RESUMEN
Photodynamic therapy (PDT) is generally based on the generation of highly reactive singlet oxygen ((1)O(2)) through interactions of photosensitizer, light, and oxygen ((3)O(2)). These three components are highly interdependent and dynamic, resulting in variable temporal and spatial (1)O(2) dose deposition. Robust dosimetry that accounts for this complexity could improve treatment outcomes. Although the 1270 nm luminescence emission from (1)O(2) provides a direct and predictive PDT dose metric, it may not be clinically practical. We used (1)O(2) 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 (3)O(2), 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 (3)O(2) 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 (1)O(2) dose.
Asunto(s)
Mediciones Luminiscentes , Fotoblanqueo , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Oxígeno Singlete/química , Radiometría , Factores de TiempoRESUMEN
To date, singlet oxygen ((1)O(2)) 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 (500-2000ns) 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 (1)O(2) measurements. Here, the impact of gating was investigated in detail, determining the rate constants from SOL and direct pO(2) 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 â¼40% 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.
Asunto(s)
Oxígeno/metabolismo , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Oxígeno Singlete/metabolismo , Relación Dosis-Respuesta en la Radiación , Humanos , Luminiscencia , Fármacos Fotosensibilizantes/farmacología , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Sondas Moleculares/química , Sondas Moleculares/síntesis química , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/síntesis química , Oxígeno Singlete/química , Automatización , Sitios de Unión , Cromatografía Líquida de Alta Presión , ADN/síntesis química , ADN/química , Interacciones Hidrofóbicas e Hidrofílicas , Compuestos Organofosforados/química , Soluciones/química , Solventes/químicaRESUMEN
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-alpha (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 > or = 5.1 x 10(13) 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 > or = 3.7 x 10(15) 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.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Oxígeno/química , Fármacos Fotosensibilizantes/química , Oxígeno Singlete , Línea Celular Tumoral , Supervivencia Celular , Colorantes/farmacología , Relación Dosis-Respuesta en la Radiación , Humanos , Lisina/química , Modelos Químicos , Fotoquímica/métodos , Fármacos Fotosensibilizantes/farmacología , Estructura Terciaria de Proteína , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Factores de TiempoRESUMEN
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) Asunto(s)
Gliosarcoma/metabolismo
, Luciferasas/metabolismo
, Transgenes/genética
, Adenosina Trifosfato/metabolismo
, Animales
, Hipoxia de la Célula
, Línea Celular Tumoral
, Gliosarcoma/genética
, Luciferasas/genética
, Mediciones Luminiscentes
, Oxígeno
, Ratas
, Transfección
RESUMEN
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 MePEG(5000)-b-PCL(4100) [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 MePEG(5000)-b-PCL(4100) diblock copolymer micelles have great potential as a drug delivery system for hydrophobic photodynamic sensitizers.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Poliésteres/química , Polietilenglicoles/química , Protoporfirinas/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Micelas , Microscopía Electrónica de Transmisión , Fotoquimioterapia , Protoporfirinas/farmacología , EspectrofotometríaRESUMEN
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.