Low-Intensity Continuous Ultrasound Enhances the Therapeutic Efficacy of Curcumin-Encapsulated Exosomes Derived from Hypoxic Liver Cancer Cells via Homotropic Drug Delivery Systems

Exosomes are extracellular nanovesicles secreted by cells that efficiently deliver therapeutic cargo for cancer treatment. However, because exosomes are present in low quantities and have limited target specificity, internal and external stress stimulation has been studied to increase exosome efficiency. Inspired by these studies, the uptake efficiency of cobalt chloride-induced hypoxic cancer cell-secreted exosomes was evaluated. Western blotting and RT-PCR data revealed increased exosome secretion and different protein compositions exhibited by hypoxic exosomes (H-Exos) compared to natural normoxic exosomes (N-Exos). Furthermore, these H-Exos were continuously stimulated using low-intensity ultrasound (LICUS) at an intensity of 360 mW/cm² and a frequency of 3 MHz in vitro and 1 MHz in vivo. Hyperthermic and mechanical stress caused by ultrasound successfully improved exosome uptake via clathrin-mediated pathways, and confocal laser microscopy showed strong internal localization near the target cell nuclei. Finally, LICUS-equipped H-Exos were loaded with hydrophobic curcumin (H-Exo-Cur) and used to treat parent HepG2 liver cancer cells. The UV-Vis spectrophotometer displayed enhanced stability, solubility, and concentration of the encapsulated drug molecules. In MTT and FACS studies, approximately 40 times higher cell death was induced, and in animal studies, approximately 10 times higher tumor sizes were suppressed by LICUS-assisted H-Exo-Cur compared to the control. In this study, the delivery platform constructed demonstrated enormous potential for liver cancer therapy.