Photodynamic Therapy Of Breast Cancer Based On Platelet Membrane Coated Red Phosphorus Photosensitizer

Objective With the development of economy and the progress of medical treatment,people have higher and higher requirements for health quality.At present,pathogenic microorganisms related to infectious diseases have become a serious killer,which kills about 17 million people every year.Pathogens,such as bacteria,enter the host through different routes and can cause tissue damage,inflammation and other pathological changes,delayed treatment can even lead to cancer.At present,surgery,radiotherapy and chemotherapy are still the main clinical treatment methods for cancer,but the traditional therapy is easy to cause harm to the body,the specificity is not strong,and the treatment effect of metastatic cancer is worse,so it is urgent to develop a safe and efficient cancer treatment method in clinic.In this study,a novel photosensitizer was constructed with red phosphorus-based carrier,which can generate a large number of reactive oxygen species by the interaction of oxygen and light energy conversion to induce the inactivation of pathogenic microorganisms.Platelet membrane coating technology is used to wrap photosensitive material in platelet membrane to avoid immune system tracking,and the homologous targeting effect of platelet is used to transport photosensitive material to tumor site for photodynamic therapy of breast cancer cells.This study realized the effective combination of photodynamic therapy and immunotherapy,and its synergistic effect on breast cancer,which is expected to provide new ideas and methods for the clinical treatment of breast cancer.Methods Red phosphate-based photosensitizer（RP-CN）was constructed by chemical vapor deposition method.The inactivation efficiency of the RP-CN on suspended bacteria was detected by time-sterilization curve method under visible light irradiation.The bactericidal effect was observed by plate colony formation unit counting.Regulating the amount of red phosphorus,looking for the optimal sterilization conditions.Scanning electron microscopy and SYIO9/PI live/dead bacteria staining kit were used to determine the bacterial state after light treatment.CBA protein kit and ATP content colorimetric test kit were used to detect the changes of protein and ATP content in the bacteria after light treatment,and then the cause of bacterial inactivation was analyzed.The mechanism of photodynamic therapy was explored by active species capture experiment,electron paramagnetic resonance and UV-vis absorption spectrum.The reusability of photosensitizer was investigated by cyclic stability test.Red phosphate-based photosensitizer（P25-RP）was constructed by vapor deposition method.In order to improve the biocompatibility of photosensitizer,platelets（PLT）membranes were extracted from PLT donated by volunteers,and P25-RP was extruded into PLT membrane by a micro-extruder to construct biotype photosensitizer P25-RP@PLT.Transmission electron microscopy,zeta potential and dynamic light scatterometer were used to confirm that the PLT membrane coated photosensitizer successfully.The functional expression of surface protein of biotype photosensitizer was detected by SDS-PAGE and Western blot.Biotoxicity of photosensitizer was tested by CCK-8 method,cell morphology observation and hemolysis rate.The uptake of photosensitizers by breast cancer cells was detected by fluorescein and confocal microscopy.CCK-8 assay,Annexin V-FITC/PI double staining apoptosis assay and flow cytometry were used to detect the effect of photosensitizer in vitro photodynamic therapy for breast cancer.A mouse breast cancer model was constructed to evaluate the effect of photodynamic therapy with photosensitizer in vivo.Results Red phosphorus-based photosensitizer（RP-CN）was constructed successfully.Photosensitizer inactivated pathogenic microorganisms’detection results:By regulating the amount of red phosphorus in photosensitizer,after visible light irradiation for 30 min,both S.aureus and E.coil can achieve 100%inhibition effect.After the light treatment,bacteria were stained with SYIO9/PI live/dead bacteria,a large number of red fluorescence appeared,indicating that the bacteria showed a state of apoptosis.Scanning electron microscope observation showed that the bacteria showed cell membrane rupture after the light treatment.CBA protein kit and ATP content colorimetric test kit found that after bacterial membrane rupture,protein and ATP leakage occurred in bacteria,resulting in the dual destruction of bacterial morphology and function.Detection results of photosensitizer inactivation mechanism of pathogenic microorganisms:The capture experiment results of active species showed that many active species were generated during photodynamic sterilization,among which superoxide radical（^·O₂^-）,photogenic hole（h⁺）and hydrogen peroxide（H₂O₂）were the most important active groups.The results of electron paramagnetic resonance and UV-vis absorption spectra further indicated that^·O₂^-and H₂O₂ played a leading role in photodynamic sterilization under visible light irradiation.Cycling stability test results of photosensitizer:After five rounds of cycling test,photosensitizer still maintains 99.89%sterilization rate,has good cycling stability,and can be reused.Successful construction of PLT membrane coated red phosphate-based photosensitizer（P25-RP@PLT）:Transmission electron microscope images showed that P25-RP surface was covered with membrane,and the increased particle size and more negative potential proved that the construction of PLT membrane coated red phosphate-based photosensitizer was successful.Surface protein functionalization of PLT membrane coated with red phosphate-based photosensitizers:SDS-PAGE results showed that the type and content of proteins in P25-RP@PLT were consistent with that of PLT,indicating that the translocation of PLT membrane protein from PLT to material was successful,and the translocation of membrane protein was still in good condition.Western blot results showed that CD47 could be expressed on P25-RP@PLT,indicating that P25-RP@PLT could resist phagocytosis of macrophages.Photosensitizer biological toxicity results:After the co-incubation of photosensitizer and breast cancer cells for 24 h,the activity of breast cancer cells was stable and the morphology was normal,photosensitizer would not lead to the occurrence of hemolysis,proved that photosensitizer has no biological toxicity.Results of cell uptake and co-localization:Fluorescein microplate detection showed that when 20μg/m L photosensitizer was co-incubated with breast cancer cells for 4 h,the maximum amount of photosensitizer was ingested into breast cancer cells.Confocal microscopy confirmed that the membrane-coated photosensitizer could be ingested by more breast cancer cells.Experimental results of in vitro photodynamic therapy for breast cancer:The apoptosis rates of cells were 12.51%and 44.7%before and after P25-RP（20μg/m L）laser irradiation,respectively.However,the apoptosis rate of the cancer cells induced by P25-RP@PLT（20μg/m L）laser irradiation for 5 min reached 51.1%.Results of in vivo photodynamic therapy for breast cancer:After light treatment,the tumor tissue of breast cancer in mice was significantly reduced,and the tumor site was scabby.HE staining showed obvious tissue damage and necrosis at the tumor site 5 min after laser irradiation.Conclusion The photosensitizer used in photodynamic therapy was successfully constructed.The photosensitizer alone has no toxicity and can produce a large number of reactive oxygen species for the treatment of infection caused by pathogenic microorganisms under light irradiation,and all pathogenic microorganisms can be removed in a short time.In order to improve the specificity of photodynamic therapy,the PLT membrane coated with red phosphate-based photosensitizer was constructed,and the PLT membrane protein antigen was used to avoid the monitoring of immune system and prolong the circulation life of photosensitizer in vivo.At the same time,the homologous adhesion of PLT is used to deliver photosensitizer to the location of breast cancer,so that it can be enriched in the cancer tissue,so as to obtain efficient photodynamic therapy effect.This study provides a new idea for the clinical transformation of photodynamic therapy combined with immunotherapy for breast cancer.