Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model.

Marina Shirmanova,Ekaterina Serebrovskaya,Konstantin Lukyanov,Vladislav Kamensky,Diana Yuzhakova,Ilya Turchin,Gregory Perelman,Pavel Subochev,Elena Zagaynova,Sergey Lukyanov,Ludmila Snopova,Robert M Hoffman

doi:10.1371/journal.pone.0144617

Abstract

The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.

Highlights

Photodynamic therapy (PDT) is a method for the treatment of oncological and some nononcological diseases based on killing pathologic cells as a result of the production of reactive oxygen species (ROS) by a photosensitizer under exposure to light
The CT26 cell line stably expressing KR was obtained by lentiviral transduction and its fluorescence was analysed using flow cytometry and fluorescence microscopy
The experiments by Momiyama et al [19] and Kimura et al [20] indicated that fluorescent proteins green fluorescent protein (GFP) and RFP increased the efficacy of ultraviolet C on cancer cells and, enhanced PDT

Summary

Introduction

Photodynamic therapy (PDT) is a method for the treatment of oncological and some nononcological diseases based on killing pathologic cells as a result of the production of reactive oxygen species (ROS) by a photosensitizer under exposure to light. PDT with Genetically Encoded Photosensitizer KillerRed a chemically synthesized cyclic tetrapyrrole administered into the body intravenously or topically. The main problems with chemical photosensitizers are associated with their heterogeneous distribution within the lesion and cells, their redistribution over time, and nonspecific drug accumulation in the skin and mucosae. This has encouraged a search for more efficacious phototoxic drugs and modified treatment regimens. As soon as the first phototoxic protein KillerRed (KR) was engineered in 2006 [1], the idea arose that it could be used as a genetically encoded photosensitizer for very specific direct cell killing. The main advantages of KR over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to the targeted cell compartment

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