Effects of 5-aminolevulinic acid-mediated sonodynamic therapy on macrophages

Abstract

Jiali Cheng,1,* Xin Sun,1,2,* Shuyuan Guo,1,* Wei Cao,1 Haibo Chen,1 Yinghua Jin,1 Bo Li,1 Qiannan Li,1 Huan Wang,1 Zhu Wang,3 Qi Zhou,3 Peng Wang,3 Zhiguo Zhang,3 Wenwu Cao,3,4 Ye Tian1,21Division of Cardiology, the First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, Harbin, People’s Republic of China; 2Division of Pathophysiology, the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education, Harbin, People’s Republic of China; 3Laboratory of Photo- and Sono-theranostic Technologies and Condensed Matter Science and Technology Institute, Harbin Institute of Technology, Harbin, People’s Republic of China; 4Materials Research Institute, The Pennsylvania State University, University Park, PA, USA*These authors contributed equally to this workBackground: Inflammatory cells exhibit an elevated level of protoporphyrin IX (PpIX) after the administration of 5-aminolevulinic acid (ALA). Here, we investigate the sonodynamic effects of ALA-derived PpIX (ALA-PpIX) on macrophages, which are the pivotal inflammatory cells in atherosclerosis.Methods and results: Cultured THP-1 macrophages were incubated with ALA. Fluorescence microscope and fluorescence spectrometer detection showed that intracellular PpIX increased with the concentration of ALA in the incubation solution in a time dependent manner; the highest level of intracellular PpIX was observed after 3-hour incubation. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays demonstrated that lower concentrations (less than 2 mM) of ALA have no influence on cell viability (more than 90% of cells survived), but sonodynamic therapy (SDT) with a low concentration of ALA significantly decreased the survival rate of cells, and the effect was increased with both ALA concentration and ultrasound exposure time. Cell apoptosis and necrosis induced by ALA-mediated SDT (ALA-SDT) were measured using Hoechst 33258 and propidium iodide assay. ALA-SDT induced both cell apoptosis and necrosis, and the maximum apoptosis/necrosis ratio was observed at 6 hours after SDT with 1 mM of ALA and 5 minutes of ultrasound exposure. Flow cytometry analysis showed that ALA-SDT significantly increased late stage apoptotic cells (about 10-fold control). Furthermore, ALA-SDT induced reactive oxygen species generation in THP-1 macrophages immediately after the treatment and a conspicuous loss of mitochondrial membrane potential (MMP) at 6 hours compared with that of the control, ALA alone, and ultrasound alone groups.Conclusion: ALA-SDT exhibited synergistic apoptotic effects on THP-1 macrophages, involving excessive intracellular reactive oxygen species generation and MMP loss. Therefore, ALA-SDT is a potential treatment for atherosclerosis.Keywords: 5-aminolevulinic acid, protoporphyrin IX, sonodynamic therapy, macrophage, atherosclerosis