Regulation Of Endocytosis By Ultrasound And Microbubble Treatment: Potential For Controlled Cellular Delivery Of Drugs To Cancer Cells

Abstract

The delivery of therapeutics across biological barriers is a limiting factor in achieving ideal pharmacologic responses in patients. Modulating endocytic mechanisms with targeted, clinically-relevant interventions can increase intracellular delivery across biological barriers, and improve the efficacy of drugs. Ultrasound-microbubble (USMB) is a novel targeted delivery strategy that has shown promising potential in both diagnostic and therapeutic applications. The collective behaviour of microbubbles in the acoustic field can increase the plasma membrane permeability of surrounding cells, and enhance the delivery of therapeutics across biological barriers. USMB achieves the intracellular delivery of drugs through sonoporation and modulation of endocytic pathways, but the type of endocytic pathways and the mechanisms of activation were not known. I identified that, under distinct regulations, USMB enhances the rate of both clathrin-mediated endocytosis, as well as a non-receptor-mediated pathway responsible for internalizing bulk fluid into cells. I discovered that lysosome exocytosis and acidsphingomyelinase are required for the regulation of the clathrin-mediated pathway but not fluidphase endocytosis following USMB treatment. Given the potential of the clathrin-independent pathway to form high capacity carries for the uptake of fluids and therapeutics into cells, I aimed to identify the molecular identity of the proteins that drive the formation of non-clathrin coated vesicles following USMB treatment. I established that flotillins contribute to the USMB-induced vesicular uptake of fluid into cells, a phenomenon that depends on palmitoyltransferase DHHC5 and the Src-family kinase Fyn. Furthermore, I confirmed that USMB treatment can enhance the intracellular delivery of chemotherapeutic drugs such as cisplatin, and improve its therapeutic efficacy in a flotillin-dependent manner. This project established that both clathrin-mediated endocytosis and flotillin-dependent endocytosis can be modulated by clinically-relevant USMB treatments to enhance drug uptake and efficacy, revealing an important new strategy for targeted drug delivery in cancer treatment.