Effect of Microbubble Size, Composition and Multiple Sonication Points on Sterile Inflammatory Response in Focused Ultrasound-Mediated Blood-Brain Barrier Opening.

Blood-brain barrier opening (BBBO) using focused ultrasound (FUS) and microbubbles (MBs) has emerged as a promising technique for delivering therapeutics to the brain. However, the influence of various FUS and MB parameters on BBBO and subsequent sterile inflammatory response (SIR) remains unclear. In this study, we investigated the effects of MB size and composition, as well as the number of FUS sonication points, on BBBO and SIR in an immunocompetent mouse model. Using MRI-guided MB+FUS, we targeted the striatum and assessed extravasation of an MRI contrast agent to assess BBBO and RNAseq to assess SIR. Our results revealed distinct effects of these parameters on BBBO and SIR. Specifically, at a matched microbubble volume dose (MVD), MB size did not affect the extent of BBBO, but smaller (1 µm diameter) MBs exhibited a lower classification of SIR than larger (3 or 5 µm diameter) MBs. Lipid-shelled microbubbles exhibited greater BBBO and a more pronounced SIR compared to albumin-shelled microbubbles, likely owing to the latter's poor in vivo stability. As expected, increasing the number of sonication points resulted in greater BBBO and SIR. Furthermore, correlation analysis revealed strong associations between passive cavitation detection measurements of harmonic and inertial MB echoes, BBBO and the expression of SIR gene sets. Our findings highlight the critical role of MB and FUS parameters in modulating BBBO and subsequent SIR in the brain. These insights inform the development of targeted drug delivery strategies and the mitigation of adverse inflammatory reactions in neurological disorders.

Effect of Poly(ethylene glycol) Configuration on Microbubble Pharmacokinetics.

Microbubbles (MBs) hold substantial promise for medical imaging and therapy; nonetheless, knowledge gaps persist between composition, structure, and in vivo performance, especially with respect to pharmacokinetics. Of particular interest is the role of the poly(ethylene glycol) (PEG) layer, which is thought to shield the MB against opsonization and rapid clearance but is also known to cause an antibody response upon multiple injections. The goal of this study was, therefore, to elucidate the role of the PEG layer in circulation persistence of MBs in the naïve animal (prior to an adaptive immune response). Here, we directly observe the number and size of individual MBs obtained from blood samples, unifying size and concentration into the microbubble volume dose (MVD) parameter. This approach enables direct evaluation of the pharmacokinetics of intact MBs, comprising both the lipid shell and gaseous core, rather than separately assessing the lipid or gas components. We examined the in vivo circulation persistence of 3 µm diameter phospholipid-coated MBs with three different mPEG2000 content: 2 mol % (mushroom), 5 mol % (intermediate), and 10 mol % (brush). MB size and concentration in the blood were evaluated by a hemocytometer analysis over 30 min following intravenous injections of 20 and 40 µL/kg MVD in Sprague-Dawley rats. Interestingly, pharmacokinetic analysis demonstrated that increasing PEG concentration on the MB surface resulted in faster clearance. This was evidenced by a 1.6-fold reduction in half-life and area under the curve (AUC) (p < 0.05) in the central compartment. Conversely, the AUC in the peripheral compartment increased with PEG density, suggesting enhanced MB trapping by the mononuclear phagocyte system. This was supported by an in vitro assay, which showed a significant rise in complement C3a activation with a higher PEG content. In conclusion, a minimal PEG concentration on the MB shell (mushroom configuration) was found to prolong circulation and mitigate immunogenicity.

Monodispersity Increases Adhesion Efficiency and Specificity for Ultrasound-Targeted Microbubbles.

Ultrasound molecular imaging with targeted microbubbles (MBs) can be used to noninvasively diagnose, monitor, and study the progression of different endothelial-associated diseases. Acoustic radiation force (Frad) can initiate and enhance MB adhesion at the target site. The goal of this study was to elucidate the effects of various MB parameters on Frad targeting. Monodisperse or polydisperse MBs with the immune-stealth cloaked (buried)-ligand architecture were conjugated with targeting RGD or nonspecific isotype control RAD peptides and then pumped through an αvß3 integrin-coated microvessel phantom at a wall shear stress of 3.5 dyn/cm2. Targeting was assessed by measuring MB attachment for varying Frad time and frequency, as well as MB concentration and size distribution. We first confirmed that primary Frad is necessary to target the cloaked-ligand MBs. MB targeting increased monotonically with αvß3 integrin density and Frad time. MB attachment and, to a lesser extent specificity, also increased when driven by Frad near resonance. MB targeting increased with MB concentration, although a shift in behavior was observed with increasing MB-MB interactions and aggregations forming from secondary Frad effects as MB concentration was increased. These secondary Frad effects reduced targeting specificity. Finally, after having validated our approach by testing different parameters with the appropriate controls, we then determined the effects of monodispersity on adhesion efficiency and specific targeting. We observed that both MB targeting efficiency and specificity were greatly enhanced for monodisperse vs polydisperse MBs. Analysis of videomicroscopy images indicated that secondary Frad effects may have disproportionally inhibited targeting of polydisperse MBs. In conclusion, our in vitro results indicate that monodisperse MBs driven near resonance and at a low concentration (∼106 MB/mL) can be used to maximize the adhesion efficiency (up to 88%) and specificity of RGD-MB targeting.