Photodynamic therapy (PDT) is generally based on the generation of highly reactive singlet oxygen (1O2) through interactions of photosensitizer, light, and oxygen (3O2). These three components are ...highly interdependent and dynamic, resulting in variable temporal and spatial 1O2 dose deposition. Robust dosimetry that accounts for this complexity could improve treatment outcomes. Although the 1270 nm luminescence emission from 1O2 provides a direct and predictive PDT dose metric, it may not be clinically practical. We used 1O2 luminescence (or singlet oxygen luminescence (SOL)) as a gold-standard metric to evaluate potentially more clinically feasible dosimetry based on photosensitizer bleaching. We performed in vitro dose-response studies with simultaneous SOL and photosensitizer fluorescence measurements under various conditions, including variable 3O2, using the photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC). The results show that SOL was always predictive of cytotoxicity and immune to PDT's complex dynamics, whereas photobleaching-based dosimetry failed under hypoxic conditions. However, we identified a previously unreported 613 nm emission from mTHPC that indicates critically low 3O2 levels and can be used to salvage photobleaching-based dosimetry. These studies improve our understanding of PDT processes, demonstrate that SOL is a valuable gold-standard dose metric, and show that when used judiciously, photobleaching can serve as a surrogate for 1O2 dose.
The development of activatable photodynamic therapy (PDT) has demonstrated a utility for effective photosensitizer quenchers. However, little is known quantitatively about Forster resonance energy ...transfer (FRET) quenching of photosensitizers, even though these quenchers are versatile and readily available. To characterize FRET deactivation of singlet oxygen generation, we attached various quenchers to the photosensitizer pyropheophorbide-α (Pyro) using a lysine linker. The linker did not induce major changes in the properties of the photosensitizer. Absorbance and emission wavelength maxima of the quenched constructs remained constant, suggesting that quenching by ground-state complex formation was minimal. All quenchers sharing moderate spectral overlap with the fluorescence emission of Pyro (J ≥ 5.1 × 1013 M−1 cm−1 nm4) quenched over 90% of the singlet oxygen, and quenchers with weaker spectral overlap displayed minimal quenching. A self-quenched double Pyro construct exhibited intermediate quenching. Consistent with a FRET deactivation mechanism, extension of the linker to a 10 residue polyproline peptide resulted in only the quenchers with spectral overlap almost 2 orders of magnitude higher (J ≥ 3.7 × 1015 M−1 cm−1 nm4) maintaining high quenching efficiency. Overall, there was good correlation (0.98) between fluorescence quenching and singlet oxygen quenching, implying that fluorescence intensity can be a convenient indicator for the singlet oxygen production status of activatable photosensitizers. Uniform singlet oxygen luminescence lifetimes of the compounds, along with minimal triplet state transient absorption were consistent with quenchers primarily deactivating the photosensitizer excited singlet state. In vitro, cells treated with well-quenched constructs demonstrated greatly reduced PDT induced toxicity, indicating that FRET-based quenchers can provide a level of quenching useful for future biological applications. The presented findings show that FRET-based quenchers can potently decrease singlet oxygen production and therefore be used to facilitate the rational design of activatable photosensitizers.
As photodynamic therapy (PDT) continues to develop and find new clinical indications, robust individualized dosimetry is warranted to achieve effective treatments. We posit that the most direct PDT ...dosimetry is achieved by monitoring singlet oxygen (1O2), the major cytotoxic species generated photochemically during PDT. Its detection and quantification during PDT have been long-term goals for PDT dosimetry and the development of techniques for this, based on detection of its near-infrared luminescence emission (1270 nm), is at a noteworthy stage of development. We begin by discussing the theory behind singlet-oxygen luminescence dosimetry (SOLD) and the seminal contributions that have brought SOLD to its current status. Subsequently, technology developments that could potentially improve SOLD are discussed, together with future areas of research, as well as the potential limitations of this method. We conclude by examining the major thrusts for future SOLD applications: as a tool for quantitative photobiological studies, a point of reference to evaluate other PDT dosimetry techniques, the optimal means to evaluate new photosensitizers and delivery methods and, potentially, a direct and robust clinical dosimetry system.
To date, singlet oxygen (1O2) luminescence (SOL) detection was predictive of photodynamic therapy (PDT) treatment responses both in vitro and in vivo, but accurate quantification is challenging. In ...particular, the early and strongest part of the time‐resolved signal (500–2000 ns) is difficult to separate from confounding sources of luminescence and system noise, and so is normally gated out. However, the signal dynamics change with oxygen depletion during PDT, so that this time gating biases the 1O2 measurements. Here, the impact of gating was investigated in detail, determining the rate constants from SOL and direct pO2 measurements during meso‐tetra(hydroxyphenyl)chlorin (mTHPC)‐mediated PDT of cells in vitro under well‐controlled conditions. With these data as input, numerical simulations were used to examine PDT and SOL dynamics, and the influence of various time gates on cumulative SOL signals. It is shown that gating can underestimate the SOL at early treatment time points by ∼40% and underestimate the cumulative SOL signal by 20–25%, representing significant errors. In vitro studies with both mTHPC and aminolevulinic acid‐photosensitizer protoporphyrin IX demonstrate that rigorous analysis of SOL signal kinetics is then crucial in order to use SOL as an accurate and quantitative PDT dose metric.
Polymeric micelles are emerging as an effective drug delivery system for hydrophobic photosensitizers in photodynamic therapy (PDT). The objective of this study was to investigate the formulation of ...hydrophobic protoporphyrin IX (PpIX) with MePEG5000‐b‐PCL4100 methoxy poly (ethylene glycol)‐b‐poly (caprolactone) diblock copolymers and to compare their PDT response to that of free PpIX. The photophysical and photochemical properties of the polymeric PpIX micelles were studied by measuring absorbance and fluorescence spectra, PpIX‐loading efficiency and stability, the micelle particle size and morphology, as well as singlet oxygen luminescence and lifetime. The spherical micelles have a high PpIX‐loading efficiency of 82.4% and a narrow size distribution with a mean diameter of 52.2 ± 6.4 nm. The cellular uptake of PpIX in RIF‐1 cells using PpIX micelles was approximately two‐fold higher than that for free PpIX. Free PpIX and PpIX formulated in micelles exhibited similar subcellular localization in or around the cellular plasma membrane, as demonstrated using fluorescence microscopy. In vitro PDT results showed that the PpIX micelles have markedly increased photocytotoxicity over that with free PpIX, by nearly an order of magnitude at the highest light dose used. The micelles alone had no evident phototoxicity or dark toxicity. These findings suggest that MePEG5000‐b‐PCL4100 diblock copolymer micelles have great potential as a drug delivery system for hydrophobic photodynamic sensitizers.
Nucleic acid photodynamic molecular beacons (PMBs) are a class of activatable photosensitizers that increase singlet oxygen generation upon binding a specific target sequence. Normally, PMBs are ...functionalized with multiple solution-phase labeling and purification steps. Here, we make use of a flexible solid-phase approach for completely automated synthesis of PMBs. This enabled the creation of a new type of molecular beacon that uses a linear superquencher architecture. The 3′ terminus was labeled with a photosensitizer by generating pyropheophorbide-labeled solid-phase support. The 5′ terminus was labeled with up to three consecutive additions of a dark quencher phosphoramidite. These photosensitizing and quenching moieties were stable in the harsh DNA synthesis environment and their hydrophobicity facilitated PMB purification by HPLC. Linear superquenchers exhibited highly efficient quenching. This fully automated synthesis method simplifies not only the synthesis and purification of PMBs, but also the creation of new activatable photosensitizer designs.
Excited-state singlet oxygen (1O2), generated during photodynamic therapy (PDT), is believed to be the primary cytotoxic agent with a number of clinically approved photosensitizers. Its relative ...concentration in cells or tissues can be measured directly through its near-infrared (NIR) luminescence emission, which has correlated well with in vitro cell and in vivo normal skin treatment responses. Here, its correlation with the response of tumor tissue in vivo is examined, using the photosensitizer meso-tetrahydroxyphenylchlorin (mTHPC) in an animal model comprising luciferase- and green fluorescent protein (GFP)-transduced gliosarcoma grown in a dorsal window chamber. The change in the bioluminescence signal, imaged pretreatment and at 2, 5 and 9 d post treatment, was used as a quantitative measure of the tumor response, which was classified in individual tumors as "non", "moderate" and "strong" in order to reduce the variance in the data. Plotting the bioluminescence-based response vs the 1O2 counts demonstrated clear correlation, indicating that 1O2 luminescence provides a valid dosimetric technique for PDT in tumor tissue.
Firefly luciferase catalyzes the emission of light from luciferin in the presence of oxygen and adenosine triphosphate. This bioluminescence is commonly employed in imaging mode to monitor tumor ...growth and treatment responses in vivo. A potential concern is that, since solid tumors are often hypoxic, either constitutively and/or as a result of treatment, the oxygen available for the bioluminescence reaction could be reduced to limiting levels, leading to underestimation of the actual number of luciferase-labeled cells during in vivo experiments. We present studies of the oxygen dependence of bioluminescence in vitro in rat 9 L gliosarcoma cells tagged with the firefly luciferase gene (9L(luc)). We demonstrate that the bioluminescence signal decreases at pO(2) <or= 5%, falling by approximately 50% at 0.2% pO(2). Further experiments showed that the critical threshold for the initiation of metabolic depression in these cells was around 5%. Below this level, the decrease of oxygen saturation was followed by a decrease in intracellular ATP due to the reduction of mitochondrial membrane potential. Hence, the data suggest that the decrease of intracellular ATP level in vitro is the limiting factor for bioluminescence reaction and so is responsible for the reduction of bioluminescence signal in 9L(luc) cells in acute hypoxia, rather than luciferase expression or oxygen itself.
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