Cholangiocarcinoma Photodynamic Therapy Experimental Study Of Innovative Photosensitizer DCFP-Cl Based On Targeting Mitochondria

Objective: To investigate the biosafety and effectiveness of a new photosensitizer,DCFP-Cl,in targeted mitochondrial photodynamic therapy for bile duct cancer cell lines through in vivo and in vitro experiments.Methods :1.DCFP-Cl was co-cultured with human hepatobiliary duct cell line RBE to detect DCFP-Cl uptake and fluorescence imaging ability of RBE cells,and Mito-Tracker Green was introduced to observe mitochondrial targeting of DCFP-Cl.2.Calcein-AM kit was used to detect the cell activity of RBE after photodynamic therapy,and DCFH-DA was used to detect the production of monomer oxygen in cells after treatment.3.CCK-8 kit was used to quantitatively evaluate the dark toxicity and photodynamic efficiency of DCFP-Cl on cells,and flow cytometry was used to observe the mode of DCFP-CL-mediated photodynamic action.4.Dynamic in vivo fluorescence imaging was performed by injecting DCFP-Cl into RBE subcutaneous tumor bearing nude mice via tail vein to observe the in vivo fluorescence imaging ability and observe the uptake effect of DCFP-Cl in subcutaneous tumor tissue.5.Photodynamic treatment was performed on subcutaneous nude mice with RBE tumor,and weight and tumor volume of nude mice in each group were measured.After the experiment,all nude mice were killed,and major organs and tumors were removed for H&E and TUNEL staining.Results:1.In the uptake experiment,distinct red fluorescence was observed in cells incubated with DCFP-Cl;in the DCFP-Cl sublocalization experiment,a strong correlation coefficient of 0.78±0.07 was found between the red fluorescence of intracellular DCFP-Cl and the green fluorescence of the mitochondrial probe.2.Cell viability test results demonstrated that after various treatments,all groups were incubated with Calcein AM.The results indicated that cells in the control group and those only incubated with DCFP-Cl displayed prominent green fluorescence,while no noticeable green fluorescence was observed after irradiation by the photodynamic therapy device.In the reactive oxygen assay,DCFH-HA was incubated in all groups,and only bile duct cancer cells exhibited significant green fluorescence following photodynamic therapy.3.In the CCK-8 cytotoxicity test,the survival rate of RBE cells incubated with DCFPCl did not change significantly with increasing concentration gradient under dark conditions and remained above 90%.Meanwhile,the survival rate of cells in the treatment group decreased with increasing concentration,with an IC50 of DCFP-Cl at 7.12μM.FACS analysis revealed that PDT-induced cell death by DCFP-Cl was primarily apoptotic.The apoptosis rate of cells treated with light for 10 min and 20 min showed considerable necrosis compared to the control group(F=119.2,P<0.001).The overall apoptosis rate increased with the duration of irradiation(t=5.726,P<0.05),reaching 44.1±1.52% after20 min of 630 nm light exposure.4.In vivo fluorescence imaging results indicated that DCFP-Cl started to accumulate in the tumor area 2 hours after tail vein injection in tumor-bearing nude mice,with the optimal aggregation effect at 8h and fluorescence at the tumor site maintained up to 24 h.5.Following DCFP-Cl ingestion and light exposure,the residual tumor size was significantly smaller than in the other three groups(F=26.82,P<0.001),with scar tissue observed after the loss of necrotic tissue.H&E staining revealed no distinct necrosis of tumors in the four groups,with only an incomplete envelope observed in the DCFP-Cl/light group.Subsequent TUNEL staining displayed evident apoptosis at the residual tumor edge solely in the DCFP-Cl/light group,while no obvious abnormalities were detected in other groups.Simultaneously,H&E staining showed no damage to the major organs in each group.Conclusion:Based on the photosensitizer DCFP-Cl’s characteristics of thermally activated delayed fluorescence and mitochondrial targeting,it demonstrated low dark biotoxicity,strong fluorescence imaging capability,stability,and outstanding anti-tumor activity of PDT in bile duct cancer cells both in vitro and in vivo.This suggests potential integration with tumor diagnosis and treatment.Therefore,this novel photosensitizer has promising clinical application prospects.