An In Vitro Investigation of 5-Aminolevulinic Acid and Acridine Orange as Sensitizers in Radiodynamic Therapy for Prostate and Breast Cancer

Abstract Purpose: Radiodynamic Therapy (RDT) is an emerging technique that enhances the therapeutic effects of radiation by using photosensitizers to amplify tumor cell damage while minimizing harm to normal tissues. This in vitro investigation compares the biocompatibility and sensitizing efficacy of two candidate photosensitizers, 5-aminolevulinic acid (5-ALA) and acridine orange (AO), in human breast adenocarcinoma (MCF7) and prostate adenocarcinoma (PC3) cell lines. Materials and Methods: MCF7 and PC3 cell lines were cultured and exposed to a range of 5-ALA and AO concentrations to assess biocompatibility using PrestoBlue viability assays. Based on these results, optimal concentrations were selected for irradiation experiments. Cells were then seeded in T-25 flasks and incubated with 5-ALA or AO prior to receiving 2 Gy or 4 Gy of megavoltage photon radiation (18 MV or 45 MV). Clonogenic assays were performed to determine the surviving fractions of the cells. Results: 5-ALA exhibited a broader biocompatibility profile than AO, remaining non-cytotoxic up to 100 µg/mL. In contrast, AO showed cytotoxic effects above 1 µg/mL. At 18 MV, limited radiosensitization was observed, except at higher 5-ALA concentrations. However, at 45 MV, both sensitizers significantly reduced cell survival, particularly at 4 Gy. The most pronounced effect was observed with 100 µg/mL 5-ALA, which consistently resulted in lower surviving fractions than AO across both cell lines. Each sensitizer demonstrated differing effectiveness depending on the cell line and photon energy used. Conclusions: Both 5-ALA and AO enhanced the cytotoxic effects of radiation, but 5-ALA demonstrated superior biocompatibility and more consistent radiosensitization across both cell lines. Notably, the effectiveness of both sensitizers increased with higher photon energy, reinforcing the importance of beam energy in RDT design. These results underscore the advantages of 5-ALA over AO and highlight the need to optimize both sensitizer selection and radiation energy in clinical applications.