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.

Comprehensive Assessment of Blood-Brain Barrier Opening and Sterile Inflammatory Response: Unraveling the Therapeutic Window.

Microbubbles (MBs) combined with focused ultrasound (FUS) have emerged as a promising noninvasive technique to permeabilize the blood-brain barrier (BBB) for drug delivery to the brain. However, the safety and biological consequences of BBB opening remain incompletely understood. This study investigates the effects of varying microbubble volume doses (MVD) and ultrasound mechanical indices (MI) on BBB opening and the sterile inflammatory response (SIR) using high-resolution ultra-high field MRI-guided FUS in mouse brains. The results demonstrate that both MVD and MI significantly influence the extent of BBB opening, with higher doses and mechanical indices leading to increased permeability. Moreover, RNA sequencing reveals upregulated inflammatory pathways and immune cell infiltration after BBB opening, suggesting the presence and extent of SIR. Gene set enrichment analysis identifies 12 gene sets associated with inflammatory responses that are upregulated at higher doses of MVD or MI. A therapeutic window is established between significant BBB opening and the onset of SIR, providing operating regimes for avoiding each three classes of increasing damage from stimulation of the NFκB pathway via TNFL signaling to apoptosis. This study contributes to the optimization and standardization of BBB opening parameters for safe and effective drug delivery to the brain and sheds light on the underlying molecular mechanisms of the sterile inflammatory response. Significance Statement: The significance of this study lies in its comprehensive investigation of microbubble-facilitated focused ultrasound for blood-brain barrier (BBB) opening. By systematically exploring various combinations of microbubble volume doses and ultrasound mechanical indices, the study reveals their direct impact on the extent of BBB permeability and the induction of sterile inflammatory response (SIR). The establishment of a therapeutic window between significant BBB opening and the onset of SIR provides critical insights for safe and targeted drug delivery to the brain. These findings advance our understanding of the biological consequences of BBB opening and contribute to optimizing parameters for clinical applications, thus minimizing potential health risks, and maximizing the therapeutic potential of this technique.

MRI-guided focused ultrasound blood-brain barrier opening increases drug delivery and efficacy in a diffuse midline glioma mouse model.

Background: Diffuse intrinsic pontine glioma (DIPG) is the most common and deadliest pediatric brainstem tumor and is difficult to treat with chemotherapy in part due to the blood-brain barrier (BBB). Focused ultrasound (FUS) and microbubbles (MBs) have been shown to cause BBB opening, allowing larger chemotherapeutics to enter the parenchyma. Panobinostat is an example of a promising in vitro agent in DIPG with poor clinical efficacy due to low BBB penetrance. In this study, we hypothesized that using FUS to disrupt the BBB allows higher concentrations of panobinostat to accumulate in the tumor, providing a therapeutic effect. Methods: Mice were orthotopically injected with a patient-derived diffuse midline glioma (DMG) cell line, BT245. MRI was used to guide FUS/MB (1.5 MHz, 0.615 MPa peak negative pressure, 1 Hz pulse repetition frequency, 10-ms pulse length, 3 min treatment time)/(25 µL/kg, i.v.) targeting to the tumor location. Results: In animals receiving panobinostat (10 mg/kg, i.p.) in combination with FUS/MB, a 3-fold increase in tumor panobinostat concentration was observed, without significant increase of the drug in the forebrain. In mice receiving 3 weekly treatments, the combination of panobinostat and FUS/MB led to a 71% reduction of tumor volumes (P = .01). Furthermore, we showed the first survival benefit from FUS/MB improved delivery increasing the mean survival from 21 to 31 days (P < .0001). Conclusions: Our study demonstrates that FUS-mediated BBB disruption can increase the delivery of panobinostat to an orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.

MRI-Guided Focused Ultrasound Blood-Brain Barrier Opening Increases Drug Delivery and Efficacy in a Diffuse Midline Glioma Mouse Model.

Diffuse intrinsic pontine glioma (DIPG) is the most common and deadliest pediatric brainstem tumor and is difficult to treat with chemotherapy in part due to the blood-brain barrier (BBB). Focused ultrasound (FUS) and microbubbles (MBs) have been shown to cause BBB disruption (BBBD), allowing larger chemotherapeutics to enter the parenchyma. Panobinostat is an example of a promising in vitro agent in DIPG with poor clinical efficacy due to low BBB penetrance. In this study, we hypothesized that using FUS to disrupt the BBB allows higher concentrations of panobinostat to accumulate in the tumor, providing a therapeutic effect. Mice were orthotopically injected with a patient-derived DMG cell line, BT-245. MRI was used to guide FUS/MB (1.5 MHz, 0.615 MPa PNP, 1 Hz PRF, 10 ms PL, 3 min treatment time) / (25 µL/kg, IV) targeting to the tumor location. In animals receiving panobinostat (10 mg/kg, IP) in combination with FUS/MB, a 3-fold increase in tumor panobinostat concentration was observed, with only insignificant increase of the drug in the forebrain. In mice receiving three weekly treatments, the combination of panobinostat and FUS/MB led to a 71% reduction of tumor volumes by MRI ( p = 0.01). Furthermore, FUS/MB improved the mean survival from 21 to 31 days ( p < 0.0001). Our study demonstrates that FUS-mediated BBBD can increase the delivery of panobinostat to an orthotopic DMG tumor, providing a strong therapeutic effect and increased survival. One Sentence Summary: FUS and microbubbles can increase the delivery of panobinostat to a patient-derived xenograft (PDX) orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.

Microbubble Size and Dose Effects on Pharmacokinetics.

Optimization of contrast-enhanced imaging and focused ultrasound therapy requires a comprehensive understanding of in vivo microbubble (MB) pharmacokinetics. Prior studies have focused pharmacokinetic analysis on indirect techniques, such as ultrasound imaging of the blood pool and gas chromatography of exhaled gases. The goal of this work was to measure the MB concentration directly in blood and correlate the pharmacokinetic parameters with the MB size and dose. MB volume dose (MVD) was chosen to combine the size distribution and number into a single-dose parameter. Different MB sizes (2, 3, and 5 µm diameter) at 5-40 µL/kg MVD were intravenously injected. Blood samples were withdrawn at different times (1-10 min) and analyzed by image processing. We found that for an MVD threshold < 40 µL/kg for 2 and 3 µm and <10 µL/kg for 5 µm, MB clearance followed first-order kinetics. When matching MVD, MBs of different sizes had comparable half-lives, indicating that gas dissolution and elimination by the lungs are the primary mechanisms for elimination. Above the MVD threshold, MB clearance followed biexponential kinetics, suggesting a second elimination mechanism mediated by organ retention, possibly in the lung, liver, and spleen. In conclusion, we present the first direct MB pharmacokinetic study, demonstrate the utility of MVD as a unified dose metric, and provide insights into the mechanisms of MB clearance from circulation.

Contrast-Enhanced Sonography with Biomimetic Lung Surfactant Nanodrops.

Nanodrops comprising a perfluorocarbon liquid core can be acoustically vaporized into echogenic microbubbles for ultrasound imaging. Packaging the microbubble in its condensed liquid state provides some advantages, including in situ activation of the acoustic signal, longer circulation persistence, and the advent of expanded diagnostic and therapeutic applications in pathologies which exhibit compromised vasculature. One obstacle to clinical translation is the inability of the limited surfactant present on the nanodrop to encapsulate the greatly expanded microbubble interface, resulting in ephemeral microbubbles with limited utility. In this study, we examine a biomimetic approach to stabilize an expanding gas surface by employing the lung surfactant replacement, beractant. Lung surfactant contains a suite of lipids and proteins that provide efficient shuttling of material from bilayer folds to the monolayer surface. We hypothesized that beractant would improve stability of acoustically vaporized microbubbles. To test this hypothesis, we characterized beractant surface dilation mechanics and revealed a novel biophysical phenomenon of rapid interfacial melting, spreading, and resolidification. We then harnessed this unique functionality to increase the stability and echogenicity of microbubbles produced after acoustic droplet vaporization for in vivo ultrasound imaging. Such biomimetic lung surfactant-stabilized nanodrops may be useful for applications in ultrasound imaging and therapy.

Acoustic Nanodrops for Biomedical Applications.

Acoustic nanodrops are designed to vaporize into ultrasound-responsive microbubbles, which presents certain challenges nonexistent for conventional nano-emulsions. The requirements of biocompatibility, vaporizability and colloidal stability has focused research on perfluorocarbons (PFCs). Shorter PFCs yield better vaporizability via their lower critical temperature, but they also dissolve more easily owing to their higher vapor pressure and solubility. Thus, acoustic nanodrops have required a tradeoff between vaporizability and colloidal stability in vivo. The recent advent of vaporizable endoskeletal droplets, which are both stable and vaporizable, may have solved this problem. The purpose of this review is to justify this premise by pointing out the beneficial properties of acoustic nanodrops, providing an analysis of vaporization and dissolution mechanisms, and reviewing current biomedical applications.

Phospholipid Oxygen Microbubbles for Image-Guided Therapy.

In recent work, oxygen microbubbles (OMB) have been shown to oxygenate hypoxic tumors, increase radio-sensitivity and improve tumor control by radiation therapy. Compared to intra-tumoral injection, intravenous delivery of adjuvant agents such as OMBs for radiotherapy offers an attractive means of achieving true theranostic function in a minimally invasive manner via contrast-enhanced ultrasound (CEUS), while reducing the risk of injury, infection or displacing tumor cells. However, short intravascular circulation times with conventional DSPC-lipid OMBs may lead to premature off-target dissolution of OMBs with an associated reduction in tumoral oxygen delivery. Prior work on microbubble stability and gas exchange suggests that increasing phospholipid acyl-chain length of the encapsulating shell and OMB size may increase circulation persistence, delivery and dissolved oxygen content. In the following studies, we investigate the effect of two phospholipid shell compositions, DSPC (C18:0) and DBPC (C22:0), as well as three size distributions (0.5-2 µm, 2-10 µm and polydisperse) on OMB circulation persistence utilizing CEUS in the kidneys of live C57B1/6 male and female mice, six weeks of age. DBPC OMB formulations demonstrated increased circulation half-lives versus DSPC formulations (2.4 ± 1.0 vs. 0.6 ± 0.5 s, p<0.01 for 2-10 µm), as well as an increased maximum intensity by over tenfold (p<0.01). Size-dependent effects remained consistent across both formulations with larger 2-10 µm microbubbles demonstrating significantly increased half-lives (2.4 ± 1.0 vs. 0.3 ± 0.2 s, p < 0.01) compared to smaller 0.5-2 µm formulations of DBPC. These studies indicate that DBPC 2-10 µm OMBs may be improved adjuvant agents for radiotherapy with significant potential for CEUS interrogation.

Ultrasound-mediated delivery of siESE complexed with microbubbles attenuates HER2+/- cell line proliferation and tumor growth in rodent models of breast cancer.

The highly tunable, noninvasive and spatially targeted nature of microbubble-enhanced, ultrasound-guided (MB+US) drug delivery makes it desirable for a wide variety of therapies. In breast cancer, both HER2+ and HER2- type neoplasms pose significant challenges to conventional therapeutics in greater than 40% of breast cancer patients, even with the widespread application of biologics such as trastuzumab. To address this therapeutic challenge, we examined the novel combination of tumor-injected microbubble-bound siRNA complexes and monodisperse size-isolated microbubbles (4-µm diameter) to attenuate tumor growth in vivo, as well as MB+US-facilitated shRNA and siRNA knockdown of ESE-1, an effector linked to dysregulated HER2 expression in HER2+/- cell line propagation. We first screened six variants of siESE and shESE for efficient knockdown of ESE in breast cancer cell lines. We demonstrated efficient reduction of BT-474 (PR+, ER+, HER2+; luminal B) and MDA-MB-468 (PR-, ER-, HER2-; triple-negative) clonogenicity and non-adherent growth after knockdown of ESE-1. A significant reduction in proliferative potential was seen for both cell lines using MB+US to deliver shESE and siESE. We then demonstrated significant attenuation of BT-474 xenograft tumor growth in Nod/SCID female mice using direct injection of microbubble-adsorbed siESE to the tumor and subsequent sonication. Our results suggest a positive effect on drug delivery from MB+US, and highlights the feasibility of using RNAi and MB+US for breast cancer pathologies. RNAi coupled with MB+US may also be an effective theranostic approach to treat other acoustically accessible tumors, such as melanoma, thyroid, parotid and skin cancer.

Reverse engineering the ultrasound contrast agent.

In this review, a brief history and current state-of-the-art is given to stimulate the rational design of new microbubbles through the reverse engineering of current ultrasound contrast agents (UCAs). It is shown that an effective microbubble should be biocompatible, echogenic and stable. Physical mechanisms and engineering calculations have been provided to illustrate these properties and how they can be achieved. The reverse-engineering design paradigm is applied to study current FDA-approved and commercially available UCAs. Given the sophistication of microbubble designs reported in the literature, rapid development and adoption of ultrasound device hardware and techniques, and the growing number of revolutionary biomedical applications moving toward the clinic, the field of Microbubble Engineering is fertile for breakthroughs in next-generation UCA technology. It is up to current and future microbubble engineers and clinicians to push forward with regulatory approval and clinical adoption of advanced UCA technologies in the years to come.

State-of-the-art of microbubble-assisted blood-brain barrier disruption.

Focused ultrasound with microbubbles promises unprecedented advantages for blood-brain barrier disruption over existing intracranial drug delivery methods, as well as a significant number of tunable parameters that affect its safety and efficacy. This review provides an engineering perspective on the state-of-the-art of the technology, considering the mechanism of action, effects of microbubble properties, ultrasound parameters and physiological variables, as well as safety and potential therapeutic applications. Emphasis is placed on the use of unified parameters, such as microbubble volume dose (MVD) and ultrasound mechanical index, to optimize the procedure and establish safety limits. It is concluded that, while efficacy has been demonstrated in several animal models with a wide range of payloads, acceptable measures of safety should be adopted to accelerate collaboration and improve understanding and clinical relevance.

Microbubble gas volume: A unifying dose parameter in blood-brain barrier opening by focused ultrasound.

Focused ultrasound with microbubbles is being developed to transiently, locally and noninvasively open the blood-brain barrier (BBB) for improved pharmaceutical delivery. Prior work has demonstrated that, for a given concentration dose, microbubble size affects both the intravascular circulation persistence and extent of BBB opening. When matched to gas volume dose, however, the circulation half-life was found to be independent of microbubble size. In order to determine whether this holds true for BBB opening as well, we independently measured the effects of microbubble size (2 vs. 6 µm diameter) and concentration, covering a range of overlapping gas volume doses (1-40 µL/kg). We first demonstrated precise targeting and a linear dose-response of Evans Blue dye extravasation to the rat striatum for a set of constant microbubble and ultrasound parameters. We found that dye extravasation increased linearly with gas volume dose, with data points from both microbubble sizes collapsing to a single line. A linear trend was observed for both the initial sonication (R2=0.90) and a second sonication on the contralateral side (R2=0.68). Based on these results, we conclude that microbubble gas volume dose, not size, determines the extent of BBB opening by focused ultrasound (1 MHz, ~0.5 MPa at the focus). This result may simplify planning for focused ultrasound treatments by constraining the protocol to a single microbubble parameter - gas volume dose - which gives equivalent results for varying size distributions. Finally, using optimal parameters determined for Evan Blue, we demonstrated gene delivery and expression using a viral vector, dsAAV1-CMV-EGFP, one week after BBB disruption, which allowed us to qualitatively evaluate neuronal health.

High Efficiency Molecular Delivery with Sequential Low-Energy Sonoporation Bursts.

Microbubbles interact with ultrasound to induce transient microscopic pores in the cellular plasma membrane in a highly localized thermo-mechanical process called sonoporation. Theranostic applications of in vitro sonoporation include molecular delivery (e.g., transfection, drug loading and cell labeling), as well as molecular extraction for measuring intracellular biomarkers, such as proteins and mRNA. Prior research focusing mainly on the effects of acoustic forcing with polydisperse microbubbles has identified a "soft limit" of sonoporation efficiency at 50% when including dead and lysed cells. We show here that this limit can be exceeded with the judicious use of monodisperse microbubbles driven by a physiotherapy device (1.0 MHz, 2.0 W/cm(2), 10% duty cycle). We first examined the effects of microbubble size and found that small-diameter microbubbles (2 µm) deliver more instantaneous power than larger microbubbles (4 & 6 µm). However, owing to rapid fragmentation and a short half-life (0.7 s for 2 µm; 13.3 s for 6 µm), they also deliver less energy over the sonoporation time. This translates to a higher ratio of FITC-dextran (70 kDa) uptake to cell death/lysis (4:1 for 2 µm; 1:2 for 6 µm) in suspended HeLa cells after a single sonoporation. Sequential sonoporations (up to four) were consequently employed to increase molecular delivery. Peak uptake was found to be 66.1 ± 1.2% (n=3) after two sonoporations when properly accounting for cell lysis (7.0 ± 5.6%) and death (17.9 ± 2.0%), thus overcoming the previously reported soft limit. Substitution of TRITC-dextran (70 kDa) on the second sonoporation confirmed the effects were multiplicative. Overall, this study demonstrates the possibility of utilizing monodisperse small-diameter microbubbles as a means to achieve multiple low-energy sonoporation bursts for efficient in vitro cellular uptake and sequential molecular delivery.

Nogo receptor deletion and multimodal exercise improve distinct aspects of recovery in cervical spinal cord injury.

We tested the ability of two plasticity-promoting approaches to enhance recovery in a mouse model of incomplete spinal cord injury (SCI). Genetically, we reduced myelin-mediated inhibition of neural plasticity through Nogo66-receptor (NgR) gene deletion. Behaviorally, we utilized a novel multimodal exercise training paradigm. Adult mice of wild-type or NgR-null genotype were subjected to partial lateral hemisection (LHx) at C3-C4 with the intent of producing anatomically and functionally mild deficits. Exercise training or control treatment proceeded for 14 weeks. Behavioral outcomes were assessed prior to tract tracing and histological analysis. Genotype and training exerted differing effects on performance; training improved performance on a test related to the training regimen (task-specific benefit), whereas genotype also improved performance on more generalized behaviors (task-non-specific benefit). There were no significant histological differences across genotype or training assignment with regard to lesion size or axonal tract staining. Thus either NgR gene deletion or exercise training benefits mice with mild cervical spinal injury. In this lesion model, the effects of NgR deletion and training were not synergistic for the tasks assessed. Further work is required to optimize the interaction between pharmacological and physical interventions for SCI.

Ibuprofen enhances recovery from spinal cord injury by limiting tissue loss and stimulating axonal growth.

The GTP-binding protein RhoA regulates microfilament dynamics in many cell types and mediates the inhibition of axonal regeneration by myelin and chondroitin sulfate proteoglycans. Unlike most other nonsteroidal anti-inflammatory drugs, ibuprofen suppresses basal RhoA activity (Zhou et al., 2003). A recent report suggested that ibuprofen promotes corticospinal axon regeneration after spinal cord injury (Fu et al., 2007). Here, we confirm that ibuprofen reduces ligand-induced Rho signaling and myelin-induced inhibition of neurite outgrowth in vitro. Following 4 weeks of subcutaneous administration of ibuprofen, beginning 3 days after spinal cord contusion, animals recovered walking function to a greater degree, with twice as many rats achieving a hind limb weight-bearing status. We examined the relative role of tissue sparing, axonal sprouting, and axonal regeneration in the action of ibuprofen. Histologically, ibuprofen-treated animals display an increase in spared tissue without an alteration in astrocytic or microglial reaction. Ibuprofen increases axonal sprouting from serotonergic raphespinal axons, and from rostral corticospinal fibers in the injured spinal cord, but does not permit caudal corticospinal regeneration after spinal contusion. Treatment of mice with complete spinal cord transection demonstrates long-distance raphespinal axon regeneration in the presence of ibuprofen. Thus, administration of ibuprofen improves the recovery of rats from a clinically relevant spinal cord trauma by protecting tissue, stimulating axonal sprouting, and allowing a minor degree of raphespinal regeneration.