Microbubble formulation influences inflammatory response to focused ultrasound exposure in the brain

The use of microbubbles in conjunction with focused ultrasound (FUS) to transiently open the blood-brain barrier (BBB) shows promise for targeted drug delivery to the brain. Clinical trials have utilized commercially available microbubbles like Definity and SonoVue, while preclinical research explores both commercial and in-house formulations. However, a detailed understanding of how microbubble characteristics beyond BBB permeability enhancement affect biological responses is lacking. Microbubble composition (lipids, proteins, or polymers), size (1-10 µm), and size distribution (polydisperse vs. monodisperse) significantly impact their response to insonation, influencing factors like circulation half-life and collapse probability. The size distribution, particularly the polydispersity of most clinically approved formulations, is crucial, as it leads to a range of behaviors within the microbubble population at pressures relevant to BBB opening. Previous studies have shown that microbubble size and total gas volume influence the initial magnitude of BBB permeability enhancement, proposed as a unifying dose parameter. While this is relevant for predicting drug delivery and treatment safety, it's unclear if different BBB permeability enhancement regimes lead to similar biological responses. Past research has found correlations between gadolinium contrast enhancement (BBB permeability indicator) and proinflammatory cytokine expression, but it's unknown if equivalent permeability increases using different FUS+MB parameters produce the same inflammatory response. This study aimed to address this gap by investigating biological responses following FUS+MB exposures, comparing outcome measures among three microbubble formulations and assessing the efficacy of a clinically relevant acoustic feedback control strategy. The study hypothesizes that acute biological responses are not solely determined by the initial magnitude of BBB permeability enhancement but also by microbubble characteristics.

The literature extensively documents the use of microbubbles as ultrasound contrast agents and their therapeutic potential for targeted drug delivery across the BBB. Studies using Definity and SonoVue in phase one clinical trials and preclinical work with Optison, SonoVue, and Definity, along with custom-developed formulations, highlight the field's progress. However, research on the influence of microbubble characteristics on biological responses beyond BBB permeability and overt tissue damage remains limited. Existing research indicates the influence of microbubble size and total gas volume on the initial magnitude of BBB permeability enhancement, suggesting total gas volume may be a suitable dose parameter. While understanding factors contributing to the initial magnitude of BBB permeability is crucial for predicting drug delivery and safety, a comprehensive investigation into whether different BBB permeability enhancement regimes yield similar biological responses is needed. Prior research has demonstrated positive correlations between gadolinium contrast enhancement and the expression of proinflammatory cytokines; however, it is unclear if equivalent BBB permeability enhancement achieved through different FUS+MB parameters result in similar inflammatory responses. The varying mechanisms of BBB leakage further complicate the issue, with each contributing to overall permeability but driven by distinct biological processes.

The study used three microbubble formulations: Definity (commercially available, polydisperse), BG8774 (research grade, polydisperse), and MSB4 (research grade, monodisperse). Microbubble size distribution was characterized using a Coulter Counter. Male Sprague Dawley rats were divided into three cohorts: Cohort 1 assessed microbubble half-life in circulation; Cohort 2 evaluated the acoustic feedback control algorithm; and Cohort 3 examined the relationship between BBB permeability enhancement and gene expression. A pre-clinical, MRI-guided FUS system with a spherically focused transducer (580 kHz) was used for sonication. Acoustic emissions were captured using a PZT hydrophone. In Cohort 1, microbubbles were administered as a bolus, and 2f emissions were monitored to estimate half-life. Cohort 2 employed an acoustic feedback control algorithm that adjusted peak negative pressure (PNP) based on ultraharmonic emissions (1.5f). Gadobutrol was used to assess BBB permeability via contrast-enhanced T1-weighted (CE-T1w) MRI. In Cohort 3, animals were sonicated at fixed PNPs (250, 350, 450 kPa), and BBB permeability was quantified using dynamic contrast-enhanced (DCE)-MRI to determine Ktrans (gadobutrol transfer constant). Evans blue dye was used to identify regions of extravasation for tissue sampling. Gene expression of 84 genes was analyzed using qRT-PCR, focusing on inflammation-related genes. Statistical analysis included ANOVA, ANCOVA, and regression analysis.

Microbubble half-life in circulation differed significantly between formulations, with Definity having a shorter half-life than MSB4 (p=0.04). The acoustic feedback control algorithm effectively limited wideband emissions, indicating reduced inertial cavitation, but the proportion of bursts with wideband emissions differed significantly between formulations (BG8774>Definity, BG8774>MSB4, p<0.05). Gadolinium contrast enhancement (BBB permeability) varied significantly between formulations, with Definity exhibiting greater enhancement than MSB4 (p<0.001). The relationship between exposure-average 2f emissions and gadolinium contrast enhancement differed significantly between formulations (p<0.001), with varying slopes for each formulation. At 24 hours post-sonication, minimal red blood cell extravasation was observed across all formulations. Analysis of Ktrans (a quantitative measure of BBB permeability) from DCE-MRI in Cohort 3 showed varying relationships between exposure-average 2f emissions and Ktrans across formulations (p<0.001). The correlation between Ktrans and relative gene expression for inflammatory mediators (e.g., TNF, CCL2, IL1β, SELE) also differed significantly between formulations (p<0.05). For targets sonicated with Definity or BG8774, strong correlations between Ktrans and several inflammation-related genes were observed (adjusted r² >0.5). However, these correlations were significantly different when MSB4 was included, indicating an influence of microbubble characteristics beyond permeability enhancement on inflammation.

The study's findings demonstrate that the inflammatory response to FUS+MB exposure is not solely dependent on the initial magnitude of BBB permeability enhancement but is significantly influenced by microbubble characteristics. The difference in inflammatory responses between MSB4 and the other two formulations, despite similar Ktrans values in some cases, highlights the importance of microbubble size distribution and composition. The observed differences in correlations between acoustic emissions and BBB permeability suggest that the acoustic feedback control strategy needs to be tailored to specific microbubble formulations. The lower Ktrans values observed with MSB4 despite similar emission levels may be attributed to altered blood flow dynamics, potentially caused by increased vessel wall stimulation due to the larger microbubble size, leading to vasoconstriction and reduced gadolinium delivery. This vasoconstriction could also exacerbate the inflammatory response due to reduced blood flow and potential hypoxia. The strong correlations between Ktrans and inflammatory gene expression with Definity and BG8774, but not MSB4, further reinforce the influence of microbubble characteristics on the inflammatory response. Optimizing therapeutic applications requires understanding these intricate interactions.

This study underscores the importance of considering microbubble characteristics (size distribution and composition) when predicting biological responses to FUS+MB-mediated BBB opening. While initial BBB permeability enhancement influences the inflammatory response, microbubble properties play a crucial role. Tailoring acoustic control strategies to specific microbubble formulations is essential to optimize treatment efficacy and safety. Future research should focus on investigating the mechanisms underlying the formulation-dependent differences in inflammatory responses and developing more refined acoustic feedback control algorithms that account for these microbubble-specific effects.

The study has several limitations. The differences in shell and gas compositions of the microbubble formulations make it challenging to isolate the effect of size distribution. Gene expression analysis was conducted at a single early time point, precluding conclusions about the peak magnitude or duration of the response. The FUS+MB parameters and acoustic feedback control algorithm were optimized for Definity, potentially not being optimal for other formulations. Future studies should use more controlled experimental conditions to isolate the effects of microbubble size and composition, investigate the temporal dynamics of inflammatory responses, and develop tailored parameters and control strategies for each microbubble formulation.