Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms.
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.
Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada.|Graduate Program in Molecular Science, Ryerson University, Toronto, Ontario, Canada.
Farnaz
F
Fekri
Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada.|Graduate Program in Molecular Science, Ryerson University, Toronto, Ontario, Canada.
Ralph Christian
RC
Delos Santos
Department of Medical Physics, Ryerson University, Toronto, Ontario, Canada.|Graduate Program in Molecular Science, Ryerson University, Toronto, Ontario, Canada.|Institute for Biomedical Engineering, Science and Technology (iBEST), a partnership between Ryerson University and St. Michael's Hospital, Toronto, Ontario, Canada.|Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, Ontario, Canada.
Raffi
R
Karshafian
Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada.|Graduate Program in Molecular Science, Ryerson University, Toronto, Ontario, Canada.|Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, Ontario, Canada.
Costin N
http://orcid.org/0000-0001-9192-6340
CN
Antonescu
D002461:Cell Line, Transformed; D002966:Clathrin / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods*; D004705:Endocytosis / Q000187:drug effects*; D005089:Exocytosis / Q000187:drug effects; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D045423:Microbubbles; D011990:Receptors, Transferrin / Q000378:metabolism; D000069453:Ultrasonic Waves
D002966:Clathrin; D006576:Heterocyclic Compounds, 4 or More Rings; D011990:Receptors, Transferrin; C503870:vacuolin-1