Based On FRET Effect Of Nanosystems Toward Two-photon Fluorescence Imaging And Photodynamic Therapy

Objective:Cancer is one of the deadliest diseases of our time and throughout the world.Cancer incidence and mortality have been increasing in China,making cancer the leading cause of death since 2010 and a major public health problem in the country.Photodynamic therapy(PDT)has become one of the important therapeutic options in management of cancer in recent year.PDT involves the application of a non-toxic and tumor-localizing photosensitizer(PS)that can be activated by focused light to generate cytotoxic reactive oxygen species(ROS),which can induce cancer cell death through multifactorial mechanisms,ultimately resulting in necrosis and/or apoptosis,the destruction of tumor microvasculature,and the stimulation of the inflammatory and immune responses that can destroy the remaining cancer cells.Compared to conventional cancer therapies,PDT is more controllable and has the potential to selectively destroy malignant cells while sparing the normal tissues.However,there are still a few problems and obstacles to gaining general clinical acceptance for PDT.One of the obstacles is that currently approved PDT PSs absorb in the visible spectral regions below 700 nm,resulting that most application of PDT are for superficial treatments.The promising approach to increase light penetration is to apply two-photon activated technique in PDT.Compared with one-photon laser,two-photon absorption can increase light penetration and reduce photo-induced damage.The synthesis of two-photon absorption materials makes it possible for deeper bio-tissue imaging.Nevertheless,less PDT photosensitizer has high two-photon section and good two-photon absorption,fluorescence resonance energy transfer(FRET)theory has been applied in the design of novel two-photon PDT systems.In this thesis,two kinds of nanosystems has been designed and developed.The FRET process in these nanosystems was investigated and cell toxicities were determined.Methods:1.A typical photosensitive drug,Rose Bengal(RB),was selected to pair up with Nitrogen-doped graphene quantum dots(N-GQD)to induce the FRET process.The two species were conjugated together to form N-GQD–RB through a simple coupling reaction,and then characterized by TEM,IR and CLSM.The FRET process was confirmed by the photo-bleaching experiment and time-resolved fluorescence spectra.The photogeneration of singlet oxygen was detected by the chemical probe,and an MTT assay was applied to evaluate the cytotoxicity of N-GQD–RB.In addition,N-GQD–RB induced cytotoxicity by two-photon excitation was investigated by CLSM with a two-photon laser.N-GQD-RB induced bio-tissue imaging and photo-toxicity also was investigated through in vivo experiment.2.Bis(pyrene)molecules(BP)was assembled into nano-aggregates as strong energy donor and coated with Rose Bengal(RB)coupled with a polyelectrolyte PAH as energy acceptor.The BP-RB nanosystem was characterized by TEM,DLS,FL and CLSM.The FRET process was confirmed by the photo-bleaching experiment and time-resolved fluorescence spectra.The penetration depth was detected by CLSM.Detection of singlet oxygen and cytotoxicity of BP-RB induced by one-/two-photon irradiation were investigated.Results:1.The diameters of N-GQD-RB are about 5 nm and of BP-RB are about 60 nm.The structures of these two nanosystems are clear and has good stability.2.RB can be excited by N-GQD or BP with a one-or two-photon laser through FRET process.3.Singlet oxygen can be generated by the laser irradiation,and N-GQD–RB and BP-RB exhibit high cytotoxicity after irradiation.4.The blood vessel is blocked with high precision utilizing only a very small amount of N-GQD-RB and a low dose of two-photon irradiation.5.The BP-RB nanosystems could be still detected until 2000 ?m generated with two-photon laser.Conclusion:1.N-GQD-RB and BP-RB nanosystems have clear structures,good photo-stability and biocompatibility.2.The FRET process can be occurred in N-GQD-RB and BP-RB nanosystems.3.N-GQD–RB and BP-RB nanosystems exhibit high cytotoxicity after irradiation under one-or two-photon laser irradiation.4.N-GQD-RB and BP-RB nanosystems present a high penetration capability,which have a potential for deep bio-tissue imaging and PDT.