Background: Compared to the excitation light in photodynamic therapy, ultrasound in sonodynamic therapy (SDT) could easily penetrate into the deep tumor in liver. However, the photosensitizer chlorin e6 (E6) activated by ultrasound has been limited in its application in clinics for the poor water solubility of E6 and poor effect of SDT. Nanoparticles as cavitation promotors may be able to amplify the E6-mediated SDT effect and also improve its water solubility.
Objective: The objective of the study was to develop an E6-based sonosensitizer with improved SDT effect and good water solubility using nanotechnology. 
Materials and methods: Polyethylene glycol (PEG)ylated iron oxide nanoparticles coated with E6 (PION@E6) was prepared by means of pyrolysis and phase transfer. Characterization of PION@E6 was performed by means of transmission electron microscopy, hydrate particle size analysis, and absorption and fluorescence spectra analysis. Uptake of PION@E6 by H22 cells (a murine hepatoma cell line) was measured by inductively coupled plasma atomic emission spectroscopy. The effect of SDT on H22 cells was studied by the combination of ultrasound treatment with PION@E6 incubation. Cell viability was measured using cell counting kit-8 assay. Cell apoptosis was analyzed by flow cytometry. ROS generation was measured using DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) probing kit.
Results: Absorption spectra of PION@E6 revealed successful loading of E6 onto the PIONs. It showed excellent water solubility and stability with a size of 37.86±12.90 nm in diameter. The fluorescence spectra of PION@E6 revealed a red-shift compared with free E6. When combined with ultrasound treatment, it showed a significantly better inhibitory effect on H22 cells and correspondingly higher level of intracellular ROS generation compared with free E6. Furthermore, either higher dose of PION@E6 or higher power intensity of ultrasound initiated significantly better SDT effect and correspondingly higher level of intracellular ROS generation compared with lower dose of PION@E6 or ultrasound, respectively.
Conclusion: PION@E6 is a superior potential sonosensitizer to E6 to treat tumors by SDT.
Keywords: iron oxide nanoparticles, chlorin e6, sonodynamic therapy, photodynamic therapy, liver cancer