Synthesis And Photodynamic/photothermal Therapy Of Meso-phosphate-substituted Pyronine Dye

Phototherapy includes photodynamic therapy（PDT）and photothermal therapy（PTT）.At present,phototherapy has become a complementary and even alternative treatment for traditional cancer therapy due to its good spatial-temporal selectivity,low side effects,negligible drug resistance and non-invasive property.PDT is a minimally invasive technique for treating tumors by using photosensitizers（PSs）to produce toxic reactive oxygen species.The reactive oxygen species（singlet oxygen,superoxide anion,hydroxyl radical,etc.）generated by PDT oxidize the biological macromolecules in tumor cells,resulting in cytotoxicity and cell death.PTT is also a minimally invasive and efficient anti-tumor method based on photothermal agents（PTAs）with high photothermal conversion efficiency.PTAs accumulate in tumor tissue by targeted recognition technology,and convert light energy into heat to kill cancer cells,thus PTT causes less damage to normal tissues.Therefore,PSs and PTAs play an important role in phototherapy.So far,various types of PSs and PTAs have been developed,mainly including inorganic nanomaterials,polymers and organic small molecules.Compared with inorganic nanoparticles or polymers,organic small molecule PSs and PTAs have the advantages of better biocompatibility and water solubility,smaller side effects and easy removal by the excretion system,so they have attracted more and more attention.Therefore,it is of great significance to develop efficient organic small molecule PSs and PTAs to achieve efficient PDT and PTT.The research contents of this thesis include the following two parts:1.Synthesis of meso phosphate substituted pyronin dye and its type I PDT research under HypoxiaAccording to the existing research,most PSs have been developed are type II,the O₂ consumption mechanism makes them impossible to achieve the desired effect in hypoxia.While type I PDT involves the consumption and regeneration of O₂.Theoretically,even low concentration of O₂ could make the PDT process continue efficiently.Therefore,in this chapter,we developed an efficient type I PS for efficient PDT study in hypoxia.The meso phosphate substituted type I PS PY-P was synthesized by Michaelis-Becker reaction using pyronin Y as the matrix.The stability study showed that PY-P tends to carry out meso nucleophilic reaction in the presence of nucleophilic reagents（alcohols,biothiols,OH^-,etc.）,resulting in the colorless of PY-P.Nanoparticles were prepared by encapsulating PY-P with Pluronic F127.The stability test showed that PY-P NPs showed reaction inertia with nucleophilic reagents,such as GSH and OH^-.The particle size of PY-P NPs was characterized by transmission electron microscopy（TEM）and dynamic light scattering（DLS）as about 200 nm.After10 days of dark storage,the DLS particle size and zeta potential（<-20 m V）of PY-P NPs almost unchanged,indicating that long-term storage can also maintain stability.Reactive oxygen species test showed that both PY-P and PY-P NPs could effectively produce O₂^·-without producing ¹O₂,indicating that both of them experienced pure type I PDT process.Using dihydroethidium（DHE）as intracellular O₂^·-indicator indicated that PY-P NPs efficiently produced O₂^·-in Hela cells with extreme hypoxia.CCK-8cytotoxicity test showed that PY-P NPs had the advantage of low dark toxicity（the viability of Hela cells incubated at 10μM for 1 h was still>90%）;however,PY-P NPs exhibits high phototoxicity under light and extreme hypoxia conditions（IC50=5.2μM）.At the same time,the excellent luminescence properties of PY-P NPs（φ_F=0.35）make it possible for fluorescence imaging-guided PDT.2.Synthesis of meso phosphate substituted xanthene dye and its research in tumor photoacoustic imaging and photothermal therapyThe meso phosphate substituted xanthene dye P7 wes synthesized with extendedπ-conjugated xanthene as the parent,and the excitation wavelengths reached to near-infrared region（693 nm in water）.We encapsulated P7 in amphiphilic polymer to form nanoparticles for subsequent PTT research.Transmission electron microscope（TEM）and dynamic light scattering（DLS）verify that the particle size of P7 NPs is 150-200nm.After 10 days’storage,the average zeta potential（<-27 e V）hardly changed,which proved the stability of P7 NPs.The photothermal stability test showed that polymer encapsulated P7 exhibited photothermal stability.Meanwhile,P7 NPs have high photothermal conversion efficiency（36%）.Cytotoxicity test showed that P7 NPs had the advantage of low dark toxicity（20μM P7 NPs incubated for 1 h still keep a survival rate>80%）.However,PY-P NPs showed high phototoxicity under light irradiation.In vivo photothermal test showed that P7 NPs had good PTT effect and could effectively inhibit tumor tissue growing.