Photodynamic Activity of Substituted Zinc Trisulfophthalocyanines: Role of Plasma Membrane Damage

Photochemistry and Photobiology, Nov/Dec 2006 by Cauchon, Nicole, Nader, Moni, Bkaily, Ghassan, van Lier, Johan E, Hunting, Darel

ABSTRACT

We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS^sub 3^C^sub n^) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS^sub 3^C^sub n^ by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS^sub 3^C^sub n^. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS^sub 3^C^sub 6^-C^sub 9^ is a major factor in their overall photodynamic activity.

INTRODUCTION

Photodynamic therapy (PDT) is an alternative treatment for cancer and several other medical conditions; it requires a photosensitizer (PS), oxygen and light (1-4). Upon illumination of the affected area with light of the appropriate wavelength (630-800 nm) the PS is excited to its triplet state, which interacts with molecular oxygen to produce singlet oxygen and various other reactive oxygen species. The main cytotoxic entity is believed to be singlet oxygen, which acts at the site of the PS because of its very short half-life (5). Depending on the distribution of the PS, tissue response may be induced either directly by cell inactivation (6,7) and/ or indirectly by destruction of the vascular microcirculation (8-10).

The importance of lipophilicity vs. hydrophilicity for the photodynamic potency of the PS has been demonstrated in several studies (11-13). Amphiphilic (14) and hydrophobic PS with fewer than two negative charges can be internalized in cells by diffusion, passive partitioning between hydrophobic and hydrophilic environments of the cell membrane, endocytosis via association with lowdensity lipoproteins (15,16), pinocytosis after binding to albumin (17) or a combination of these processes. Hydrophilic PS, or hydrophobic PS with more than two negative charges, cannot diffuse across the plasma membrane; following noncovalent binding to serum proteins such as albumin and globulins they are transported by endocytosis only (1,18). In general, amphiphilic PS exhibit the highest in vitro and in vivo photodynamic activity (1,13,15,19,20).

We recently reported that variations in cellular phototoxicity among a series of trisulfonated zinc phthalocyanines (ZnPcS^sub 3^C^sub n^) correlate with their amphiphilic properties, which facilitate cell uptake and intracellular localization at photosensitive sites (Scheme 1) (13). However, our data also indicated that the intracellular localization does not constitute the sole determining parameter in the overall photodynamic potency of the PS. Other factors, such as differences in membrane permeability and cell uptake kinetics, may play an equally important role. This was in particular suggested by the selective localization of the most amphiphilic dyes in the series (ZnPcS^sub 3^C^sub 6-9^) in the lysosomes and Golgi apparatus, which is indicative of an endocytic pathway. Such a pathway proceeds via initial, rapid plasma membrane localization resulting in extensive plasma membrane damage after PDT at early incubation times. Several other classes of PS have been shown to target the plasma membrane during PDT (21-23).

The hydrophobic fluorescent bulk membrane probe 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), with its well-documented properties, was used to visualize membrane internalization of a PS as well as plasma membrane damage after PDT (24-26). This cationic dye does not cross the intact cell membrane, but enters the membrane leaflets by endocytosis (27). TMA-DPH does not fluoresce in aqueous medium; however, upon adhesion and internalization into the plasma membrane, its photophysical properties change because of interaction with the lipid environment, resulting in the emission of strong fluorescence after photoactivation (quantum yield 0.7). TMA-DPH is not cytotoxic and resists metabolic transformation, and the noninternalized fraction is easily removed from the membrane periphery by washing. As a result, this fluorescent probe has been used extensively to evaluate plasma membrane fluidity and damage, as well as endocytosis and exocytosis.