| Background and Objective Breast cancer,a prevalent malignancy among women,has a high mortality rate due to its rapid progression and recurrence following treatment.Early-stage breast cancer patients undergo surgery and receive adjuvant therapies such as radiotherapy and chemotherapy based on their tumor stage.However,these treatments are not sufficiently effective and pose a serious threat to women’s life and health.Chlorin e6,(Ce6)is an algal-derived second-generation photosensitizer with a high singlet oxygen generation efficiency.It has been extensively applied in photodynamic therapy for various tumors.Nevertheless,its clinical use is hampered by its hydrophobicity.Cytarabine(Cytarabine,Ara-C)is the first marine natural anticancer agent isolated from deep-sea sponges,but it exhibits poor stability,rapid metabolism in vivo,and low efficacy against solid tumors.Therefore,developing novel strategies to overcome the drawbacks of Ce6 and Ara-C is crucial for expanding the therapeutic options for breast cancer.Pyroptosis is a newly discovered mode of cell death that has pro-inflammatory effects and can remodel the tumor immune suppressive microenvironment.Breast cancer cells tend to acquire resistance to apoptosis during prolonged chemotherapy,which facilitates the establishment of an immunosuppressive milieu in the tumor site.Hence,inducing pyroptosis in breast cancer cells and converting them from “cold tumors” to “hot tumors” is a promising approach to reverse the immunosuppression and enhance the therapeutic outcome of breast cancer.In this project,we aim to use nanotechnology to develop a nanoplatform that can overcome the limitations of Ce6 and Ara-C and synergize their chemotherapeutic and photodynamic effects.This nanoplatform can induce pyroptosis in breast cancer cells and activate anti-tumor immunity both intrinsically and extrinsically,thereby achieving immunotherapy and inhibiting the rapid growth and recurrence of breast cancer.Materials and methodsPart 1(1)MTT method was used to detect the effect of Ce6 on breast cancer cell viability.(2)Cloning formation experiments were applied to clarify the effect of Ce6 on the proliferation of breast cancer cells in vitro.(3)4T1 breast cancer-bearing mouse model was constructed to observe tumor size,detect tumor volume,tumor weight and tumorinhibition rate to clarify the in vivo anti-tumor effect of Ce6.Part 2(1)A-C/NPs were prepared with appropriate concentrations by screening different mass ratios of Ce6 and Ara-C.(2)The size and zeta potential of A-C/NPs were measured by dynamic light scattering(DLS),and their morphology and structure were observed by transmission electron microscopy(TEM).The UV-Vis absorption spectra of A-C/NPs were recorded by a UV-Vis spectrophotometer.(3)The release behavior of A-C/NPs was investigated under different p H conditions.(4)MDA-MB-231,MCF-7,and 4T1 breast cancer cells were used as cell models to evaluate their uptake of AC/NPs.(5)The in vitro photodynamic ability of A-C/NPs was assessed by using DPBF as a probe.(6)The in vitro photothermal potential of A-C/NPs was verified by an infrared thermal imaging system.Part 3The effects of A-C/NPs on breast cancer cells were evaluated as follows:(1)The cell viability was measured by MTT assay.(2)The cell proliferation was assessed by Ed U incorporation assay and colony formation assay.(3)The cell killing ability was detected by AM/PI live/dead cell staining.(4)The cell apoptosis was evaluated by Annexin VFITC/PI staining and flow cytometry analysis.Part 4The following experiments were conducted:(1)The morphological changes of breast cancer cells were observed by inverted microscope and SYTOX staining.(2)The levels of pro-inflammatory cytokines NLRP3,IL-18,and IL-1β in the cell supernatants were measured by ELISA after A-C/NPs treatment.(3)The lactate release was detected by LDH assay kit.(4)The expression of pyroptosis-related key protein GSDME was detected by western blotting.(5)The subcellular localization of A-C/NPs was verified by Mito Tracker staining.(6)The ROS level was detected by fluorescence quantification and flow cytometry.(7)The role of GSDME as a key molecule for pyroptosis induction was confirmed by si RNA rescue experiment.(8)The immunogenic cell death induced by A-C/NPs was confirmed by immunofluorescence and ATP detection.(9)The splenocytes from mice were extracted and the differentiation of CD4 and CD8 T cells was determined by flow cytometry.Part 5The following experiments were conducted:(1)The in vivo distribution and targeting of A-C/NPs were determined by small animal imaging.(2)The in vivo anti-tumor effect of A-C/NPs was verified by establishing a mouse model of breast cancer orthotopic tumor.(3)The morphological changes of tumor tissues and major organs were observed by H&E staining after A-C/NPs treatment.(4)The levels of pro-inflammatory cytokines NLRP3,IL-18,and IL-1β in the serum were measured by ELISA.(5)The effects of A-C/NPs on cell pyroptosis and proliferation were determined by immunohistochemistry(IHC).(6)The liver and kidney function biochemical indicators were detected to evaluate the safety of A-C/NPs.Part 6The following experiments were conducted:(1)A mouse model of breast cancer bilateral tumor was established to verify the inhibitory effect of A-C/NPs on distant tumor.(2)The stimulation effect of A-C/NPs on CD4 and CD8 T cells in the tumor tissue and spleen was detected.(3)A mouse model of breast cancer recurrence was established to confirm the role of A-C/NPs in inhibiting breast cancer recurrence.(4)The ratio of central memory T cells and effector memory T cells in the splenocytes was detected to confirm the role of A-C/NPs in activating immune memory and inhibiting tumor recurrence.ResultsPart 1The following results were obtained:(1)Ce6 exhibits a certain degree of inhibitory effect on the in vitro viability and proliferative capacity of breast cancer cells.(2)Ce6 is capable of suppressing the in vivo growth of tumor cells in the 4T1 tumor-bearing mouse model,but the efficacy is limited.Part 2The following results were obtained:(1)A-C/NPs were self-assembled at an AraC:Ce6 ratio of 5:1.(2)A-C/NPs had a quasi-spherical shape,a particle size of 142 nm,a negative potential,and a UV absorption wavelength at 660 nm.(3)A-C/NPs exhibited p H-responsive characteristics.(4)Breast cancer cells showed good uptake of A-C/NPs,reaching a peak at 4 h.(5)A-C/NPs had excellent singlet oxygen generation capacity and did not produce extra heat under laser irradiation.Part 3The following results were obtained:(1)A-C/NPs inhibited the viability and induced the in vitro death of breast cancer cells under near-infrared light irradiation,and also inhibited their in vitro proliferation and induced apoptosis.Part 4The following results were obtained:(1)A-C/NPs induced morphological changes in breast cancer cells,such as swelling and foaming,and promoted the release of inflammatory cytokines NLRP3,IL-18,IL-1β and LDH,triggering the cleavage of pyroptosis key protein GSDME and thus inducing cell pyroptosis.(2)A-C/NPs colocalized with mitochondria and induced oxidative stress in cells,causing mitochondrial dysfunction.(3)A-C/NPs triggered cell pyroptosis by increasing intracellular ROS levels.(4)A-C/NPs promoted ATP release and CRT translocation,causing immunogenic cell death of breast cancer cells.(5)A-C/NPs promoted the differentiation of CD4 and CD8 T cells and activated anti-tumor immunity.Part 5The following results were obtained:(1)A-C/NPs accumulated in the tumor site and prolonged their retention time in the tumor.(2)A-C/NPs inhibited the growth of mouse orthotopic tumors.(3)A-C/NPs inhibited tumor cell proliferation and triggered tumor pyroptosis in vivo.(4)A-C/NPs had good biosafety.Part 6The following results were obtained:(1)A-C/NPs inhibited the growth of mouse distal tumors.(2)A-C/NPs activated anti-tumor immunity in mice.(3)A-C/NPs prevented breast cancer recurrence.(4)A-C/NPs enhanced immune memory and thus prevented breast cancer recurrence.Conclusion1.The self-assembly synthesis method of A-C/NPs improved the stability of Ara-C and the hydrophobicity of Ce6,increased bioavailability,and reduced toxic side effects.2.A-C/NPs promoted ROS accumulation and triggered GSDME-mediated cell pyroptosis,thus showing efficient inhibition effects on breast cancer in vitro and in vivo.3.A-C/NPs activated anti-tumor immunity by releasing DAMPs through cell pyroptosis,thus inhibiting breast cancer growth in vitro and in vivo.4.A-C/NPs inhibited the progression and recurrence of bilateral tumors by activating anti-tumor immunity and immune memory. |
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