Mitochondria And Plasma Membrane Dual-targeted Chimeric Peptide For Single-agent Synergistic Photodynamic Therapy

Photodynamic therapy?PDT?is a promising anti-epidermal tumor interventional treatment strategy.Due to its minimally invasive,low toxicity,high efficiency and controllable characteristics,it shows great advantages in tumor collaborative treatment.In the process of photodynamic therapy,singlet oxygen?¹O₂?produced by the action of photosensitizer and oxygen is widely regarded as a key factor in photodynamic therapy.It is well known that whether a pharmacologically active molecule interacts with a target is a key factor for a drug to fully exert its therapeutic effect.The subcellular organelle distribution of drugs affects the therapeutic effect of drugs,and precise treatment can be achieved through the regulation of the subcellular organelle distribution of drugs.Similarly,the subcellular organelle distribution of photosensitizers will also affect the effect of drugs.Because of the short half-life of ¹O₂?<40 ns?and limited diffusion distance?<20nm?,it can only act at the site of formation.The traditional photosensitizer has no targeting,which reduces the efficacy of photodynamic therapy and even causes damage to normal tissues.Therefore,it is particularly important to achieve precise tumor treatment by regulating the distribution of sub-organelles of photosensitizers.However,no matter whether the reported cell membrane targeted photodynamic therapy or mitochondrial targeted photodynamic therapy,these single subcellular organelle targeting strategies have not exerted the greatest therapeutic effect.Obviously,the combination of cell membrane targeted photodynamic therapy and mitochondrial targeted photodynamic therapy may be a more effective strategy to eliminate tumors,and this simple dual-targeted combination strategy is rarely reported.Objective:In this study,a mitochondrial and cell membrane dual-targeted self-delivery chimeric peptide?PpIX-KrFxrFxrFxr-PEG8,M-ChiP?was prepared for the study of tumor photodynamic therapy.To investigate whether the dual subcellular organelle targeting function of the chimeric peptide cell membrane and mitochondria can achieve tumor cooperative photodynamic therapy to achieving the role of precise tumor treatment in vitro and at animal level.Methods:1.Characterize the structure of M-Chi P by measuring its particle size,electron microscope and ultraviolet.2.Singlet oxygen sensor green?SOSG?was used to determine the ¹O₂ production capacity of M-ChiP,and the fluorescent dye DCFH-DA was used as the detection indicator to detect the generation of ROS.3.Using cell membrane green fluorescent probe?Dio?,lysosomal green fluorescent dye?Lyso Tracker Green?,mitochondrial green fluorescent dye?Mito Tracker Green?and nuclear dye?Hoechst 33342?to study the distribution characteristics of M-ChiP in 4T1cells.4.Use trypan blue as the detection indicator to observe the integrity of the cell membrane with an inverted microscope,use rhodamine 123 as the detection indicator with a confocal laser microscope to observe the membrane potential of mitochondria,and quantitatively analyze it by flow cytometry.In this way,the subcellular destruction ability induced by the photodynamic therapy of M-ChiP under laser irradiation was evaluated.5.The anti-tumor effect of dual-targeted M-ChiP was studied in vitro using MTT method,flow cytometry and double-stained detection kit for living and dead cells.6.Construct an animal model of breast cancer in mice and study the in vivo distribution and antitumor activity of M-ChiP.Results:1.Dynamic light scattering results show that the nanoparticles have a hydrodynamic size of approximately 167.9 nm.Electron microscopy results showed that the nanoparticles were evenly dispersed.The ultraviolet spectrum shows that M-ChiP has a sharp peak at400 nm and characteristic absorption peaks at 530 nm,580 nm and 630 nm.2.SOSG detects the generation of singlet oxygen ¹O₂,and the fluorescence intensity of SOSG in the M-Chi P+light group receiving light is significantly enhanced.Similarly,using DCFH-DA as a ROS sensor to detect the production of ROS in cells,similar results were found.3.DiO,MitoTracker Green's green fluorescence and M-Chip's red fluorescence can coincide well and most LysoTracker Green's green fluorescence and M-ChiP's red fluorescence overlap to a certain extent,while M-ChiP's red fluorescence There is almost no overlap with the blue fluorescence of Hoechst 33342.4.The cells in the blank group and the cells in the M-ChiP group that were not irradiated with laser light did not stain with trypan blue,while the cells in the M-ChiP+Light group that received light turned blue.In the absence of laser irradiation,M-ChiP-treated4T1 cells showed obvious green fluorescence,while the laser-irradiated M-Chi P group showed almost no fluorescence in 4T1 cells in this group.5.MTT results showed that M-ChiP had concentration-dependent phototoxicity to 4T1cells and showed significantly lower dark toxicity.The results of flow cytometry showed that after 30 s of laser irradiation,more than half of 4T1 cells were in early or late apoptosis.The results of live/dead cell detection showed that after 30s of laser irradiation,4T1 cells incubated with M-ChiP showed obvious PI red fluorescence.6.At 12 hours,the accumulation of red fluorescence of M-ChiP at the tumor site reached the highest level,and the fluorescence at the tumor site gradually decreased because of metabolic reasons.The tumors of mice in the M-Chi P+Light group receiving laser irradiation were significantly inhibited and showed good anti-tumor effects.Blood routine examination found no abnormalities and no significant difference in biochemical test results.H&E staining results showed that the main organs of mice after receiving M-ChiP photodynamic therapy showed no obvious physiological morphological changes.Conclusion:1.Transmission electron microscopy observation and dynamic light scattering results prove that we have successfully prepared uniformly dispersed nanoparticles.The ultraviolet spectrum results show that M-ChiP has the ultraviolet absorption properties of the photosensitizer PpIX.2.Compared with the free photosensitizer PpIX,M-ChiP can produce more 1O2 under laser irradiation.Using DCFH-DA as a ROS sensor to detect the generation of ROS in cells,similar results were found,indicating the great potential of M-ChiP in tumor photodynamic therapy.3.M-ChiP has the targeting ability of cell membrane and mitochondria.4.The ROS produced by M-ChiP anchored on the cell membrane and mitochondria under laser irradiation will destroy the integrity of the cell membrane and mitochondria.5.M-ChiP significantly amplifies the degree of photodamage through the dual targeted positioning effect of cell membrane and mitochondria,and obtains a highly effective in vitro synergistic anti-tumor effect.6.6.M-ChiP can be enriched in tumor tissues through the EPR effect,and tumor growth inhibition can be effectively achieved through photodynamic therapy.At the same time,M-ChiP also has good biocompatibility and biosecurity.