Co-delivery Of Gambogic Acid And Indocyanine Green By Nanostructured Lipid Carriers Induces Pyroptosis For The Treatment Of Cervical Cancer

Objective:Programming pyroptosis exhibites great potential for clinical application in the treatment of malignant tumors.In this study,photodynamic therapy（PDT）combined with natural active ingredients was used to trigger pyroptosis of cervical cancer and discover new strategies for cervical therapy.In order to overcome the limitation of drug application in clinical application and develop a robust pyroptosis inducer,nanostructured lipid carriers（NLC）were used to co-encapsulate gambogic acid（GA）and indocyanine green（ICG）,and modified tumor-targeting peptide c TMTP1,thus the c TMTP1-GA/ICG-NLC（referred as TP-NLC）nano-carriers were constructed.By combination of TP-NLC+laser irradiation（TP-NLC+L）with anti-programmed cell death-1（anti-PD-1）antibody,an immune checkpoint site inhibitor,this study aimed to explore new strategies for cervical cancer therapy.Methods:The morphological alterations of a total of 18 cells from 7 cancer types including ovarian,breast,lung,liver,cervical,colorectal and pancreatic cancers after exposure to GA were monitored by light microscopy,and intracellular adenosine triphosphate（ATP）levels,lactate dehydrogenase（LDH）release and the number of Annexin V-FITC/PI positive cells were also measured in the colorectal cancer cell lines,HCT116 and CT26.RNA sequencing was performed to detect the effect of GA treatment on cellular gene expression profiles.The expression levels of pyroptotic executive proteins（GSDMD and GSDME）and apoptosis-related protein（cleaved caspase-3）were assessed by western blotting analysis.The si RNA technology was used to interfere with the expression of GSDME,and its effect on GA-induced pyroptosis was further observed.HCT116 and CT26 cells cells were pretreated with Z-DEVD-FMK,a caspase-3 inhibitor,to evaluate the effect of inhibition of caspase-3 activation on GA-induced pyroptosis.CT26 tumor-bearing mouse models were established to evaluate the anticancer activity of GA on colorectal cancer in vivo.The c TMTP1-modified TP-NLC was prepared for co-delivery GA and ICG by the emulsification-solvent evaporation method,and the physicochemical properties of TP-NLC were measured.Near infrared absorption and anti-photobleaching of TP-NLC were determined by ultraviolet visible（UV-Vis）spectrophotometer.The morphological alterations were monitored by light microscopy to assessed the effect of TP-NLC on cervical cancer cells under 808 nm near-infrared（NIR）laser irradiation,and intracellular ATP levels and LDH release were also assessed by ATP Assay Kit and LDH assay kit.The number of Annexin V-FITC/PI positive cells was monitored by flow cytometer.The GSDME and cleaved-caspase-3 protein expressions and intracellular reactive oxygen species（ROS）levels were also measured.TC-1tumor-bearing mice models were established to evaluate the anticancer activity of TP-NLC+L.CD3⁺CD8⁺and CD8⁺PD-1⁺T cells in the tumor tissues,dendritic cells（DCs）in the tumor draining lymph nodes（TDLNs）and effector memory T cells(T_EM)（CD3⁺CD8⁺CD44⁺CD62L^-）in the spleen were assessed and analysed.The subcutaneous tumor model of mice was established to evaluate therapeutic effect on cervical cancer by combination of TP-NLC+L with anti-PD-1 antibody,and the immune cells in the tumor tissues and spleen were detected.The bilateral subcutaneous tumor model of mice was constructed to evaluate the therapeutic effect on distant tumors by combination of TP-NLC+L with anti-PD-1 antibody.Results:GA could induce pyroptosis of multiple cancer cells,and the expression of GSDME-N was observed in these cells.The expression of GSDME-N and cleaved caspase-3 were detected in the colorectal cancer cells（HCT116 and CT26）after exposure to GA.RNA-seq results showed that apoptosis-and pyroptosis-related genes were found in the differentially expressed genes（DEGs）,and enrichment analysis of DEGs revealed that they could be enriched in cell cycle,DNA damage,apoptosis and apoptosis-related signaling pathways such as P53 and MAPK signaling pathway.Knockdowning GSDME expression or inhibiting caspase-3 activation by Z-DEVD-FMK could reverse GA-induced pyroptosis in the colorectal cancer cells,indicating that GA-induced pyroptosis was dependent on the both activation of caspase-3 and execution of GSDME.The results of animal experiments demonstrated that the treatment with GA at both the low-dose and high-dose significantly reduced the volumes and weights of colorectal cancer.The TP-NLC of about 70 nm was successfully synthesized,and the entrapment efficiencies of GA and ICG in TP-NLC were above 95%.Moreover,TP-NLC under 808 nm NIR laser irradiation kept the stability of ICG against photo-bleaching.TP-NLC+L could enhance generation of ROS to trigger pyroptosis via caspase-3/GSDME signal pathway in He La and TC-1 cells,and increase cleaved caspase-3 and GSDME-N proteins expression.TP-NLC+L could inhibit tumor growth,promote an increase in the proportion of CD3⁺CD8⁺T cells in tumor tissues,induce maturation of DCs in tumor-draining lymph nodes,and stimulate an increase in the ratio of T_EM in the spleen under 808 nm NIR light irradiation in vivo.The combination of TP-NLC+L and anti-PD-1 antibody significantly inhibited the growth of cervical cancer,increased the proportion of CD3⁺CD8⁺T cells in tumor tissues.Moreover,the combination of TP-NLC+L with anti-PD-1 antibody could inhibite distant tumors of cervical cancer,and increased the proportion of CD3⁺CD8⁺T cells in distant tumors,suggesting that the strategy could activate systemic anti-tumor immune responses.Conclusions:GA induced pyroptosis of cancer cells through the caspase-3/GSDME dependent pathway.TP-NLC,which actively targeted TC-1-bearing subcutaneous tumors,could trigger pyroptosis of cervical cancer cells under 808 nm NIR laser irradiation,inhibit the progression of cervical cancer,and activate anti-tumor immune responses.Moreover,the combination of TP-NLC+L with anti-PD-1 antibody significantly inhibited distant tumors via activating systemic anti-tumor immune responses.In summary,the combination of TP-NLC+L mediated pyroptosis with anti-PD-1 antibody offers an attractive therapeutic strategy for cervical cancer therapy.