Reactive Oxygen Species and Folate Receptor-Targeted Nanophotosensitizers Composed of Folic Acid-Conjugated and Poly(ethylene glycol)-Chlorin e6 Tetramer Having Diselenide Linkages for Targeted Photodynamic Treatment of Cancer Cells.

Seong-Won Yang,Young-IL Jeong,Min-Suk Kook,Byung-Hoon Kim

doi:10.3390/ijms23063117

Abstract

Folic acid-conjugated nanophotosensitizers composed of folic acid (FA), poly(ethylene glycol) (PEG) and chlorin e6 (Ce6) tetramer were synthesized using diselenide linkages for reactive oxygen species (ROS)- and folate receptor-specific delivery of photosensitizers. Ce6 was conjugated with 3-[3-(2-carboxyethoxy)-2,2-bis(2-carboxyethoxymethyl)propoxy]propanoic acid (tetra acid, or TA) to make Ce6 tetramer via selenocystamine linkages (TA-sese-Ce6 conjugates). In the carboxylic acid end group of the TA-sese-Ce6 conjugates, FA-PEG was attached again using selenocystamine linkages to make FA-PEG/TA-sese-Ce6 conjugates (abbreviated as FAPEGtaCe6 conjugates). Nanophotosensitizers were fabricated by a dialysis procedure. In the morphological observations, they showed spherical shapes with small diameters of less than 200 nm. Stability of the aqueous FAPEGtaCe6 nanophotosensitizer solution was maintained (i.e., their particle sizes were not significantly changed until 7 days later). When H2O2 was added to the nanophotosensitizer solution, the particle size distribution was changed from a monomodal pattern to a multimodal pattern. In addition, the fluorescence intensity and Ce6 release rate from the nanophotosensitizers were also increased by the addition of H2O2. These results indicated that the nanophotosensitizers had ROS-sensitive properties. In an in vitro cell culture study, an FAPEGtaCe6 nanophotosensitizer treatment against cancer cells increased the Ce6 uptake ratio, ROS generation and light-irradiated cytotoxicity (phototoxicity) compared with Ce6 alone against various cancer cells. When the folic acid was pretreated to block the folate receptors of the Y79 cells and KB cells (folate receptor-overexpressing cells), the intracellular Ce6 uptake, ROS generation and thereby phototoxicity were decreased, while the MCF-7 cells did not significantly respond to blocking of the folate receptors. These results indicated that they could be delivered by a folate receptor-mediated pathway. Furthermore, an in vivo pulmonary metastasis model using Y79 cells showed folate receptor-specific delivery of FAPEGtaCe6 nanophotosensitizers. When folic acid was pre-administered, the fluorescence intensity of the lungs was significantly decreased, indicating that the FAPEGtaCe6 nanophotosensitizers had folate receptor specificity in vitro and in vivo. We suggest that FAPEGtaCe6 nanophotosensitizers are promising candidates for a targeted photodynamic therapy (PDT) approach against cancer cells.

Highlights

Photodynamic therapy (PDT) has been extensively investigated in the last two decades because it is regarded as a safe candidate for the treatment of cancer patients [1–5]
These results indicate that the FAPEGtaCe6 nanophotosensitizers had reactive oxygen species (ROS)-sensitive properties, and they could be disintegrated according to the oxidative stress
Blocking of the folate receptors of the cancer cells by folic acid (FA) pretreatment was less effective in the folate receptor negative cells, such as MCF-7 cells. These results indicate that the FAPEGtaCe6 nanophotosensitizers could be intracellularly delivered through a folate receptor mediated pathway

Summary

Introduction

Photodynamic therapy (PDT) has been extensively investigated in the last two decades because it is regarded as a safe candidate for the treatment of cancer patients [1–5]. PDT and its output are normally composed of a photosensitizer, light and oxygen species, and photosensitizers are only activated in a specific wavelength of light and produce reactive oxygen species (ROS) (i.e., PDT produces excessive ROS in the site of irradiation and inhibits the viability of disease cells) [6]. 5-aminolevulinic acid (5-ALA)-based PDT for brain cancer is efficient and safe for highgrade glioma [8]. They argued that glioma patients receiving 5-ALA PDT recorded longer survival times than those without PDT. PDT induces a response rate higher than 90% and no local recurrence in oral squamous carcinoma cells [8]. In spite of these advantages, some drawbacks to photosensitizers still limit the clinical application of PDT

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