Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

Abstract

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

Targeted enhancement of flotillin-dependent endocytosis augments cellular uptake and impact of cytotoxic drugs

“…The role of DHHC5 in USMB-triggered enhancement of fluid-phase endocytosis suggests that this endocytic mechanism may be similar to MEND 26 . MEND can be triggered by a signaling pathway initiated by opening of mitochondrial permeability transition pore channels that leads to DHHC5-dependent enhancement of endocytosis of lipid-ordered membrane domains 26 , which is consistent with our observation that USMB stimulates flotillin internalization 48 . Our work is novel in that we find DHHC5-dependent endocytosis to require flotillin, and we demonstrate that stimulation of this pathway can lead to enhanced drug delivery into cells, which had not been examined in previous studies.…”

“…Most experimental strategies that assess the ability of USMB to enhance drug uptake and efficacy involve pre-treatment of cells with the drug in question, and then ongoing incubation in this drug solution following USMB stimulation. Hence, these strategies do not distinguish between access of drugs to the cellular interior through transient pores that form and re-seal during or immediately after USMB (<1 min), or the sustained enhancement of fluid-phase endocytosis as we 48 and others [44][45][46][47] have observed.…”

“…We previously reported that the acid sphingomyelinase (ASMase) inhibitor desipramine synergizes with USMB to enhance fluid-phase endocytosis yet we excluded ASMase as a target for desipramine in the control of endocytosis upon USMB 48 . Since desipramine is a clinicallyapproved drug, the possibility of a therapeutic combination of desipramine and USMB could be very promising, so we based our study here of the effects of USMB treatment on fluid phase endocytosis on the combined use of USMB and desipramine.…”

“…ARPE-19 human retinal pigment epithelial cells (RPE herein) and MDA-MB-231 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured as previously described 48 . Mycoplasma testing was routinely performed by staining with DAPI, approximately every 2 months.…”

“…Indeed, we previously reported that USMB enhances the rate of both clathrin-mediated endocytosis and fluid-phase uptake into cells by distinct signaling mechanisms 48 . However, the molecular mechanisms and intracellular signals by which USMB triggers enhanced fluid-phase endocytosis and whether this phenomenon can contribute to targeted drug delivery to control cancer cell survival had not been addressed, and must be understood in order for this drug delivery strategy to move closer to clinical use.…”