Abstract
Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue® MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US-mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis.
Highlights
In oncology, microbubble (MB)-assisted ultrasound (US) was introduced as a promising method to improve therapeutic efficacy of drugs by increasing their local delivery
Selection of the inhibitor concentration—cell viability assessment At high concentrations, both endocytosis inhibitors have an effect on the viability of C6 cells, which were for these experiments not exposed to US and MBs
According to the XTT absorbance data, a loss of cell viability was noticed from 20 μM of chlorpromazine, followed by a continuous decrease, until reaching a significant loss below 50% at 50 μM (MW, p < 0.05)
Summary
Microbubble (MB)-assisted ultrasound (US) was introduced as a promising method to improve therapeutic efficacy of drugs by increasing their local delivery. Many studies have found that US-mediated plasma membrane permeabilization results from the formation of transient pore-like structures in the plasma membrane, facilitating the delivery of molecules of different molecular weights into the intracellular compartment [10,11,12] This phenomenon was named sonoporation [13]. Cavitation can be categorized either as (1) inertial cavitation, which involves rapid growth and collapse of MBs [14], or as (2) stable cavitation with an oscillatory motion of bubbles with fluid microstreamings exerting shear forces on the plasma membrane [14] Both induce transient and local cell membrane permeabilization. In a clinical case study using combined US and SonoVue® MBs to enhance gemcitabine delivery in a patient suffering from pancreatic cancer [4], 0.5 mL of clinically approved MBs followed by 5 mL of saline were injected intravenously every 3.5 min throughout the 1 h treatment
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