Photobleaching Kinetics of Optically Trapped Multilamellar Vesicles Containing Verteporfin Using Two-photon Excitation (original) (raw)
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Quantitative In Vitro Demonstration of Two-Photon Photodynamic Therapy Using Photofrin®and Visudyne®
Photochemistry and Photobiology, 2007
Photodynamic therapy (PDT), the combined action of a photosensitizer and light to produce a cytotoxic effect, is an approved therapy for a number of diseases. At present, clinical PDT treatments involve one-photon excitation of the photosensitizer. A major limitation is that damage may be caused to healthy tissues that have absorbed the drug and lie in the beam path. Two-photon excitation may minimize this collateral damage, as the probability of absorption increases with the square of the light intensity, enabling spatial confinement of the photosensitizer activation. A potential application is the treatment of the wet-form of age-related macular degeneration, the foremost cause of central vision loss in the elderly. Herein, the commercial photosensitizers Visudyne Ò and Photofrin Ò are used to demonstrate quantitative in vitro two-photon PDT. A uniform layer of endothelial cells (YPEN-1) was irradiated with a Ti:sapphire laser (300 fs, 865 nm, 90 MHz) using a confocal scanning microscope. Quantification of the two-photon PDT effect was achieved using the permeability stain Hoechst 33258 and a SYTOX Ò Orange viability stain. Visudyne was found to be around seven times more effective as a two-photon photosensitizer than Photofrin under the conditions used, consistent with its higher two-photon absorption cross-section. We also demonstrate for the first time the quadratic intensity dependence of cellular two-photon PDT. This simple in vitro method for quantifying the efficacy of photosensitizers for two-photon excited PDT will be valuable to test specifically designed twophoton photosensitizers before proceeding to in vivo studies in preclinical animal models.
2012
Photodynamic therapy (PDT) exploits the cytotoxic effects of light-activated compounds to achieve spatially selective tissue eradication. It is used in treating a wide range of tumors (Lou et al., 2003), localized infections (Hamblin & Hasan, 2004), and diseases like the wet form of age-related macular degeneration (Bressler & Bressler, 2000). The treatment involves application of a non-toxic photosensitizer that is preferentially taken up by the target cells/tissue. Optical excitation of the photosensitizer produces reactive oxygen species that cause localised, apoptotic cell death. Herein we review the application of a new modalitytwo-photon excitation-PDT (TPE-PDT)-to the treatment of wet age-related macular degeneration (wet-AMD). We show that the application of TPE-PDT, in conjunction with newly developed photosensitizers, has the potential to greatly improve therapy of wet-AMD. 1.1 Why two-photon photodynamic therapy (TPE-PDT)? 1.1.1 Photodynamic therapy (PDT) Wet-AMD is characterised by generation of blood vessels in the normally avascular retinal macula. The newly formed blood vessels leak fluid and/or blood under the macula, leading to rapid vision loss through damage to the photoreceptors (Rattner & Nathans, 2006). PDT, using single photon activation of the photosensitizer Verteporfin (trade name Visudyne), has been used for the treatment of wet-AMD since 2000 (Bressler & Bressler, 2000). In the clinic, verteporfin is first administered to patients through systemic injections (Soubrane & Bressler, 2001), and the photosensitizer accumulates in areas of high cellular reproduction like the neovasculature in the retinal tissue. Photo-irradiation of the photosensitizer leads to a localised, Type II photoreaction associated with singlet oxygen generation (Schmidt-Erfurth & Hasan, 2000). The photosensitizer absorbs a photon and is promoted to the excited singlet state that converts to an excited triplet state through intersystem crossing. An energy transfer between the triplet excited state of photosensitizer and naturally occurring triplet oxygen then produces reactive singlet oxygen. While Type I reactions involving radicals are also possible, PDT is generally accepted as occurring predominantly through the singlet oxygen mechanism. www.intechopen.com Age Related Macular Degeneration-The Recent Advances in Basic Research and Clinical Care 214 The PDT-induced vessel occlusion, in vivo, is generally attributed to singlet oxygen mediated direct vascular damage of blood vessel endothelium. This initiates a cascade of responses which include platelet aggregation, leukocyte adhesion, vascular permeabilization and vasoconstriction (Krammer, 2001). These, in turn, are expected to cause vascular occlusion. The short lifetime of singlet oxygen (3.5µs in aqueous environment (Pervaiz, 2001)) ensures that the area affected by it is spatially confined to a small volume. It is estimated that singlet oxygen can diffuse to a distance of around 100nm or less (Skovsen et al., 2005) in vivo. PDT, thus, offers a relatively selective and non-invasive method to occlude the abnormal vascularization characteristic of wet-AMD. The stages in PDT for treatment of wet-AMD are diagrammed in Figure 1.
System identification of the intracellular photoreaction process induced by photodynamic therapy
2008 16th Mediterranean Conference on Control and Automation, 2008
Photodynamic therapy (PDT) is an alternative treatment for cancer that involves the administration of a photosensitizing agent, which is activated by light at a specific wavelength. This illumination causes a sequence of photoreactions, which-in the presence of molecular oxygen-is supposed to be responsible for the death of the tumor cells. The PDT efficiency stems from the optimal interaction between these three factors (light, drug and oxygen). In this paper, a new approach is proposed to estimate photophysical parameters which characterize the ability of a photosensitizing drug to produce singlet oxygen. This approach is based on system identification techniques. This model-based method would allow biologists to estimate all the photophysical parameters from spectro-fluorescence data generated by only one experiment. Secondly, contrary to usual techniques which are restricted to in vitro studies, this approach can be directly applied to in vivo data.
Journal of Biomedical Optics, 2003
Two-photon excitation photodynamic therapy (TPE-PDT) is being investigated as a clinical treatment for age-related macular degeneration (AMD). TPE-PDT has the potential to provide a more specific and therefore advantageous therapy regime than traditional onephoton excitation PDT. The highly vascularized 8 to 9-day-old chicken chorioallantoic membrane (CAM) is used to model the rapid growth of blood vessels in the wet form of AMD. Using an ex ovo model system for the CAM, ablation studies were successful in mimicking the leaky vessels found in AMD. In addition, the distribution and localization of liposomal Verteporfin were investigated in order to characterize the photosensitizing drug in vivo. Localization of the photosensitizer appears to be greatest on the upper vessel wall, which indicates a potentially strong treatment locale for TPE-PDT. © 2003 Society of Photo-Optical Instrumentation Engineers.
Photochemistry and Photobiology, 2005
We present a quantitative framework to model a Type II photodynamic therapy (PDT) process in the time domain in which a set of rate equations are solved to describe molecular reactions. Calculation of steady-state light distributions using a Monte Carlo method in a heterogeneous tissue phantom model demonstrates that the photon density differs significantly in a superficial tumor of only 3 mm thickness. The time dependences of the photosensitizer, oxygen and intracellular unoxidized receptor concentrations were obtained and monotonic decreases in the concentrations of the ground-state photosensitizer and receptor were observed. By defining respective decay times, we quantitatively studied the effects of photon density, drug dose and oxygen concentration on photobleaching and cytotoxicity of a photofrin-mediated PDT process. Comparison of the dependences of the receptor decay time on photon density and drug dose at different concentrations of oxygen clearly shows an oxygen threshold under which the receptor concentration remains constant or PDT exhibits no cytotoxicity. Furthermore, the dependence of the photosensitizer and receptor decay times on the drug dose and photon density suggests the possibility of PDT improvement by maximizing cytotoxicity in a tumor with optimized light and drug doses. We also discuss the utility of this model toward the understanding of clinical PDT treatment of chest wall recurrence of breast carcinoma.
Modeling of Oxygen Transport and Cell Killing in Type-II Photodynamic Therapy
Photochemistry and Photobiology, 2012
Photodynamic therapy (PDT) provides an effective option for treatment of tumors and other diseases in superficial tissues and attracts attention for in vitro study with cells. In this study, we present a significantly improved model of in vitro cell killing through Type-II PDT for simulation of the molecular interactions and cell killing in time domain in the presence of oxygen transport within a spherical cell. The self-consistency of the approach is examined by determination of conditions for obtaining positive definitive solutions of molecular concentrations. Decay constants of photosensitizers and unoxidized receptors are extracted as the key indices of molecular kinetics with different oxygen diffusion constants and permeability at the cell membrane. By coupling the molecular kinetics to cell killing, we develop a modeling method of PDT cytotoxicity caused by singlet oxygen and obtain the cell survival ratio as a function of light fluence or initial photosensitizer concentration with different photon density or irradiance of incident light and other parameters of oxygen transport. The results show that the present model of Type-II PDT yields a powerful tool to quantitate various events underlying PDT at the molecular and cellular levels and to interpret experimental results of in vitro cell studies.
Photodynamic Efficiency: From Molecular Photochemistry to Cell Death
International Journal of Molecular Sciences, 2015
Photodynamic therapy (PDT) is a clinical modality used to treat cancer and infectious diseases. The main agent is the photosensitizer (PS), which is excited by light and converted to a triplet excited state. This latter species leads to the formation of singlet oxygen and radicals that oxidize biomolecules. The main motivation for this review is to suggest alternatives for achieving high-efficiency PDT protocols, by taking advantage of knowledge on the chemical and biological processes taking place during and after photosensitization. We defend that in order to obtain specific mechanisms of cell death and maximize PDT efficiency, PSes should oxidize specific molecular targets. We consider the role of subcellular localization, how PS photochemistry and photophysics can change according to its nanoenvironment, and how can all these trigger specific cell death mechanisms. We propose that in order to develop PSes that will cause a breakthrough enhancement in the efficiency of PDT, researchers should first consider tissue and intracellular localization, instead of trying to maximize singlet oxygen quantum yields in in vitro tests. In addition to this, we also indicate many open questions and challenges remaining in this field, hoping to encourage future research.
The role of photodynamic therapy (PDT) physics
Medical Physics, 2008
Photodynamic therapy ͑PDT͒ is an emerging treatment modality that employs the photochemical interaction of three components: light, photosensitizer, and oxygen. Tremendous progress has been made in the last 2 decades in new technical development of all components as well as understanding of the biophysical mechanism of PDT. The authors will review the current state of art in PDT research, with an emphasis in PDT physics. They foresee a merge of current separate areas of research in light production and delivery, PDT dosimetry, multimodality imaging, new photosensitizer development, and PDT biology into interdisciplinary combination of two to three areas. Ultimately, they strongly believe that all these categories of research will be linked to develop an integrated model for real-time dosimetry and treatment planning based on biological response.
Fluence-rate effects upon m-THPC photobleaching in a formalin-fixed cell system
Photodiagnosis and Photodynamic Therapy, 2004
We have applied a micro-spectroscopic technique in order to record the laser-induced fluorescence emission of the PDT photosensitiser m-THPC (Foscan) from micron-scale locations within individual formalin-fixed keratinocytes. We demonstrate that m-THPC is highly photolabile in this cellular environment, and that the process of photobleaching can be monitored via the depletion in fluorescence emission during continuous irradiation with 410 nm laser light. The progressive reduction of the characteristic 652 nm m-THPC fluorescence peak can be described with bi-exponential decay kinetics, consistent with a singlet oxygen-mediated process. The rate of photobleaching, when plotted as a function of light dose, shows inverse fluence-rate dependence. Specifically, the rate of photobleaching induced by the higher laser powers appears to be limited by oxygen availability, as demonstrated by an increase in the (1/e) bleaching dose. Fractionated irradiation provides evidence of intracellular re-oxygenation. These results are in qualitative agreement with previous in vitro and in vivo studies, which indicate that the photodynamic dose delivered during light irradiation is critically dependent upon local fluence rate and oxygen partial pressure.