Huiyun Lin

Photosensitized singlet oxygen generation and detection: Recent advances and future perspectives in cancer photodynamic therapy

Photodynamic therapy (PDT) uses photosensitizers and visible light in combination with molecular oxygen to produce reactive oxygen species (ROS) that kill malignant cells by apoptosis and/or necrosis, shut down the tumor microvasculature and stimulate the host immune system. The excited singlet state of oxygen (1 O2 ) is recognized to be the main cytotoxic ROS generated during PDT for the majority of photosensitizers used clinically and for many investigational new agents, so that maximizing its production within tumor cells and tissues can improve the therapeutic response, and several emerging and novel approaches for this are summarized. Quantitative techniques for 1 O2 production measurement during photosensitization are also of immense importance of value for both preclinical research and future clinical practice. In this review, emerging strategies for enhanced photosensitized 1 O2 generation are introduced, while recent advances in direct detection and imaging of 1 O2 luminescence are summarized. In addition, the correlation between cumulative 1 O2 luminescence and PDT efficiency will be highlighted. Meanwhile, the validation of 1 O2 luminescence dosimetry for PDT application is also considered. This review concludes with a discussion on future demands of 1 O2 luminescence detection for PDT dosimetry, with particular emphasis on clinical translation. Eye-catching color image for graphical abstract.

Singlet oxygen detection during photosensitization

Singlet oxygen (1O2) is a highly reactive oxygen species involved in numerous chemical and photochemical reactions in different biological systems and in particular, in photodynamic therapy (PDT). However, the quantification of 1O2 generation during in vitro and in vivo photosensitization is still technically challenging. To address this problem, indirect and direct methods for 1O2 detection have been intensively studied. This review presents the available methods currently in use or under development for detecting and quantifying 1O2 generation during photosensitization. The advantages and limitations of each method will be presented. Moreover, the future trends in developing PDT-1O2 dosimetry will be briefly discussed.

SINGLET OXYGEN QUANTUM YIELDS OF PORPHYRIN-BASED PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY

The major cytotoxic agent with most current photosensitizers used in photodynamic therapy (PDT) is widely believed to be singlet oxygen (1O2). Determination of the 1O2 quantum yields for porphyrin-based photosensitizers, including hematoporphyrin derivative (HiPorfin), hematoporphyrin monomethyl ether (HMME) and photocarcinorin (PsD-007) in air-saturated dimethylformamide (DMF) solutions were performed by the direct measurement of their near-infrared luminescence. In addition, 1O2 quencher sodium azide was employed to confirm the 1O2 generation from the investigated photosensitizers. The maximal 1O2 luminescence occurs at about 1280 nm with full width at half maximum of 30 nm. The 1O2 quantum yields were found to be 0.61 ± 0.03, 0.60 ± 0.02 and 0.59 ± 0.03 for HiPorfin, HMME and PsD-007, respectively. These results provide that these porphyrin-based photosensitizers produce 1O2 under irradiation, which is of significance for the study of their photodynamic action in PDT.

Detection system for singlet oxygen luminescence in photodynamic therapy

Singlet oxygen (1O2) is widely considered to play a major role in photodynamic therapy (PDT), and thus an increasing attention has been focused on the direct detection of 1O2 near-infrared luminescence around 1270 nm for PDT dosimetry. A new sensitive detection system is developed to directly measure the temporal and spectral resolved 1O2 luminescence spectra. The triplet state and 1O2 lifetimes of Rose Bengal as a model photosensitizer in different solvents are determined, and the obtained results agree well with the published data. Our detection system has the potential application in 1O2 luminescence-based PDT dosimetry.

Light-Emitting Diode-Based Illumination System for In Vitro Photodynamic Therapy

The aim of this study is to develop a light-emitting diode- (LED-) based illumination system that can be used as an alternative light source for in vitro photodynamic therapy (PDT). This illumination system includes a red LED array composed of 70 LEDs centered at 643 nm, an air-cooling unit, and a specific-designed case. The irradiance as a function of the irradiation distance between the LED array and the sample, the homogeneity and stability of irradiation, and the effect of long-time irradiation on culture medium temperature were characterized. Furthermore, the survival rate of the CNE1 cells that sensitized with 5-aminolevulinic acid after PDT treatment was evaluated to demonstrate the efficiency of the new LED-based illumination system. The obtained results show that the LED-based illumination system is a promising light source for in vitro PDT that performed in standard multiwell plate.

Influence of pulse-height discrimination threshold for photon counting on the accuracy of singlet oxygen luminescence measurement

Direct measurement of near-infrared (NIR) luminescence around 1270?nm is the golden standard of singlet oxygen (1O2) identification. In this study, the influence of pulse-height discrimination threshold on measurement accuracy of the 1O2 luminescence that is generated from the photoirradiation of meso-tetra (N-methyl-4-pyridyl) morphine tetra-tosylate (TMPyP) in aqueous solution was investigated by using our custom-developed detection system. Our results indicate that the discrimination threshold has a significant influence on the absolute 1O2 luminescence counts, and the optimal threshold for our detection system is found to be about ? 41.2?mV for signal discrimination. After optimization, the derived triplet-state and 1O2 lifetimes of TMPyP in aqueous solution are found to be 1.73 ? 0.03 and 3.70 ? 0.04??s, respectively, and the accuracy of measurement was further independently demonstrated using the laser flash photolysis technique.

Kinetic analysis of singlet oxygen generation in a living cell using Singlet Oxygen Sensor Green

Singlet oxygen (1O2) can be generated in a living cell upon focused laser irradiation of an intracellular photosensitizer. In this study, 1O2 generation from the plasma membrane-targeted protoporphyrin IX (PpIX) in human nasopharyngeal carcinoma CNE2 cells was monitored indirectly by using the fluorescence probe Singlet Oxygen Sensor Green agent (SOSG). The confocal images indicate that the fluorescence of SOSG in the vicinity of the cells that incubated with PpIX was dramatically enhanced with the increased irradiation time, while there is no significant enhancement for the control cells. Moreover, the fluorescence of SOSG is dramatically enhanced with the increase of the intracellular PpIX in CNE2 cells for the same photoirradiation time. These observations imply that the 1O2 generated from the plasma membrane-targeted PpIX in the CNE2 cells can be escaped into the extracellular medium and to react with the SOSG to produce SOSG-EP, and the fluorescence enhancement of SOSG around the cells mainly depends on the intracellular PpIX. Our findings may be useful for further monitored the 1O2 that can be escaping from the living cells.

Direct imaging of singlet oxygen luminescence generated in blood vessels during photodynamic therapy

Singlet oxygen (1O2) is commonly recognized to be a major phototoxic component for inducing the biological damage during photodynamic therapy (PDT). In this study, a novel configuration of a thermoelectrically-cooled near-infrared sensitive InGaAs camera was developed for imaging of photodynamically-generated 1O2 luminescence. The validation of 1O2 luminescence images for solution samples was performed with the model photosensitizer Rose Bengal (RB). Images of 1O2 luminescence generated in blood vessels in vivo in a well-controlled dorsal skinfold window chamber model were also recorded during PDT. This study demonstrated the capacity of the newly-developed imaging system for imaging of 1O2 luminescence, and the first reported images of 1O2 luminescence in blood vessels in vivo. This system has potential for elucidating the mechanisms of vascular targeted PDT.

Effects of pulse width and repetition rate of pulsed laser on kinetics and production of singlet oxygen luminescence

Pulsed and continuous-wave (CW) lasers have been widely used as the light sources for photodynamic therapy (PDT) treatment. Singlet oxygen (1O2) is known to be a major cytotoxic agent in type-II PDT and can be directly detected by its near-infrared luminescence at 1270nm. As compared to CW laser excitation, the effects of pulse width and repetition rate of pulsed laser on the kinetics and production of 1O2 luminescence were quantitatively studied during photosensitization of Rose Bengal. Significant difference in kinetics of 1O2 luminescence was found under the excitation with various pulse widths of nanosecond, microsecond and CW irradiation with power of 20mW. The peak intensity and duration of 1O2 production varied with the pulse widths for pulsed laser excitation, while the 1O2 was generated continuously and its production reached a steady state with CW excitation. However, no significant difference (P>0.05) in integral 1O2 production was observed. The results suggest that the PDT efficacy using pulsed laser may be identical to the CW laser with the same wavelength and the same average fluence rate below a threshold in solution.

Imaging singlet oxygen luminescence in blood vessels

Photodynamic therapy (PDT) is a minimally invasive treatment for cancer and other diseases that uses light-activated compounds to attack malignant cells and tissues. Using a photosensitizer (an agent that transfers energy from incident light), PDT produces reactive oxygen species—predominantly singlet oxygen (O2)—that are toxic to the targeted cells. Direct imaging of singlet oxygen near-IR (NIR) luminescence at around 1270nm reveals the spatial and temporal heterogeneity of tumors and their response to PDT. However, the imaging process is technically challenging because of the extremely high reactivity of singlet oxygen and its short lifetime in biological microenvironments.1–3 This can limit the chances of luminescence emission. Furthermore, current NIR detectors have very low quantum efficiency (the ratio of incident photons to converted electrons). During the past decade, there have been two main approaches for imaging singlet oxygen luminescence and attempting to correlate it with the biological response during PDT.4 The first method uses an NIR photomultiplier tube or an indium gallium arsenide (InGaAs) photodiode linear array combined with a