Abstract 4922: The feasibility of evaluation method for the newly developed Photomultiplier-tube-based singlet oxygen detection system in photodynamic therapy

Abstract

Abstract Purpose: Singlet oxygen (1O2) generated in photodynamic therapy (PDT) plays a major role in killing tumor cells. Therefore, a direct measurement of 1O2 production can specify the dose of photosensitizer or the irradiation for the PDT to be an effective modality. The purpose of this study was to investigate the feasibility for the newly developed Photomultiplier-tube (PMT)-based singlet oxygen detection (SOD) system, which uses a monolithic fiber optic cable to direct the diode laser to the target tissues or cells and collects the 1O2 emission. Methods: PDT was performed using a diode laser emitting red light at 670 nm wave length with total radiation dose of 4 J/cm2. We measured the production of 1O2 with the two photosensitizers, chlorin e6 (Che6) or Pyropheophorbide-a (PPa) in the four cancer cell lines, SW480, HT29, AsPC-1, and MiaPaCa-2 cells. In the cell-free experiments, produced 1O2 level was measured with the PMT-based SOD system as well as the NaN3 quenching experiments after incubation of Che6 or PPa and the results of the each method were compared. Cellular uptake of Che6 or PPa was measured by fluorescence meter. 1O2 photon counts were compared with the fluorescence intensity at a variety of photosensitizer concentrations. To see the association between the production of 1O2 and the tumor cell killing, cell viability was determined by MTT assay. Cells were incubated with various concentrates Che6 or PPa. They were exposed to a diode laser emitting at 670 nm wave length with total radiation dose of 4∼6 J/cm2. Results: The standard curve was drawn using 1O2 photon count and FL-meter values, enabling respective analysis of the photosensitizer concentration. Cell-free studies showed that 1O2 concentration generated by irradiation was positively correlated with the concentration of Che6 or PPa. Lifetime and photon count of 1O2 was decreased as NaN3 concentration was increased, showing that PMT-SOD system could accurately measure 1O2 concentrationàphoton count. Cell viability after treatment with the different photosensitizer concentrations showed positive correlation with 1O2 concentrationàphoton count. The cellular uptake of the photosensitizer was evaluated according to time, which also showed positive correlation with 1O2 photon count. MTT assay, performed in a 96 well plate (<1 x 105 in each well), showed a relationship between the two results, indicating the PMT-SOD system could accurately measure 1O2 production even in a small number of cells. Conclusion: The PMT-SOD system detected the 1O2 production more directly and accurately in evaluation of photosensitizer efficacy than the previously used indirect methods. Especially, this system could detect the 1O2production in small cell numbers, which enabled simultaneous analysis of various cell lines or diverse photosensitizers. Note: This abstract was not presented at the meeting. Citation Format: In-Wook Kim, Ju hee Kim, Jae Myung Park, Myung-Gyu Choi. The feasibility of evaluation method for the newly developed Photomultiplier-tube-based singlet oxygen detection system in photodynamic therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4922. doi:10.1158/1538-7445.AM2014-4922