(623a) Efficacy of 5-Aminolevulinic Acid (5-ALA)-Mediated Photodynamic Therapy (PDT) Using Cold Atmospheric Plasma (CAP) As a Light Source for Anti-Tumor Applications

Introduction:Photodynamic therapy (PDT) is a treatment modality that takes advantage of the cytotoxic effects induced by a light source and a photosensitizer to generate singlet oxygen species [1]. It is well known that 5-ALA has been widely used in photodynamic therapy, which is a natural biochemical precursor for heme synthesis in living mammalian cells. In this study, cold atmospheric plasma (CAP) was used for the first time as a light source in photodynamic therapy. CAP is an ionized gas where the ion temperature is close to room temperature. It contains electrons, reactive oxygen species (ROS), reactive nitrogen species (RNS), radicals, and UV light [2]. The wavelengths for CAP are widely covered from 250 nm to 800 nm, which makes CAP a possible light source for photodynamic therapy.

Materials and Methods: 5-ALA and gold nanoparticles 2-4 nm in diameter were encapsulated inside polymersomes by self-assembly. After being encapsulated, TEM, DLS and SEM were used to measure particle size, zeta potential and particle morphologies. Both fibroblasts (ATCC, PCS-201-011) and melanoma A375 (ATCC, CRL-1619) cells were seeded at 2*10⁴ cells/ml on a 96-well plate maintained in DMEM (ATCC) supplemented with 10% FBS (ATCC) and 1% penicillin/streptomycin (Sigma). After 24 hours of incubation, polymersomes at different concentrations (from 100 to 500 μg/ml) were added to the cells and were further co-cultured overnight. Then, both cells with nanoparticles were exposed under a CAP light source for 90s. Cell viability of the two cell lines was quantified by an MTS assay, and cell morphology was observed under confocal microscopy. All cells were incubated in a standard incubator at 5% CO₂at 37 °C. The cellular experiments were run in triplicate and repeated three times for each group.

Results and Discussion: Cell viability results with the 5-ALA mediated photodynamic therapy indicated that using CAP as light source can significantly decrease melanoma cell number when compared with non-CAP treatment. Specifically, at concentrations of 200 μg/ml, the melanoma cell number reduced by more than 50% when compared to controls. More importantly, this treatment was non-cytotoxic to fibroblasts. In addition, confocal images indicated that the 5-ALA/PDT treated melanoma cells lost their cellular protrusions and showed a polygonal, nearly epithelial cell-like appearance in contrast to the elongated cells with multiple protrusions in untreated controls. Fibrillar actin formation in growth cones was reduced after ALA/PDT treatment, which showed a rearrangement of the actin cytoskeleton, resulting in a loss of cellular polarization. Also, focal adhesions decreased significantly observed from confocal images, which is related with cell integrin adhesion, signaling and actin cytoskeleton formation after ALA/PDT treatment.

Conclusions: 5-ALA/PDT with CAP as a light source treatment induced melanoma cancer cell apoptosis as well as suppression of cell growth and cell morphological changes. All of these results showed that 5-ALA/ PDT with CAP as a light source is a novel effective PDT method for anti-tumor applications deserving further study.

Acknowledgement: This study was supported by the Northeastern University.

References: [1] L. M. Rossi, P. R. Silva, L. R. Vono, et al. Langmuir, 2008, 24, 12534-12538.

[2] M. Wang, X. Cheng, W. Zhu, et al. Tissue engineering: part A, 2014, 5(20), 1060-1071.