John W. Snyder

Choice of Oxygen-Conserving Treatment Regimen Determines the Inflammatory Response and Outcome of Photodynamic Therapy of Tumors

The rate of light delivery (fluence rate) plays a critical role in photodynamic therapy (PDT) through its control of tumor oxygenation. This study tests the hypothesis that fluence rate also influences the inflammatory responses associated with PDT. PDT regimens of two different fluences (48 and 128 J/cm2) were designed for the Colo 26 murine tumor that either conserved or depleted tissue oxygen during PDT using two fluence rates (14 and 112 mW/cm2). Tumor oxygenation, extent and regional distribution of tumor damage, and vascular damage were correlated with induction of inflammation as measured by interleukin 6, macrophage inflammatory protein 1 and 2 expression, presence of inflammatory cells, and treatment outcome. Oxygen-conserving low fluence rate PDT of 14 mW/cm2 at a fluence of 128 J/cm2 yielded ∼70–80% tumor cures, whereas the same fluence at the oxygen-depleting fluence rate of 112 mW/cm2 yielded ∼10–15% tumor cures. Low fluence rate induced higher levels of apoptosis than high fluence rate PDT as indicated by caspase-3 activity and terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. The latter revealed PDT-protected tumor regions distant from vessels in the high fluence rate conditions, confirming regional tumor hypoxia shown by 2-(2-nitroimidazol-1[H]-yl)-N-(3,3,3-trifluoropropyl) acetamide staining. High fluence at a low fluence rate led to ablation of CD31-stained endothelium, whereas the same fluence at a high fluence rate maintained vessel endothelium. The highest levels of inflammatory cytokines and chemokines and neutrophilic infiltrates were measured with 48 J/cm2 delivered at 14 mW/cm2 (∼10–20% cures). The optimally curative PDT regimen (128 J/cm2 at 14 mW/cm2) produced minimal inflammation. Depletion of neutrophils did not significantly change the high cure rates of that regimen but abolished curability in the maximally inflammatory regimen. The data show that a strong inflammatory response can contribute substantially to local tumor control when the PDT regimen is suboptimal. Local inflammation is not a critical factor for tumor control under optimal PDT treatment conditions.

Control and Selectivity of Photosensitized Singlet Oxygen Production: Challenges in Complex Biological Systems

Singlet molecular oxygen is a reactive oxygen species that plays an important role in a number of biological processes, both as a signalling agent and as an intermediate involved in oxidative degradation reactions. Singlet oxygen is commonly generated by the so‐called photosensitization process wherein a light‐absorbing molecule, the sensitizer, transfers its energy of excitation to ground‐state oxygen to make singlet oxygen. This process forms the basis of photodynamic therapy, for example, where light, a sensitizer, and oxygen are used to initiate cell death and ultimately destroy undesired tissue. Although the photosensitized production of singlet oxygen has been studied and used in biologically pertinent systems for years, the photoinitiated behaviour is often indiscriminate and difficult to control. In this Concept, we discuss new ideas and results in which spatial and temporal control of photosensitized singlet oxygen production can be implemented through the incorporation of the sensitizer into a conjugate system that selectively responds to certain triggers or stimuli.

5,10,15,20-Tetrakis(N-Methyl-4-Pyridyl)-21H,23H-Porphine (TMPyP) as a Sensitizer for Singlet Oxygen Imaging in Cells: Characterizing the Irradiation-dependent Behavior of TMPyP in a Single Cell†

Abstract Singlet molecular oxygen, a1Δg, can be detected from a single cell by its weak 1270 nm phosphorescence (a1Δg→X3Σg−) upon irradiation of the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) incorporated into the cell. The behavior of this sensitizer in a cell, and hence the behavior of the associated singlet oxygen phosphorescence signal, depends on the conditions under which the sample is exposed to light. Upon irradiation of a neuron freshly incubated with TMPyP, the intensity of TMPyP fluorescence initially increases and there is a concomitant increase in the singlet oxygen phosphorescence intensity from the cell. These results appear to reflect a photoinduced release of TMPyP bound to DNA in the nucleus of the cell, where TMPyP tends to localize, and the subsequent relocalization of TMPyP to a different microenvironment in the cell. Upon prolonged irradiation of the cell, TMPyP photobleaches and there is a corresponding decrease in the singlet oxygen phosphorescence intensity from the cell. The data reported herein provide insight into key factors that can influence photosensitized singlet oxygen experiments performed on biological samples.

Direct Optical Detection of Singlet Oxygen from a Single Cell

Abstract Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1Δg→X3Σg−) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single-cell level.

Two-Photon Singlet Oxygen Microscopy: The Challenges of Working with Single Cells

Abstract A microscope is described in which singlet molecular oxygen, O2(a1Δg), is produced in a femtoliter focal volume via a nonlinear two-photon photosensitized process, and the 1270 nm phosphorescence from this population of O2(a1Δg) is detected in a photon counting experiment. Although two-photon excitation of a sensitizer is less efficient than excitation by a one-photon process, nonlinear excitation has several distinct advantages with respect to the spatial resolution accessible. Pertinent aspects of this two-photon O2(a1Δg) microscope were characterized using bulk solutions of photosensitizers. These data were compared to those obtained from a single biological cell upon linear one-photon excitation of a sensitizer incorporated in the cell. On the basis of the results obtained, we outline the challenges of using nonlinear optical techniques to create O2(a1Δg) at the single cell level and to then optically detect the O2(a1Δg) thus produced in a time-resolved experiment.

Application of a dithered sampling technique to increase the spatial resolution of singlet oxygen images

Singlet molecular oxygen (a1Δg) is an intermediate in many important oxidative reactions in heterogeneous biological and polymeric systems. By using a custom-made microscope to detect the 1270nm phosphorescence of singlet oxygen (a1Δg→X3Σg−), singlet oxygen images of such systems can be created. The microscope uses an InGaAs linear photodiode array to detect this extremely weak near infrared phosphorescence. In this article, the effects of the microscope’s modulation transfer function and the detector’s spatial sampling frequency on the resolution of the images are examined. It is shown that a dithered sampling technique can increase the accessible resolution. In this approach, data are repeatedly acquired from the sample after it has been systematically moved on the microscope stage.

Rapid Communication: Direct Optical Detection of Singlet Oxygen from a Single Cell ¶

Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1Δg→X3Σg−) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single‐cell level.

The imaging of singlet oxygen in single cells

The lowest excited state of molecular oxygen, singlet molecular oxygen (a1Δg), is generally regarded to be the active cytotoxic species in photodynamic therapy (PDT). As a result, the direct detection of singlet oxygen in biological systems should be of great value in elucidating the mechanisms underlying the observed effects of PDT. We have recently shown that singlet oxygen can be detected by its weak 1270 nm phosphorescence (a1Δg→X3Σg-) from a single nerve cell upon irradiation of a photosensitizer incorporated into the cell. In this paper, we discuss issues pertinent to the direct optical detection and imaging of singlet oxygen from single cells.