Using Perfluorocarbon To Supply Oxygen And Cerenkov Luminescence As An Internal Excitation Source For Photodynamic Anticancer Research

Photodynamic therapy(PDT)has been applied to treat a wide range of medical conditions.PDT involves the administration of a tumor-localizing photosensitizer(PS)followed by local illumination of the tumor with light of a specific wavelength to activate the PS.The excited PS transfers its energy to oxygen,thus generating reactive oxygen species(ROS),such as singlet oxygen(1O2)that can leading to tumor cell death.PDT usually consists of three components:photosensitizer(PS),light,and tissue oxygen.Oxygen and light are two key factors in PDT.Tumor hypoxia and the tissue penetration depth of light has become a major obstacle for PDT.In order to overcome tumor hypoxia,perfluorotributylamine(PFTBA)were co-loaded with IR780(NIR-PS)into human serum albumin nanoparticles(HSA@IR780@PFTBA).Owing to the high oxygen capacity of PFTBA,it can provide sufficient O2 for IR780 to produce 1O2 in photodynamic.In order to overcome the tissue penetration limitation of excitation light.We used Cerenkov luminescence as a depth-independent self-luminescence source for PDT.Using Cerenkov luminescence from FDG activate Protoporphyrin IX(PpIX)to treat deep Tumor.This paper includes the following four parts:In the first chapter,we synthesed and characterized the nanoparticles(HSA@IR780@PFTBA),and detected Singlet Oxygen in Vitro.The nanoparticles was formed by the ultrasonic emulsification method.The particles of PFTBA@IR780@HSA dispersed as individual NPs with well-defined spherical shape were characterized byTEM.The average particle size of PFTBA@IR780@HSA was measured by dynamic light scattering(DLS).The nanoparticles showed excellent stability of size.The existence of IR780 was confirmed by the absorption peak in the NIR region.The existence of PFTBA was confirmed by GC-MS.The 1O2 generation was determined by the fluorescence intensity of oxidized SOSG.The results indicated that IR780 surrounded the core oxygen self-enriched PFTBA can successfully accelerate 1O2 generation to enhance PDT effect.Moreover,Upon NIR laser irradiation(20s),the temperature was increased only 8?.In the second chapter,we evaluated the therapeutic efficacy of PFTBA@IR780@HSA based on PDT in cell assays.Cytotoxicity of PFTBA@IR780@HSA be assessed by calcein-AM/PI staining and cell counting kit(CCK-8)assays.To verify the enhancment of PDT with PFTBA,the quantitative treatment effect was examined by CCK-8 on CT26 cells and Renca cells.ROS generation inside cells were detected by H2DCFDA.The results indicated that PFTBA affect IR780 to increase more ROS to kill cancer cells.The cell uptake of PFTBA@IR780@HSANPs was investigated by confocal microscopy,a large number of NPs entered into cells and localized in the cytoplasm and lysosome.Cell uptake of nanoparticles is mediated by endocytosis and cell membrane ligands of HSA.From these cell experiments,all results provide a fact that the oxygen-enriched PFTBA as core supply more oxygen than microenvironment to produce more 1O2 to enhance PDT effect.In the third chapter,we started a preliminary investigation on that the Cerenkov luminescence(CL)serve as a depth-independent light source for PDT.The absorption spectra,stability and self-quenching concentration of PpIX were measured.The absorption spectrum of protoporphyrin(PpIX)indicated that PpIX have the strongest absorption at 360nm,matched well with the Cerenkov luminescence emission spectrum(250-600nm).The stability of PpIX results indicated that the absorbance of the PpIX decreases with time.The results of the self-quenching experimental results indicated that the concentration of PpIX and Ce6 in the vicinity of 0.6 ?g/mL is the maximum fluorescence intensity,respectively.We need to further experiments to verify the feasibility of FDG as a PDT excitation source.In the fourth chapter,we evaluated the efficacy of PpIX excited by Cerenkov luminescence in cellular experiments.The cell uptake experiments demonstrated the synthesis of PpIX in cells after incubation with 5-ALA.The results showed that intracellular could produce a large amount of ROS by the 425nm laser which as an alternative light for Cerenkov luminescence.Meanwhile,cytotoxicity of the PpIX is better than the traditional 630 nm.The intracellular ROS detection demonstrated that FDG could activate PpIX generating ROS.The results of cytotoxicity of PpIX excited by FDG showed that there is no killing of cancer cells.In summary,the oxygen self-enriched PFC encapsulated to nanoparticles formed the nano-emulsion(PFTBA@IR780@HSA).The nanoparticles could be available in clinic achieving sufficient 1O2 to gain maximum PDT efficacy,which improve tumor hypoxia.The oxygen self-enriched nano-emulsion as an agent of improved PDT may be promise in cancer theranostics.In the study of Celenkov luminescence activate photosensitizer,the experimental results indicated that the Celenkov luminescence can activate PpIX to generate ROS.While the energy of Cerenkov luminescence is too low to kill of cells.Therefore,the use of self-luminous excitation PDT still need further study.