Objective To study the cell killing effect on isolated sarcoma 180 cells by ultrasound activating Protoporphyrin IX and to explore its biological mechanism.Methods The sonodynamical effect was investigated on S180 tumor cells exposed to the combination of 120 mol/L protoporphyrin Ⅸ (PPⅨ) and focused ultrasound at the frequency of 2.2 MHz and an intensity of 3W/cm2. The livability of cells was evaluated by trypan blue staining. Scanning electron microscope (SEM) observation of the surface of cells was performed to evaluate the morphological changes induced by ultrasonic irradiation. The generation of oxygen free radicals in cell suspensions was immediately detected after treatment by the active oxygen detection kit. Oxidative stress was assessed by measuring the activities of key antioxidant enzymes (ie, Superoxide dismutase[SOD], Glutathione peroxidase [GSH-PX], Catalase [CAT]) in S180 cells after SDT.Results The cell damage rate of ultrasound combined with PPIX was significantly higher than that treated with ultrasound alone only, and PPIX alone had no killing effect on S180 cells. Enzymatic chemical methods showed the content of MDA significantly increased after treatment, while the activities of key antioxidant enzymes in tumor cells all decreased at different levels, and was associated to the generation of oxygen free radicals in cell suspension after treatment. Conclusion Oxygen free radical may play an important role inimproving the membrane lipid peroxidation, decreasing the activities of key antioxidant enzymes in cells, and the biological mechanism might be involved in mediating the killing effect of S180 cells in SDT.

Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.