Bubble-Based Microrobots with Rapid Circular Motions for Epithelial Pinning and Drug Delivery.

Remotely powered microrobots are proposed as next-generation vehicles for drug delivery. However, most microrobots swim with linear trajectories and lack the capacity to robustly adhere to soft tissues. This limits their ability to navigate complex biological environments and sustainably release drugs at target sites. In this work, bubble-based microrobots with complex geometries are shown to efficiently swim with non-linear trajectories in a mouse bladder, robustly pin to the epithelium, and slowly release therapeutic drugs. The asymmetric fins on the exterior bodies of the microrobots induce a rapid rotational component to their swimming motions of up to ≈150 body lengths per second. Due to their fast speeds and sharp fins, the microrobots can mechanically pin themselves to the bladder epithelium and endure shear stresses commensurate with urination. Dexamethasone, a small molecule drug used for inflammatory diseases, is encapsulated within the polymeric bodies of the microrobots. The sustained release of the drug is shown to temper inflammation in a manner that surpasses the performance of free drug controls. This system provides a potential strategy to use microrobots to efficiently navigate large volumes, pin at soft tissue boundaries, and release drugs over several days for a range of diseases.

Targeting diffuse midline gliomas: The promise of focused ultrasound-mediated blood-brain barrier opening.

Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma, have among the highest mortality rates of all childhood cancers, despite recent advancements in cancer therapeutics. This is partly because, unlike some CNS tumors, the blood-brain barrier (BBB) of DMG tumor vessels remains intact. The BBB prevents the permeation of many molecular therapies into the brain parenchyma, where the cancer cells reside. Focused ultrasound (FUS) with microbubbles has recently emerged as an innovative and exciting technology that non-invasively permeabilizes the BBB in a small focal region with millimeter precision. In this review, current treatment methods and biological barriers to treating DMGs are discussed. State-of-the-art FUS-mediated BBB opening is then examined, with a focus on the effects of various ultrasound parameters and the treatment of DMGs.

Arabidopsis Root Microbiome Microfluidic (ARMM) Device for Imaging Bacterial Colonization and Morphogenesis of Arabidopsis Roots.

Imaging the spatiotemporal dynamics of host-microbiota interactions is of particular interest for augmenting our understanding of these complex systems. This is especially true of plant-microbe interactions happening around, on, and inside plant roots where relatively little is understood about the dynamics of these systems. Over the past decade, a number of microfluidic devices have been developed to grow plants hydroponically in gnotobiotic conditions and image morphogenesis of the root and/or dynamics with fluorescently labeled bacteria from the plant root microbiome. Here we describe the construction and use of our Arabidopsis Root Microbiome Microfluidic (ARMM) device for imaging fluorescent protein expressing bacteria and their colonization of Arabidopsis roots. In contrast to other plant root imaging devices, we designed this device to have a larger chamber for observing Arabidopsis root elongation and plant-microbe interactions with older seedlings (between 1.5 and 4 weeks after germination) and a 200 µm chamber depth to specifically maintain thin Arabidopsis roots within the focal distance of the confocal microscope. Our device incorporates a new approach to growing Arabidopsis seedlings in screw-top tube caps for simplified germination and transfer to the device. We present representative images from the ARMM device including high resolution cross section images of bacterial colonization at the root surface.

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) has emerged as a promising noninvasive technique to permeabilize the blood-brain barrier (BBB) for drug delivery into the brain. However, the safety and biological consequences of BBB opening (BBBO) remain incompletely understood. This study aims to investigate the effects of two parameters mediating BBBO: microbubble volume dose (MVD) and mechanical index (MI). High-resolution MRI-guided FUS was employed in mouse brains to assess BBBO by manipulating these two parameters. Afterward, the sterile inflammatory response (SIR) was studied 6 h post-FUS treatment. Results demonstrated that both MVD and MI significantly influenced the extent of BBBO, with higher MVD and MI leading to increased permeability. Moreover, RNA sequencing revealed upregulation of major inflammatory pathways and immune cell infiltration after BBBO, indicating the presence and extent of SIR. Gene set enrichment analysis identified 12 gene sets associated with inflammatory responses that were significantly upregulated at higher MVD or MI. A therapeutic window was established between therapeutically relevant BBBO and the onset of SIR, providing operating regimes to avoid damage from stimulation of the NFκB pathway via TNFɑ signaling to apoptosis. These results contribute to the optimization and standardization of BBB opening parameters for safe and effective drug delivery to the brain and further elucidate the underlying molecular mechanisms driving sterile inflammation.

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.

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.

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.

Cavitation Characterization of Size-Isolated Microbubbles in a Vessel Phantom Using Focused Ultrasound.

Pharmaceutical delivery can be noninvasively targeted on-demand by microbubble (MB) assisted focused ultrasound (FUS). Passive cavitation detection (PCD) has become a useful method to obtain real-time feedback on MB activity due to a FUS pulse. Previous work has demonstrated the acoustic PCD response of MBs at a variety of acoustic parameters, but few have explored variations in microbubble parameters. The goal of this study was to determine the acoustic response of different MB size populations and concentrations. Four MB size distributions were prepared (2, 3, 5 µm diameter and polydisperse) and pulled through a 2% agar wall-less vessel phantom. FUS was applied by a 1.515 MHz geometrically focused transducer for 1 ms pulses at 1 Hz PRF and seven distinct mechanical indices (MI) ranging from 0.01 to 1.0 (0.0123 to 1.23 MPa PNP). We found that the onset of harmonic (HCD) and broadband cavitation dose (BCD) depends on the mechanical index, MB size and MB concentration. When matched for MI, the HCD and BCD rise, plateau, and decline as microbubble concentration is increased. Importantly, when microbubble size and concentration are combined into gas volume fraction, all four microbubble size distributions align to similar onset and peak; these results may help guide the planning and control of MB + FUS therapeutic procedures.

Correction: Martinez et al. Cavitation Characterization of Size-Isolated Microbubbles in a Vessel Phantom Using Focused Ultrasound. Pharmaceutics 2022, 14, 1925.

In the original publication [...].

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.

Messenger RNA enrichment using synthetic oligo(T) click nucleic acids.

Enrichment of mRNA is a key step in a number of molecular biology techniques, particularly in the rapidly growing field of transcriptomics. Currently, mRNA is isolated using oligo(thymine) DNA (oligo(dT)) immobilized on solid supports, which binds to the poly(A) tail of mRNA to pull the mRNA out of solution through the use of magnets or centrifugal filters. Here, a simple method to isolate mRNA by complexing it with synthetic click nucleic acids (CNAs) is described. Oligo(T) CNA bound efficiently to mRNA, and because of the insolubility of CNA in water, >90% of mRNA was readily removed from solution using this method. Simple washing, buffer exchange, and heating steps enabled mRNA's enrichment from total RNA, with a yield of 3.1 ± 1.5% of the input total RNA by mass, comparable to the yield from commercially available mRNA enrichment beads. Further, the integrity and activity of mRNA after CNA-facilitated pulldown and release was evaluated through two assays. In vitro translation of EGFP mRNA confirmed the translatability of mRNA into functional protein and RT-qPCR was used to amplify enriched mRNA from total RNA extracts and compare gene expression to results obtained using commercially available products.