In Vitro And In Vivo Monitoring Of Singlet Oxygen Using Chemiluminescence

In addition to conventional surgery, ionizing radiation and chemotherapy,Photodynamic Therapy (PDT) has become an important cancer treatment modality.In past 30 years, PDT has been developed into a unique tumor therapy aiming notonly carcinoid but also certain malignant tumors. The modality has been approved asa routine clinical application in many countries. With its excellent characteristics oftarget selectivity, low cytotoxicity, and proved clinical results, PDT is recognized asan invaluable alternative, and in some specific cases primary, modality in cancertherapy.Given a particular tissue target with its intrinsic sensitivity to PDT, theeffectiveness of a treatment depends on the interplay of three main factors: thephotosensitizer in the target, the light absorption by the photosensitizer molecules,and the availability of molecular oxygen. Similar to ionizing radiation therapy, properdosimetry is required to warrant a successful PDT treatment. With the complexinterplay among these treatment parameters and the inhomogeneities in both physicaland phamacokinetical of biological tissues, it is difficult to control the dosimetryusing only explicitly measurable treatment parameters. Singlet oxygen (¹O₂) hasbecome a key of PDT dosimetry due to its center role in PDT cytotoxicity. It hasbeen well established that the PDT photochemical process is mainly via typeâ…¡reaction, i.e., the ¹O₂ is a key product of PDT. Various methods to detect ¹O₂ duringPDT are currently under development.In this study, chemiluminescence (CL) was utilized to detect ¹O₂ produced duringPDT. The chemiluminescence probe used in photochemical reaction is fluoresceinylcypridina luciferin analog (FLCA). It can specifically react with ¹O₂ to produce aneasily detectable luminescence signal. The reaction kinetics has proved that CL can beused to quantify the ¹O₂ production. An array of CL probes, e.g., FCLA,MCLA,Luminol, have been developed.The technique using chemiluminescence probe to monitor ¹O₂ production during PDT as a means to project the treatment outcome, is referred in this thesis aschemiluminescence detection (CLD). Recent development of more highly selectiveand sensitive CL probes and the the improvement in optical detection have provided astrong base for this research. In our study, the probe 3, 7-dihydro-6-{4-[2-(N'-(5-fluoresceinyl)thioureido)etho-xy]phenyl}-2-methylimidazo{1,2-a}pyrazin-3-one(FCLA) was used. FCLA can selectively react with singlet oxygen and superoxide toproduce 532nm luminescence that is readily detectable by conventional photon-multiplier tube (PMT). In addition, FCLA has minimal cytotoxicity and canpenetrate into intracellular space.This paper includes four sections. The first section is to introduce the backgroundknowledge about photophysics and photochemical kinetics for understanding theprinciple and the value of CLD technique. The second section introduces theexperimental system and the experiments of CLD. Labview was used to controlboth PDT treatment laser and the CL detection. The experiment results show that theCL has a much longer lifetime compared to that of singlet oxygen luminescence.The exponentially decreasing signal can be recorded, with an excellent signal-to-noiseratio, in a duration of several hundred ms after irradiation. In the third section, the invivo and real-time CLD experiments are discussed. The CL detection collected fromin vivo mouse skin during PDT proves that CL can be used quantify the ¹O₂production and provide a reliable dosimetry for PDT. In the last section, thefluorescence characteristics of FCLA and its application are investigated. The resultsindicate that the fluorescence of FCLA can change over time during the oxide reaction.The resultant of reaction, de-excitated FCLA, has a stronger fluorescence, comparedto that of fresh FCLA. The change in the fluorescence can be observed at single celllevel. In addition, monitoring fluorescence during a PDT treatment as a means tocorrect CL for quantitative study is also studied in this section. The study is tocompensate the variables associated with both PDT and FCLA pharmacokinetics and,thus, improve the accuracy in quantitative CLD.