SG nanosystem combined the optimal size for EPR-mediated tumor targeting, stable photothermal conversion, and the ability to trigger drug release under specific conditions, addressing the key obstacles encountered in ROS-driven ICD321 induction in TNBC treatment by simulating 320 tumor conditions.

[0120] The efficacy of the AIE780-SG construct depends on its efficient ADC internalization, given the inherent SG resistance of the BT-20 cell line,

[0121] Western blotting and flow cytometry analysis Figure 2 A-C) showed that the TROP2 level of BT-20 cells was significantly higher than that of MCF-10A control cells. In the drug resistance test of five TNBC cell lines, it was confirmed that cells with high TROP2 expression (especially BT-20) showed obvious resistance to SG monotherapy Figure 2 D) Fluorescence microscopy revealed that AIE780-SG NPs penetrated the cytoplasmic membrane and accumulated within the mitochondria, emitting strong red fluorescence, co-localized with DAPI and MitoTracker Green signals Figure 2E). In contrast, TROP2-low MCF-10A cells showed little nanoparticle uptake. Real-time intravital imaging captured the progressive internalization and mitochondrial localization of AIE780-SG in BT-20 cells after 4 hours ( Figure 2 F) These results demonstrate the ability of AIE780-SG to exploit tumor TROP2 expression to promote targeted delivery and precise mitochondrial localization. By leveraging the hRS7-mediated ADC internalization mechanism, AIE780-SG selectively targets TROP2-high-expressing TNBC cells, overcomes SG resistance, and achieves mitochondrial accumulation, as evidenced by expression analysis, drug resistance testing, and dynamic imaging.

[0122] Biological transmission electron microscopy analysis showed that 350 cell samples showed obvious morphological damage under AIE780-SG laser irradiation, including loss of membrane integrity, reduced mitochondrial volume, mitochondrial shrinkage and membrane condensation, which are characteristics of cell apoptosis ( Figure 3 A). To quantify ROS generation, a DCFH-DA probe was used. After BT-20 cells were treated with AIE780-SG+laser, their intracellular ROS levels were significantly increased compared to the control group ( Figure 3 B, Figure S2).

[0123] The cytotoxic mechanism of SG involves SN-38-mediated inhibition of topoisomerase I, induction of DNA strand breaks, and cell cycle arrest, ultimately leading to apoptosis. By combining photothermal therapy with the chemotherapeutic effects of SG, enhanced tumor killing effects were observed. Previous studies have reported that BT-20 and MDA-MB-231 cells may exhibit greater resistance to IMMU-132 compared with other triple-negative breast cancer (TNBC) cell lines. Flow cytometric analysis confirmed that BT-20 cells experienced a significantly increased rate of apoptosis after exposure to AIE780-SG+laser compared with SG alone or AIE780+laser. Figure 3 C). Western blot further revealed the apoptotic cascade process, showing that AIE780-SG irradiated the cells upregulated pro-apoptotic markers and downregulated BCL-2 ( Figure 3 D). Furthermore, assessment of damage-associated molecular patterns (DAMPs) associated with immunogenic death, such as HMGB1, HSP70, and HSP90, showed a significant increase in their release following AIE780-SG + laser treatment (compared to AIE780 + laser alone). Correspondingly, enhanced phosphorylation of eIF2-α—a key ICD signaling event—confirmed the role of ROS-driven mitochondrial stress in enhancing tumor immunogenicity.

[0124] Cytotoxicity assays revealed that there was little synergistic effect when free 372SG or AIE780 was combined with laser irradiation (Figure S3), whereas the AIE780-SG + laser group elicited strong cell death, highlighting the necessity of NP-mediated synergistic delivery. Finally, by laser-triggered SN-38 release, y-H2AX immunofluorescence (a marker of DNA double-strand breaks) was significantly elevated in cells treated with AIE780-SG + laser, indicating exacerbated DNA damage (Figure S4). Taken together, these data demonstrate that AIE780-SG NPs synergistically exploit both photodynamic and chemotherapeutic modalities to maximize tumor cell elimination while mitigating systemic toxicity.

[0125] To further validate the targeting accuracy of AIE780-SG, 384 triple-negative breast cancer PDX models (TNBC-PDOs) were employed, which faithfully recapitulate the tumor microenvironment in vivo and are invaluable for precision medicine, drug screening, and tumor immunology research. We established TNBC-PDOs and divided them into two subgroups, SG-sensitive and SG-resistant TNBC-PDOs (TNBC-PDO-S and TNBC-PDO-R, respectively) Figure 4 A-B) The successful generation of SG-resistant TNBC-PDOs (TNBC-PDO-R) was confirmed by HE staining and TROP2 immunohistochemistry Figure 4 C-D) TNBC-PDO-Rs showed significantly enhanced red fluorescence when co-cultured with AIE780-SG NPs, compared to organoids treated with AIE780 alone, indicating selective accumulation of NPs Figure 4 E) To assess the anti-tumor effect, TNBC-PDO-Rs were exposed to AIE780-SG for 432 h, followed by Annexin V / PI staining. Quantitative analysis showed a significant increase in early apoptotic (Annexin V+, green) and necrotic (PI+, red) cells, with the highest proportion of PI+ cells in the AIE780-SG + laser group Figure 4 F) AIE780-SG NPs selectively localized in TNBC-PDO-Rs and caused apparent apoptosis and necrosis under near-infrared activation, highlighting their robust anti-tumor activity in physiologically relevant three-dimensional models.

[0126] The enhanced tissue penetration provided by near-infrared luminescence for in vivo bio-distribution, tumor inhibition capability, and toxicity evaluation. Due to its emission in the near-infrared window, AIE780-SG was chosen for subsequent tumor visualization and therapy. BT-20 xenograft tumors were established by subcutaneous inoculation, followed by intravenous injection of AIE780-SG or AIE780 (at a concentration of 0.2 mg / mL). Luminescence imaging showed that AIE780-SG selectively accumulated at the tumor site, forming a sharp contrast with the surrounding tissue within 48 h. While unbound AIE780 NPs did not produce a detectable signal in the tumor area within 24 h (red dotted circles). Ex vivo luminescence analysis of removed tumors and organs confirmed that AIE780-SG was more inclined to remain at the tumor site than AIE780 Figure 5 A).

[0127] After verifying tumor targeting and photothermal conversion in vivo, the anti-tumor effect of BT-20 bearing mice was evaluated, which were randomly divided into six groups: PBS, SG, AIE780, AIE780 + laser, AIE780-SG, and AIE780-SG + laser. The maximum intratumoral NP concentration was observed 24 h after injection. Body weight and tumor size were monitored twice a day, and laser treatment was performed on the 7th and 15th days. Tumor growth curves and endpoint weights showed that the inhibitory effect of the SG, AIE780, and AIE780 + laser groups was relatively mild compared to the PBS control group. Importantly, the AIE780-SG + laser group exhibited significant tumor regression, confirming the therapeutic potential of combining photothermal therapy. After 21 days, tumor tissue sections from the AIE780-SG + laser group were subjected to

[0128] HE staining showed extensive necrosis and cell fragmentation. Immunohistochemical analysis showed a significant decrease in Ki-67 positive proliferating cells and an increase in TUNEL positivity, indicating enhanced apoptosis. Taken together, these results indicate that AIE780-SG is able to strongly inhibit the growth of TNBC by suppressing proliferation and promoting tumor cell death under near-infrared (NIR) activation. Therefore, AIE780-SG achieved selective NIR-I tumor imaging, sustained tumor retention, and provided a powerful photothermal and chemotherapeutic effect when combined with 660 nm irradiation, significantly inhibiting TNBC progression by reducing proliferation and enhancing apoptosis. Finally, a comprehensive safety evaluation was performed during the 21-day treatment regimen, including HE staining of major organs and serum biochemistry (ALB, ALT, ALP, BUN, CREA, UA), and no pathological abnormalities or deviations from normal reference ranges were found. Therefore, AIE780-SG NPs exhibit excellent safety features, demonstrating their great potential as a photothermal therapeutic agent. AIE780-SGNPs combined with photodynamic therapy to reduce the hypoxic state of tumors, enhance natural killer cell-mediated anti-tumor immune effects, and show significant effects in in vivo experiments. The combination of nanomaterials and phototherapy can reprogram the tumor microenvironment by regulating immune cell infiltration, thereby recruiting effector lymphocytes and eliminating immunosuppressive populations. Through light-induced hyperthermia and ROS generation, this strategy enhances the accumulation of cytotoxic T cells and NK cells inside the tumor. MICA / B expression was significantly up-regulated in AIE780-SG+Laser-treated tumors, indicating a mechanism for enhancing NK cell homing. CD16 (FcyRI I I), the most potent activating receptor for NK cells, can autonomously trigger degranulation after IgG binding. Specific antibody blockade of CD16 attenuated NK-mediated cytotoxicity, which was observed in both the SG and AIE780-SG groups, while CD107a up-regulation was reduced, while PBS and AIE780 treatment were not affected. In addition to NK cells, dendritic cells (DCs) also contribute to anti-tumor immunity. Bone marrow-derived DC analysis showed that CD80, CD86, and MHCII expression increased after AIE780-SG+Laser treatment, suggesting DC-mediated NK cell activation support. Indeed, analysis revealed the highest proportion of CD49b+NK cells in this population. Activated NK cells execute their killing function by secreting perforin, granzyme B, and interferon-y (IFN-Y)21,41AIE780-SG+Laser treatment not only expanded the NK cell population, but also increased the levels of these effector factors. NK cell activation is regulated by balanced signals between inhibitory and activating receptors, including NKp30, NKp44, NKp46, DNAM-1, and NKG2D. Confocal imaging confirmed increased NK cell surface expression of NKG2D and CD49b within the AIE780-SG+Laser group, and supported enhanced killing ability. Immunohistochemistry showed that AIE780-SG+Laser significantly reduced HIF-1a levels while up-regulating its pro-angiogenic target CD3, indicating that hypoxia was alleviated and vascular remodeling was improved.

[0129] The above description of disclosed embodiments allows a person skilled in the art to implement or use the invention. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to accord with the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A quinoline derivative for treating triple-negative breast cancer, characterized in that: The preparation of the quinoline derivative comprises the following steps: Step 1: Preparation of AIE780-SG NPs: 500 μg of AIE780COOH was placed in 9 mL of deionized water and shaken in an ultrasonic cell disruptor. THF was then evaporated with stirring at 25°C. After filtration, 2 mL of the NP suspension was diluted to 8 mL and reacted with 500 μg of SG using Sulfo-NHS and EDC as coupling agents at 25°C for 4 hours. The product was centrifuged to remove unbound reagents, followed by two additional PBS wash-centrifugation cycles. The final NPs were resuspended in 8 mL of PBS as a stock solution. Step 2, cell culture, breast cancer cell lines were collected and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, and BT-20 cells were cultured in a dedicated CM-0324 medium. The culture conditions included growing each cell line in an incubator with 95% humidity at 37° C. and an atmosphere of 5% carbon dioxide and 95% air; Step 3: Construct a 3D model by culturing tissue-specific adult stem cells or pluripotent stem cells in a 3D culture system to establish a 3D patient-derived organoid model; Step 4: Effect of endogenous AIE780-SGNPs in TNBC-PDOs. After TNBC-PDOs grew, the tissue spheres were collected and the Cell Recovery Solution was used to disaggregate Matrigel. 100 μl of AIE780 and AIE780-SG nanoparticles were added to the system and suspended in phosphate buffer. Mitochondria and nuclei were stained with the fluorescent dyes MitoFluorTM and DAPI, respectively, for 20 minutes. The internalization process of AIE780-SG nanoparticles was dynamically observed using a tabletop spinning disk scanning confocal microscope and its Z-stack model. Step 5, TNBC-PDOs viability detection. In the activity assay, TrypLE Express was used to dissociate the tissue spheres and suspended in Matrigel at a concentration of 2000 cells / well in each 96-well plate, with 4 μL Matrigel per well. After culture for 7 days, PDOs were treated with SG at concentrations of 0, 0.032, 0.16, 0.8, 4, 20, and 100 μM for 6 days. The images of the 96-well plates were captured using a microscope, the PDO spheres were thoroughly mixed, and 100 μL samples were extracted from each well for quantitative analysis. Using the CellCounting-Lite3D cell viability assay, 100 μL PDO sphere samples were dispensed into each well of a white opaque 96-well plate, and an equal volume of assay reagent was added to each well. The 96-well plate was then placed on a shaker for vigorous mixing for 5 minutes to promote cell lysis. After incubation at room temperature for 30 minutes to allow stabilization, the luminescence signal was measured. Step 6, TUNEL imaging: After dehydration, the mouse tissue was embedded in paraffin and cut into 4 μm sections. The sections were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% TritonX-100 for 20 minutes, and incubated with a reaction mixture containing terminal transferase at 37°C. Step 7, cytotoxicity test, NK cell cytotoxicity assessment was performed by flow cytometry detection for 6 hours; Step 8: Cytotoxicity assay. TNBC-PDOs and cells were exposed to 1 μg / mL SG, 1 μg / mL AIE780, or 1216 μg / mL AIE780-SGNPs, respectively, followed by PBS washing and irradiation with or without white light for 30 minutes. The survival rate of these cells relative to untreated cells was measured using a standard CCK-8 assay. Step 9: Flow cytometry was used to detect apoptosis. Cells were exposed to 1 μg / mL SG, 1 μg / mL AIE780, or 1 μg / mL AIE780-SG nanoparticles (NPs), respectively, and then washed with PBS. 2 Annexin V-FITC / PI and Annexin V-FITC / 7-AAD were used to quantitatively detect apoptosis and evaluate organoid damage. Step 10: Protein detection and quantification using Western blotting.

2. A quinoline derivative for treating triple-negative breast cancer according to claim 1, characterized in that: The ultrasonic cell disrupter used in the preparation of the AIE780-SG NPs was model SCIENTZ-11D, with a power of 80%, a vibration time of 1 minute, a pore size of the filter membrane of 0.2 μm, a weight of Sulfo-NHS of 17.4 μg, and a weight of EDC of 15.3 μg.

3. A quinoline derivative for treating triple-negative breast cancer according to claim 1, characterized in that: The size and ζ potential of AIE780 nanoparticles and AIE780-SG nanoparticles in the preparation of AIE780-SG NPs were evaluated using a potentiometer at 25°C, the structure of IT-PEG-RGD nanoparticles was examined by using a FEITecnai transmission electron microscope (TEM) and operated at 120 kV, and the fluorescence spectrum of AIE780-SG nanoparticles was recorded by a Horiba Fluorolog-3 fluorometer using a tetrad cuvette.

4. A quinoline derivative for treating triple-negative breast cancer according to claim 1, characterized in that The cells in the cell culture include MCF10A, MDA-MB-231, Hs578T, BT549 and BT-20.

5. A quinoline derivative for treating triple-negative breast cancer according to claim 1, characterized in that: The construction of the three-dimensional model comprises the following steps: Step 31: Fresh tumor tissue obtained during surgery was immediately immersed in DMEM supplemented with 2% penicillin / streptomycin. The tissue was cut into 1 mm thick slices and enzymatically digested in DMEM / F12 medium containing 3 mg / ml type I and type IV collagenase at 37°C for one hour. Step 32: After digestion, the cell suspension was filtered through a 70 μm sieve and washed twice with culture medium; In step 33, 15,000 cells and 30 μl of Matrigel were evenly distributed into each well of a 24-well plate. The plate was then inverted and incubated in a column oven for 0.5 h. After the Matrigel solidified, 500 μl of culture medium was added. The culture medium was subsequently changed every two days for one week.

6. A quinoline derivative for treating triple-negative breast cancer according to claim 1, characterized in that The CellCounting-Lite3D used in the TNBC-PDOs viability assay needs to be thawed at room temperature.

7. A quinoline derivative for treating triple-negative breast cancer according to claim 1, characterized in that: The cytotoxicity test comprises the following steps: Step 71: Label the tumor cells with CellTracker CM-Di 1 fluorescent dye and culture them overnight; Step 72: Co-culture with NK cells in duplicate at different effector to target ratios. After 4 hours of co-culture, measure cell viability using Live / DeadFixable V500-Aqua dead cell staining reagent. Step 73: Data were collected on a Canto II flow cytometer and analyzed using FlowJo software version 10.

8. The quinoline derivative for treating triple-negative breast cancer according to claim 7, characterized in that: The different effector to target ratios were 0.25:1, 0.5:1, 1:1 and 2:

1.

9. The quinoline derivative for treating triple-negative breast cancer according to claim 1, characterized in that The protein detection and quantification comprises the following steps: Step 101, BT-20 cells were seeded in a 6-well plate and left for 12 hours. After incubation with 1 μg / mL SG, 1 μg / mL AIE780NPs, or 1 μg / mL AIE780-SGNPs, the cells were washed with PBS and irradiated with 3 mW / cm2 white light for 30 minutes. After 30 minutes, the cells of each group were collected and lysed with RIPA buffer, and the total protein concentration in these lysates was determined by BCA assay; Step 102: 20 μg of protein is loaded onto an SDS-PAGE gel and the proteins are separated by gel electrophoresis. Step 103: Transfer to a Millipore PVDF membrane, incubate with 5% skim milk in 1xTBST for 60 minutes, and then incubate the membrane with specific primary antibodies in 5% bovine serum albumin in 1xTBST for 12 hours at 4°C. Step 104: After washing five times with 1×TBST, add 1×TBST containing HRP-conjugated secondary antibody and incubate at room temperature for 2 hours; Step 105: After washing three times with PBS, the protein bands on the membrane were visualized by ECL on a BioRad imaging system and quantified using ImageJ software.

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