Real-time Imaging of Vascular Changes and Extravasation in the Chicken Chorioallantoic Membrane (CAM) Model Exposed...

The chicken chorioallantoic membrane (CAM) is used to study biological processes, including drug delivery, and provides an in ovo model with vasculature available for intravital microscopy. Ultrasound and microbubbles have been shown to be a promising strategy for improving drug delivery to solid tumors and across the blood-brain barrier. Acoustic cluster therapy (ACT) based on large microbubbles with a volume 50-100 times larger than regular contrast agent microbubbles is of particular interest. The objective of this study was to expand our understanding of the mechanisms underlying ACT and its role in drug delivery, particularly in blood flow and extravasation.

The CAMs were cultured ex ovo and used at embryonic day 13-14, with the vascular effects and extravasation of a co-injected macromolecule (2 MDa fluorescein isothiocyanate-dextran) recorded during ultrasound exposure (1 MHz, mechanical index 0.4 or 0.8, 10,000 cycles) in combination with the microbubble Sonazoid or ACT (activation 2.7 MHz, mechanical index 0.4 to form the large bubble, enhancement 0.5 MHz, mechanical index 0.2 to oscillate the large bubble).

Extravasation of 2 MDa dextran demonstrated two different kinetics, rapid and slow, and were observed both for ACT and ultrasound and Sonazoid. ACT bubbles were easily visible in the vasculature, and vascular occlusion or opening and changing in the direction of blood flow were observed.

Slow and fast extravasation kinetics could be due to diffusion or ultrasound-induced convection. This study demonstrates that the CAM model is highly useful for studying bioeffects induced by sonopermeation at a vascular level.