The Study Of AIE Mitochondrial Targeting Probe For Anti-breast Cancer Immunotherapy

Chapter One:Comparison of three commercial photosensitizers for inducing ICD in anti-breast cancer immunotherapyObjective To investigate the photosensitive properties,Photodynamic therapy(PDT)and Immunogenic Cell Death(ICD)induction capability of Chlorin e6(Ce6),Neutral Red(NR),Rose Bengal Sodium salt(RB),the most efficient ICD inducer was selected for antitumor immunotherapy.At the same time,it provides reference for evaluation of the merits and demerits of aggregation induced emission(AIE)mitochondrial targeting probes.Methods Reactive Oxygen Species(ROS)probes were used to evaluate the photosensitivity of the three photosensitizers in the solution and tumor cells.The cytotoxicity test of CCK-8compared the cytotoxicity effect.The ICD-inducing ability was evaluated by measuring the content of DAMPs.BALB/c mouse models bearing 4T1 cell derived subcutaneous tumor were given intratumoral injection of Ce6 or NR solution(30μL,5 mg/m L),followed 2 h later by white light irradiation for 10 min(400 m W/cm~2).Body weight and tumor size changes of the mice were monitored,and the percentage of CD8~+T cells in the tumor and IFN-γ~+CD8~+T cells in the spleen were analyzed by flow cytometry 14 days after the treatment.HE and TUNEL staining was used to analyze tumor cell apoptosis in the mice.Results Among the 3 photosensitizers,Ce6 exhibited the strongest ROS-inducing capability and killing effect on the tumor cells.The results of ecto CRT,HMGB1 and ATP level detection all demonstrated a stronger ICD induction ability of Ce6.In the tumor-bearing mice,the tumor growth in Ce6 and NR groups was significantly inhibited after the treatment.The percentages of CD8~+T cells and IFN-γ~+CD8~+T cells were 12.7%and 7.1%in Ce6 group,respectively,significantly higher than those in NR group(6.1%and 2.8%,respectively;P<0.05).HE and TUNEL staining revealed obvious tumor cell apoptosis in the tumor tissues in both Ce6 and NR groups,but the therapeutic effect was more prominent in Ce6 group.Conclusion Among the 3 photosensitizers,Ce6 has the highest efficiency for inducing ROS production with the strongest PDT efficacy and ICD induction capability.Ce6 can also increase the number and function of CD8~+T cells in anti-tumor immunotherapy to initiate robust adaptive immune response.Therefore,Ce6 can also be used as a mature ICD inducer to evaluate the properties of AIE mitochondrial targeting probes.Chapter Two:The study of AIE mitochondrial targeting probe of inducing ICD for anti-breast cancer immunotherapyObjective To compare the photosensitive properties,PDT effect and ICD inducing ability of AIE mitochondrial targeting probe and Ce6,in order to find a better ICD inducer for anti-breast cancer immunotherapy.Methods Solution experiment:The photosensitizing effects of AIE probe and Ce6 were evaluated using different ROS fluorescence probes.Cell experiment:The ROS production capacity in breast cancer cells was detected by the probe;cytotoxicity assay of CCK-8 was used to compare the cytotoxic effect and biocompatibility to evaluate its PDT effect;the AIE probe and mitochondria were colocalizated;extracellular exposure to ecto-CRT,HMGB1 and ATP secreted in the supernatant was determined to evaluate the ICD-inducing ability of the two drugs.Results The solution experiment showed that AIE probe had better ROS generation ability than Ce6.The cytotoxicity of CCK-8 showed that the phototoxicity of AIE was weaker but the dark toxicity was smaller than that of Ce6.The results of co-localization with mitochondria showed that AIE probe could target mitochondria,while Ce6 had no targeting ability.Ecto-CRT,HMGB1 fluorescence imaging and extracellular ATP content showed that AIE probe had stronger ICD induction ability than Ce6.Conclusion AIE probe has better ROS generation ability,weaker cytotoxicity and higher biocompatibility than Ce6,and has better ICD induction effect.