Abstract
Blood-brain barrier (BBB) is an unique structure in mammals’ brain. Although BBB can prevent harmful substances from entering brain tissues, it makes treatments of brain diseases difficult. Recently, a lot of methods have been discovered to enhance the permeability of BBB, among which traditional phospholipid-shelled microbubbles (MBs) combined with focused ultrasound (FUS) have been approved to induce non-invasive and local BBB disruption by the oscillation and destruction of MBs under insonification of acoustic field, facilitating vascular permeability and drug delivery efficiency. However, the destruction effects of phospholipid MBs may also accompany inertial cavitation (IC) that cause unwanted hemorrhage and damage in vivo. Therefore, in this study, a novel MBs (phospholipid-coated polymer MBs, PP-MBs) was developed by polymerization of hydrophobic monomer and cross-linker inside the phospholipid monolayer of MBs using a radical mechanism, forming a two-dimensional (2D) polymer network inside phospholipid shell. During the fabrication of PP-MBs, phospholipids, monomer and cross-linker were intensively shook, followed by polymerization via adding initiator and accelerator for 3 hours at room temperature. Then, physicochemical and acoustic properties of PP-MBs, including resonance frequency, nonlinear resonance capacity, inertial cavitation (IC) dose and destruction threshold were measured. Beside, in order to avoid the attenuation effect of high frequency ultrasound (10-MHz) in tissues, PP-MBs with larger size were fabricated for BBB disruption with low frequency ultrasound (2-MHz), and the BBB disruption efficiency and hemorrhage levels were assessed. Cryo-transmission electron microscopy provided clear evidence that the 2D polymer network structure was indeed incorporated inside the hydrophobic phase of phospholipid monolayer. In the aspects of PP-MBs with smaller size, PP-MBs and unpolymeried MBs behaved not only the same ultrasound resonance frequencies (measured as 12-MHz), but also similar subharmonic intensity characterized at 5-MHz under low acoustic pressure. However, under high acoustic pressure, the IC dose of PP-MBs was 1.3-fold lower than unpolymerized MBs. In addition, the destruction threshold of PP-MBs was about 800 KPa lower than unpolymerized MBs. These results suggested that the polymer network shell of PP-MBs could reduce the inertial cavitation with no influence on oscillation properties of phospholipid shell. On the other hand, PP-MBs with larger size showed similar acoustic properties with the smaller-sized PP-MBs, except for their lower IC dose under ultrasound pulse repetition frequency of >20 Hz than that of smaller-sized PP-MBs. Finally, PP-MBs were applied to disrupt BBB in vivo with 2-MHz FUS. PP-MBs were demonstrated to exhibit more Evans blue leakage (i.e. enhance the permeability of BBB more effectively) but less hemorrhage damage than unpolymerized MBs did. In this study, we first developed a novel MBs which had great potential to provide safer application for BBB disruption in the combination with FUS. However, there were still some parameters and different materials to adjust and attempt. For example, optimizing the composition of phospholipid, monomer and cross-linker, or using stiff materials such as thermosetting polymers to form more rigid polymer shell to achieve better effectiveness.