Self-assembly Nanomedicine For Synergistic Photodynamic Therapy Cancer Therapy By Induced Autophagy

Autophagy refers to the self-digestion process in which cells use lysosomes to degrade damaged,denatured or aging macromolecules and organelles under the influence of external environmental factors.Autophagy enables these substances to be converted into reusable nutrients,thus reducing all kinds of cell damage.In the process of using photodynamic therapy（PDT）to treat cancer,cancer cells are stimulated by reactive oxygen species（ROS）,which will lead to autophagy.This protective autophagy will promote the survival of cancer cells and reduce the therapeutic effect of PDT.However,excessive activation of autophagy can promote cell apoptosis.In this paper,the photosensitizer pyropheophorbide-a（Ppa）was used to produce a large amount of reactive oxygen species to achieve the effect of killing cancer cells.At the same time,icaritin（Ica）,an autophagy inducer,was used to over-activate autophagy,which transformed the protection of cancer cells into the promotion of cancer cell apoptosis,so as to improve the effect of photodynamic therapy.However,the use of conventional carriers to deliver Ppa and Ica simultaneously to the tumor site will inevitably bring about some toxic side effects.Self-assembled nanomaterials rely on the interaction between small molecules of drugs to assemble into nano particles,and can deliver drugs to the tumor region without additional carriers,with high drug loading capacity and high biological safety.In this study,the interaction force between Ica and Ppa was exploited to successfully construct a self-assembled nanomedicine IP with good stability and high drug load.The synthesis method is simple,through using the drug itself as a carrier,and the loading efficiency（LE）of Ica and Ppa can be increased to 83.53%and 16.45%without introducing potential biosafety risks of nanocarriers.Compared with free Ppa,self-assembled nanomedicine IP showed superior performance in cellular uptake and reactive oxygen species production.In addition,the self-assembled nanomedicine IP can reverse the protective autophagy induced by PDT by activating the autophagy of tumor cells,and facilitate apoptosis and antitumor coordination,which significantly improves the antitumor activity of PDT.Objective:In this study,the autophagy inducer Icaritin and the photosensitizer Pyropheophorbide-a were prepared into nanomedicines through self-assembly,so as to induce excessive autophagy in cancer cells and synergically enhance the anti-tumor effect of photodynamics.First,focus on whether the magnesium Icaritin and Pyropheophorbide-a can form nanomedicines for testing,and detect the assembly mechanism between the two.Exploring the stability of nanomedicines and the drug load rate,it clarifies that the self-assembled nanomedicines has high stability and high-loading rate compared to free drugs.Investigation of nanomedicine in the aspects of cell intake and the ability to produce activated oxygen highlights that nanomedicines have the advantages of being susceptible to cell intake and high-efficiency active oxygen.Exploring the promotion effect of nanomedicines on the autophagy of cancer cells,as well as the killing effect of tumor cells after coordinated treatment of nanomedicines treatment and chemotherapy.Focus on clarify the synergy relationship between tumor cell optical dynamics and excessive autophagy,and then reveal the new ideas and methods of treating tumors.Methods:1.The preparation and characterization of nanomedicine IP:first,the solvent-antisolvent precipitation method was used to synthesize the self-assembled drug IP.Through ultraviolet-visible spectrophotometer and high-performance liquid chromatograph,Pyropheophorbide-a,and Icaritin drug charge in the IP are detected.Observe the micromorphology of the IP using transmission electron microscopy and detect its stability using a Malvern particle sizer.2.Exploring the self-assembly mechanism of nanomedicine IP:The change of the maximum absorption peak at 660 nm of nanomedicine IP in different solutions was compared by an ultraviolet-visible spectrophotometer.3.The ability of nanomedicine IP uptake by cells:The fluorescence intensity of mouse breast cancer cells treated with nanomedicine IP was measured by laser confocal microscopy and flow cytometry to test the ability of nanomedicine IP to be taken up by cells.4.ROS generation capacity of nanomedicine IP:A fluorescence spectrophotometer was used to detect ROS generation of nanomaterials IP in an aqueous solution,in which SOSG was used as a detection reagent for singlet oxygen.The fluorescence intensity of mouse breast cancer cells stained with CellROX^TMGreen probe and DCFH-DE probe after IP treatment was observed by laser confocal microscopy.5.The ability to induce autophagy:Mitochondrial morphology of mouse breast cancer cells treated with nanomaterial IP was studied using Rhodamine 123 under laser confocal microscopy.The autophagy performance of mouse breast cancer cells induced by nanomedicine IP was investigated by Western blot analysis.6.The synergistic therapeutic ability of nanomedicine IP:MTT colorimetric assay,live/dead cell staining and flow cytometry were used to detect cell apoptosis to investigate the growth inhibitory effect of nanomaterial IP on mouse breast cancer cells,so as to clarify the antitumor effect of nanomaterial IP.Results:1.The particle size of the nanomedicine IP measured by dynamic light scattering method was 176.5±2.17 nm,and the multiple diffusion coefficient was0.171±0.015,and remained stable within 7 days.Transmission electron microscopy（TEM）showed that the nanomedicine IP had a uniform short rod-like structure and was uniformly dispersed in water.The results of HPLC showed that the encapsulation rates of Ppa and Ica were 24.52%and 61.11%,respectively,and the drug loading rates were 16.45%and 83.53%,respectively.2.The UV-VIS absorption spectrum of the nanomedicine IP showed a characteristic absorption peak at 660 nm,and the hydrophobic sodium dodecyl sulfate would have a phase effect on the absorption spectrum of the nanomedicine IP,indicating that the nanomedicine IP self-assembly through the hydrophobic force.3.Laser confocal observation of the uptake of nanomedicine IP showed that the red fluorescence of IP group was stronger than that of free drug Ppa in both time gradient and concentration gradient.These results indicated that nanomedicine IP was more easily taken up by mouse breast cancer cells than free Ppa.4.SOSG detected the production of singlet oxygen ¹O₂in nanomedicine IP.Compared with free drug Ppa,the fluorescence intensity of nanomedicine IP was significantly enhanced after exposure to light.At the cellular level,the ROS production was detected by CellROX^TMGreen probes or DCFH-DA as ROS sensors,indicating that nanomedicine IP can produce a large number of ROS after exposure to light and has a stronger ROS production capacity than free Ppa.This suggests that photodynamic therapy can produce a large number of ROS by activating the photosensitizer Ppa.5.Rhodamine 123 staining results showed that Ica could induce obvious aggregation of mitochondria in cells,indicating that Ica could induce autophagy in mouse breast cancer cells,and the statistical results of Western blot analysis showed that after IP treatment of nanomedicines,autophagy related protein 5（ATG5）was significantly up-regulated in mouse breast cancer cells.The changes of microtubule-associated protein 1 light chain 3 beta（LC3B）also showed that nanomaterials IP could induce autophagy in mouse breast cancer cells.6.MTT colorimetric assay showed that nano drug IP had concentration-dependent phototoxicity on mouse breast cancer cells.Live/dead cell staining experiments showed that the red fluorescence of cells treated with free drug Ppa and nano drug IP was significantly enhanced after exposure to light,and the nano drug IP was more obvious.Flow cytometry showed that the apoptosis rate of cells treated with IP was much higher than that of other groups.Conclusion:1.Dynamic Light Scattering and Transmission Electron Microscopy（TEM）shows with Ppa and Ica as raw materials of the carrier free of self-assembled nano drug IP（IP for short）can form good stability,form uniform short rodlike nanoparticles.The results showed that the self-assembled nanomedical IP was successfully constructed,forming nanomedical with strong stability,good water solubility and high drug loading.2.The uptake experiment of mouse breast cancer cells showed that nanomedicine IP enhanced the uptake of photosensitizer.The results of SOSG experiments were compared with those of DCFH-DA and CellROX^TMGreen experiments,indicating that nanomedicine IP can improve ROS production by enhancing photosensitizer intake.3.Rhodamine 123 staining and WB analysis showed that nanomedicine IP could induce autophagy in mouse breast cancer cells.nanomedicine IP synergistic photodynamic therapy over activates autophagy.4.The cytotoxicity experiment proved that nanomedicine IP had obvious anti-mouse breast cancer cells after exposure to light,indicating the great potential of nanomedicine IP in photodynamic therapy.