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.

Highlights

  • Regulation of endocytosis by ultrasound and microbubble treatment: potential for controlled cellular delivery of drugs to cancer cells Farnaz Fekri Doctor of Philosophy, 2019 Molecular Science, Ryerson University

  • 1.6.7.1 Flotillin’s role in endocytosis The idea that flotillins could participate in clathrin and caveolae-independent uptake of specific cargoes was first proposed by Glebov et al, who used live total internal reflection fluorescence (TIRF) imaging and particle tracking software to follow the dynamic behaviour of flotillin-1-GFP on the plasma membrane

  • 3.2 USMB treatment rapidly enhances the rate of clathrin-mediated endocytosis To investigate whether USMB may regulate the rate of Clathrin-mediated endocytosis (CME), I first examined the cell surface levels of transferrin receptors (TfRs), a well-established cargo of CME, and compared the cell surface levels of TfR in control cells to that of cells 5 min after USMB treatment

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Summary

Bibliography viii

Dynamic palmitoylation regulates the localization of proteins at the plasma membrane and can influence membrane-associated processes and signalling events. USMB treatment rapidly reduces cell surface TfR levels. USMB treatment results in a delayed increase in fluid-phase internalization. USMB treatment increases the cell surface abundance of the lysosomal marker. Vacuolin-1 treatment impairs the reduction in cell surface TfR levels by USMB treatment. Desipramine treatment impairs the reduction in cell surface TfR levels by USMB. Desipramine enhances the rate of fluid-phase uptake in USMB-treated cells. USMB treatment regulates flotillin cell surface levels, dynamics and internalization. DHHC5 is required for USMB-triggered flotillin and fluid-phase internalization. Expression of a phosphorylation-defective mutant of DHHC5 impairs flotillin internalization elicited by USMB treatment. Fyn is required for USMB-triggered flotillin and fluid-phase internalization.

Drug delivery strategies: a new era in cancer treatment
Pharmacokinetics and drug efficacy
Drug delivery strategies to improve pharmacokinetics
Liposomal drug delivery systems
Active targeting of nanoparticles using magnetic force
Transducers
Natural cavitation
Microbubble responses in the acoustic field
Microbubble safety
The biomedical applications of microbubbles
Ultrasound and microbubble mechanisms of drug delivery
Endocytosis as the entry pathway of drugs to cells upon USMB treatment
Clathrin-mediated endocytosis
Complexity in defining CIE highlighted by GPI-APs studies
Diversity of the molecular mechanisms of CIE subclasses
Endocytic pathway mediated by Rho family of GTPases The human
GEEC pathway
Caveolae-mediated endocytosis First observed by
Macropinocytosis
Flotillin-dependent endocytosis
Flotillin’s role in endocytosis
Flotillins as a signalling platform
Calcium-induced massive endocytosis
DHHC5, a key regulator of MEND pathways
Membrane repair following injury activates endocytosis
Ceramide as a modulator of endocytosis
Materials
Ultrasound treatment
Inhibitor and drug treatments
Immunofluorescence staining
Fluorescent transferrin uptake and EEA1 immunofluorescence staining
2.10 Fluorescence microscopy For
2.11 Image analysis
2.12 Immunoblotting Whole cell lysates were prepared in
2.13 Cell viability measurements To perform measurement of cell viability as shown in
Chapter 3 rationale
USMB treatment rapidly enhances the rate of clathrin-mediated endocytosis
USMB treatment alters the properties of clathrin-coated pits
Lysosome exocytosis occurs after USMB treatment
Desipramine inhibits USMB-stimulated reduction of cell surface TfR
Desipramine synergizes with USMB treatment to enhance fluid-phase uptake
Chapter 4 rationale
USMB treatment triggers flotillin-dependent fluid-phase endocytosis
Enhanced flotillin-dependent fluid-phase uptake induced by USMB requires DHHC5
Enhanced flotillin-dependent fluid-phase uptake induced by USMB requires Fyn
Enhanced flotillin-dependent uptake induced by USMB enhances drug uptake and action
Lysosome exocytosis upon USMB treatment A seminal study by
Regulation of fluid-phase endocytosis by USMB treatment
DHHC5-dependent control of fluid-phase endocytosis
Flotillin-dependent endocytosis is tuneable by a
Enhanced endocytosis by USMB treatment is an effective strategy for targeted drug delivery
Detailed mechanism of flotillin-dependent endocytosis
Identifying the intracellular target of desipramine
Increasing the delivery of trastuzumab using USMB and desipramine treatment
Full Text
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