Photobleaching Kinetics of Optically Trapped Multilamellar Vesicles Containing Verteporfin Using Two-photon Excitation§

Abstract

Two-photon excitation photodynamic therapy (TPE-PDT) is being developed as an improved treatment for retinal diseases. TPE-PDT has advantages over one-photon PDT, including lower collateral damage to healthy tissue and more precise delivery of PDT. As with one-photon PDT, there can be local photochemical depletion of oxygen during TPE-PDT. Here, we investigate model systems and live cells to measure local photosensitizer photobleaching and through it, infer local oxygen consumption in therapeutic volumes of the order 1 microm3. Multilamellar vesicles (MLV) and African green monkey kidney (CV-1) cells were used to study the TPE photobleaching dynamics of the photosensitizer, Verteporfin. It was found that in an oxygen-rich environment, photobleaching kinetics could not be modeled using a mono-exponential function, whereas in hypoxic conditions a mono-exponential decay was adequate to represent photobleaching. A biexponential was found to adequately model the oxygen-rich conditions and it is hypothesized that the fast part of the decay is oxygen-dependent, whereas the slower rate constant is largely oxygen-independent. Photobleaching recovery studies in the CV-1 cells support this hypothesis.

Complete blood vessel occlusion in the chick chorioallantoic membrane using two-photon excitation photodynamic therapy: implications for treatment of wet age-related macular degeneration

“…13,[16][17][18][19][20][21][22][23][24][25] We have confirmed using Verteporfin that, as would be expected, the photophysical processes remain the same after the molecule has reached its first excited state ͑Fig. 1͒, regardless of whether excitation occurs through one-or two-photon absorption.…”

“…13,[16][17][18]23,25,54,55 In particular, Karotki et al Table 1. Although the irradiation setup for TPE-PDT irradiation ͑which was done using a confocal laser-scanning microscope͒ was very different from that used in these CAM studies, the average and peak irradiances were of the same order of magnitude and also similar to those of King and Oh ͑2004͒, 55 who reported singlet oxygen production using 5-͑and 6͒-chloromethyl-2Ј,7Ј-dichlorodihydronuorescein diacetate, acetyl ester with a peak power density ͑or peak irradiance͒ of 3 ϫ 10 11 W/cm 2 .…”

“…1͒, regardless of whether excitation occurs through one-or two-photon absorption. 13,25 Verteporfin has a larger two-photon cross section ͑31 GM units at 865 nm, where GM is units of Goppert-Meyer and 1 GM=1 ϫ 10 −49 cm 4 s / molecule͒ compared to other clinical photosensitizers such as Photofrin ® ͑7.5 GM units at 865 nm͒. 12,24,26 At 780 nm, the wavelength used here, the value is 61 GM units.…”

Quantitative In Vitro Demonstration of Two‐Photon Photodynamic Therapy Using Photofrin® and Visudyne®

“…Alternatively, if we fit the results from each experiment separately and then average the k values, we obtain n 2 = 1.97 ± 0.31 and n 1 = 0.86 ± 0.05. The slightly lower gradient for 1‐ γ excitation may be due to ground state depletion, photobleaching of the photosensitizer or oxygen depletion (35).…”

Two-Photon Excitation Photodynamic Therapy: Working Toward a New Treatment for Wet Age-Related Macular Degeneration

“…It should be noted that the excited states achieved by one and two-photon absorption are identical. The photophysical and photochemical properties of the photosensitizer are thus, unaffected by the mode of excitation (Goyan & Cramb, 2000;Samkoe et al, 2006). Typically, excitation volumes of a few femtoliters can be achieved with two-photon absorption.…”