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

The study examines how microbubble (MB) formulation affects biological responses to focused ultrasound (FUS)-mediated increases in blood-brain barrier (BBB) permeability. Although FUS+MB can transiently open the BBB for targeted drug delivery, the relationship between the initial magnitude of BBB permeability enhancement and subsequent inflammatory responses has not been fully decoupled. Microbubble characteristics (shell composition, gas, size distribution) influence acoustic behavior, cavitation, and vascular wall stresses. Prior work shows MB size and total gas volume affect BBB opening, but it is unclear whether equivalent BBB permeability achieved with different MB formulations/parameters yields similar inflammatory gene expression. The authors investigate (1) correlations between BBB permeability (MRI-derived Ktrans or contrast enhancement) and transcription of inflammatory mediators 4 h after sonication, and (2) the performance of an acoustic feedback control algorithm based on ultraharmonic emissions across three MB formulations: Definity (polydisperse), BG8774 (polydisperse), and MSB4 (monodisperse).

Background literature indicates: MB shells (lipid/protein/polymer) and size distributions affect circulation half-life, resonance, and collapse probability, thereby modulating cavitation and stresses on vascular walls. Clinical and preclinical BBB opening studies have used Definity, SonoVue/Optison, and in-house MBs. Prior studies show MB size and total gas volume influence the magnitude of BBB opening; gas volume has been suggested as a unifying dose parameter. Positive correlations have been reported between gadolinium enhancement and proinflammatory cytokine expression hours after sonication. Multiple BBB leakage routes exist, potentially producing similar macroscopic permeability but different biological responses. These motivate evaluating whether equivalent BBB opening produced by different MB formulations leads to similar inflammatory transcriptional responses and whether acoustic control can be generalized across formulations.

Study design included three rat cohorts (male Sprague Dawley, n=45 total). Microbubbles tested were Definity (octafluoropropane; polydisperse), BG8774 (C3F7/N2; polydisperse), and MSB4 (C4F10; monodisperse ~4 µm). Size distributions were quantified with a Coulter Counter. All in vivo dosing was normalized to a Definity-equivalent gas volume (20 µL liquid volume/kg Definity equivalent), administered IV via bolus (cohort 1) or infusion (cohorts 2–3 at 0.5 mL/min, 0.5 mL total). Anesthesia used isoflurane with medical air carrier gas. FUS system: MRI-guided preclinical setup with a 580 kHz spherically focused transducer, 10 ms bursts. Acoustic emissions were recorded each burst with a PZT hydrophone. FFTs were computed to analyze harmonics/ultraharmonics (1.5f, 2f, 2.5f) and wideband (inertial cavitation) emissions. Cohort 1 (n=9; 3/MB): Fixed PNP 250 kPa, BRF 0.17 Hz, 110 bursts per target, 8 targets per animal; MB bolus dosing. Second harmonic (2f) decay over time was used to infer circulation half-life (bi-exponential fit; terminal phase). Cohort 2 (n=24): Acoustic feedback control testing. PNP ramp starting at 128 kPa, increasing by 8 kPa per burst until ultraharmonic (1.5f or 2.5f) emissions exceeded baseline+10 SD (trigger), then PNP reduced by 50% for remainder. Two targeting schemes: 4 targets (130 bursts, 1 Hz) or 6 targets (130 bursts, 0.5 Hz). Gadobutrol administered before sonication; CE-T1w MRI ~10 min post-FUS to assess relative contrast enhancement vs non-sonicated region. Histology (H&E) at 24 h or 7 days assessed RBC extravasation. Cohort 3 (n=12; 4/MB): Fixed PNPs (250, 350, 450 kPa), 6 targets per animal (two per PNP across brain quadrants, with one quadrant as non-sonicated control). DCE-MRI began ~15 min after start of sonication: pre-contrast T1 mapping followed by FLASH DCE for 15 min with gadobutrol bolus. Ktrans was computed using a modified Tofts-Kermode model with a reference tissue arterial input function. Evans blue was administered post-DCE, animals sacrificed at 4 h. Tissue from targeted regions and control was collected for RNA extraction. qRT-PCR profiled 84 endothelial-related genes; low-expression genes were excluded; relative expression calculated via ΔΔCt normalized to 5 housekeeping genes; log2 fold changes referenced to within-animal control. Statistics: ANOVA/ANCOVA with post-hoc tests and FDR correction as appropriate. Relationships between emissions and imaging metrics (contrast enhancement or Ktrans), and between Ktrans and gene expression were modeled with linear regressions or ANCOVAs allowing unequal slopes across MB formulations. Significance threshold p=0.05.

- Microbubble characterization: Definity and BG8774 were polydisperse with >80% <2 µm; MSB4 was monodisperse with ~85% between 3–5 µm. - Circulation half-life (cohort 1, mean ± SD): Definity 79 ± 25 s; BG8774 222 ± 73 s; MSB4 206 ± 46 s. Definity was significantly shorter than MSB4 (p=0.04) and trended shorter than BG8774 (p=0.06). - Acoustic feedback control (cohort 2): - Triggering PNP for ultraharmonics (mean ± SD): Definity 370 ± 60 kPa; BG8774 405 ± 51 kPa; MSB4 410 ± 59 kPa. Definity significantly lower than BG8774 (p=0.03) and MSB4 (p=0.01). - CE-T1w relative contrast enhancement (mean ± SD): Definity 145% ± 30%; BG8774 134% ± 19%; MSB4 124% ± 15%. Definity > MSB4 (p<0.001). Variance in enhancement greater with Definity than BG8774 or MSB4 (p<0.01). - Wideband (inertial cavitation) bursts ≤1% for all groups, but proportion higher with BG8774 vs Definity (p=0.02) or MSB4 (p=0.005). - Relationship between exposure-average 2f emissions and contrast enhancement differed by formulation (ANCOVA p<0.001), with significant pairwise differences among all three. - Histology: At 7 days, no RBC extravasation. At 24 h, small RBC extravasations (<300 µm) in <50% of targets across formulations. - Fixed PNP sonications (cohort 3): - Exposure-average 2f vs Ktrans showed semi-log relationships with adjusted r2: Definity 0.59; BG8774 0.56; MSB4 0.42. ANCOVA indicated different relationships (p<0.001); MSB4 differed from Definity and BG8774 (p<0.001 each). - At higher PNPs (350, 450 kPa), mean Ktrans was lower with MSB4 than with Definity or BG8774 (p<0.05). Sustained inertial cavitation occurred mainly when Ktrans > 0.03 min−1 for Definity/BG8774; MSB4 showed significantly lower wideband emissions than the other two (p<0.05). - Gene expression vs Ktrans (4 h post-FUS, cohort 3): - Across all formulations pooled, significant positive correlations (adjusted r2 ≥ ~0.3) with Ktrans for inflammatory/vascular genes including Ccl2, Cxcl1, Il6, Il1b, Icam1, Serpine1, Selp, Fas, Itga5, Timp1, Tnf, Hmox1. - Pooling only Definity+BG8774 strengthened correlations: Cxcl1 (adj r2 0.73), Ccl2 (0.63), Il1b (0.63), Tnf (0.55), Il6 (0.58), Icam1 (0.50), Serpine1 (0.53), etc., all with positive slopes. - Considering formulation, ANCOVA showed significant formulation effects on Ktrans–expression relationships for key inflammatory genes (Tnf, Ccl2, Il1b, Sele). Slopes were significantly steeper for MSB4 vs Definity/BG8774, indicating greater transcriptional response per unit Ktrans with MSB4. Overall, while initial BBB permeability magnitude influenced acute inflammatory transcription, MB formulation (notably monodisperse larger MSB4 vs polydisperse smaller Definity/BG8774) significantly modulated these relationships and acoustic control outcomes.

The study demonstrates that microbubble formulation shapes the link between initial BBB permeability enhancement and subsequent inflammatory gene transcription. Although increases in Ktrans or contrast enhancement generally correlated positively with proinflammatory mediators (e.g., Il1b, Ccl2, Tnf), the strength and slope of these relationships depended on MB characteristics. MSB4, a monodisperse ~4 µm formulation, produced lower Ktrans at comparable 2f emission levels and elicited steeper Ktrans–inflammation slopes than polydisperse, smaller-sized Definity/BG8774. Potential mechanisms include MB-size-driven vascular responses such as vasoconstriction and transient blood flow reductions that could limit tracer delivery (lower Ktrans) yet exacerbate hypoxia-sensitive inflammatory pathways, amplifying cytokine transcription. Acoustic feedback based on ultraharmonic detection effectively minimized inertial cavitation and microhemorrhage across MBs, but thresholds, enhancement magnitude, and variability differed by formulation, indicating that a one-size-fits-all control strategy may not yield consistent biological outcomes. The findings underscore that predicting or controlling inflammation using acoustic emissions and permeability metrics should account for MB size distribution and composition, beyond gas-volume-normalized dose.

Initial BBB permeability enhancement clearly influences acute transcription of proinflammatory cytokines after FUS+MB, but microbubble characteristics (size distribution, shell/gas composition) also modulate this relationship. A monodisperse, larger MB (MSB4) produced stronger inflammatory transcriptional responses per unit Ktrans compared to polydisperse, smaller MBs (Definity, BG8774). An ultraharmonic-based acoustic feedback algorithm effectively limited inertial cavitation and RBC extravasation for all MBs, yet MB formulation affected thresholds, BBB leakage magnitude, and emissions–permeability correlations. Future work should develop formulation-specific acoustic control parameters and investigate temporal dynamics of inflammatory responses, disentangling effects of MB size distribution versus shell/gas composition, and assessing hemodynamic contributions (e.g., vasoconstriction, hypoxia) to biological outcomes.

- MB formulations differed in both size distribution and shell/gas composition, confounding the isolation of size vs composition effects, though comparisons (Definity vs BG8774 size similarity; BG8774 vs MSB4 composition similarity) suggest size distribution substantially influenced Ktrans–inflammation relationships. - Gene expression was assessed at a single early time point (4 h), limiting conclusions about peak magnitude and temporal evolution of responses. - FUS parameters and control algorithm were developed with Definity and may not be optimal for BG8774 or MSB4; formulation-specific optimization could alter safety/efficacy balances.