High-Resolution Imaging of Intracellular Calcium Fluctuations Caused by Oscillating Microbubbles

Abstract

Ultrasound insonification of microbubbles can locally enhance drug delivery, but the microbub-bleÀcell interaction remains poorly understood. Because intracellular calcium (Ca 2þ i) is a key cellular regulator, unraveling the Ca 2þ i fluctuations caused by an oscillating microbubble provides crucial insight into the underlying bio-effects. Therefore, we developed an optical imaging system at nanometer and nanosecond resolution that can resolve Ca 2þ i fluctuations and microbubble oscillations. Using this system, we clearly distinguished three Ca 2þ i uptake profiles upon sonoporation of endothelial cells, which strongly correlated with the microbubble oscillation amplitude, severity of sonoporation and opening of cellÀcell contacts. We found a narrow operating range for viable drug delivery without lethal cell damage. Moreover, adjacent cells were affected by a calcium wave propagating at 15 mm/s. With the unique optical system, we unraveled the microbubble oscillation behavior required for drug delivery and Ca 2þ i fluctuations, providing new insight into the microbubbleÀcell interaction to aid clinical translation.

Cytosolic Ca2+ transients during pulsed focused ultrasound generate reactive oxygen species and cause DNA damage in tumor cells

“…pFUS-induced DNA damage depended on cytosolic Ca 2+ entry, but it remains unclear specifically how pFUS affects intracellular Ca 2+ dynamics. Cytosolic Ca 2+ increases during sonoporation by ultrasound with MB contrast agents have been widely reported 37 - 39 . Sonoporation allows diffusion of Ca 2+ into the cytosol through indiscriminate openings of the plasma membrane and can induce Ca 2+ -dependent bioeffects 38 .…”

Opening doors with ultrasound and microbubbles: Beating biological barriers to promote drug delivery

“…The direct impact of microbubble cavitation on cellular barriers has been investigated in vitro on various cell lines including cancer cell lines and primary endothelial cells [8,[28][29][30][31][32][33][34][35][36][37]. The latter is the most biologically relevant as microbubbles cannot extravasate from the blood stream after intravenous injection.…”

“…The endothelial barrier is an important barrier for intravenously administered drugs as most drug targets lie beyond the vasculature and require extravasation [29,33]. Microbubbles have great potential for drug delivery across the endothelial layer: although they are retained in the bloodstream because of their size, they can be easily targeted to the endothelium and affect the vascular integrity by breaking the tight junctions between endothelial cells (Fig.…”

“…The brain is one of the most difficult regions in the human body to access [219]. The blood brain barrier (BBB), a special form of the previously mentioned endothelium, is almost impermeable in order to protect the brain from toxicity caused by hazardous substances, so-called neuroprotection [33,[220][221][222]. The BBB embeds selective trans-endothelial transporter molecule [219,223].…”

“…This leads to an exceptionally controlled transport of molecules to the brain and ensures a healthy brain homeostasis. The BBB has a tremendous impact on drug delivery to the brain, since it blocks the extravasation of about 98% of small molecule drugs (<600 Da) and 100% of macromolecules [33,219,220,224]. As a way to overcome this, drugs have been specifically designed to bind to brain-specific transport carriers in order to allow transcytosis towards the brain [219,220,222,225,226] and although successful, the delivery efficiency across the BBB remains quite low [227].…”