Intravital Imaging of Ultrasound-mediated Macromolecule Delivery Through the Blood Tumor Barrier in a Murine Glioma Model with Two-Photon Microscopy

Low-intensity ultrasound combined with microbubbles (US/MB) has been proven to be able to enhance the delivery outcome of many substances across the blood-brain barrier (BBB) or the blood-tumor barrier (BTB) to the brain by using magnetic resonance imaging (MRI) or post-mortem examination. Real-time observation of the dynamics of macromolecules passing through the BTB at microscopic level is important to understand the characteristics of US/MB-mediated drug delivery to the brain tumors. However, high temporal and spatial resolution are mutually exclusive in the two assessment methods. Therefore, in this study, by using two-photon fluorescent microscopy (2PFM), dynamic delivery process of macromolecules across the BBB/BTB induced by US/MB was realized in a murine glioma model at the scale of the micrometer. First, glioma cranial window model of mice was established. A ring-shaped piezoelectric transducer was used to achieve the US/MB-mediated destruction of BTB, and concurrent time sequential images were acquired through intravital 2PFM. Dextran labeled with Texas-Red were injected through the tail vein catheter before US/MB treatment and imaging. After the treatment of US/MB, a significant increase in fluorescence intensity of dextran was observed in the interstitium both around and inside the tumor, indicating that a large amount of dextran diffused across the BTB into the brain tumor tissue under the action of ultrasound. This preliminary study provided clear evidence that US/MB enabled deep penetration of macromolecules into the glioma center, which is critical to brain drug delivery strategies.