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Diffuse intrinsic pontine glioma (DIPG) is the most common and deadliest brainstem tumor in children. Focused ultrasound combined with microbubble-mediated BBB opening (FUS-BBBO) is a promising technique for overcoming the frequently intact blood-brain barrier (BBB) in DIPG to enhance therapeutic drug delivery to the brainstem. Since DIPG is highly diffusive, large-volume FUS-BBBO is needed to cover the entire tumor region. The objective of this study was to determine the optimal treatment strategy to achieve efficient and homogeneous large-volume BBBO at the brainstem for the delivery of an immune checkpoint inhibitor, anti-PD-L1 antibody (aPD-L1).Two critical parameters for large-volume FUS-BBBO, multi-point sonication pattern (interleaved vs. serial) and microbubble injection method (bolus vs. infusion), were evaluated by treating mice with four combinations of these two parameters. 2D Passive cavitation imaging (PCI) was performed for monitoring the large-volume sonication.Interleaved sonication combined with bolus injection of microbubbles resulted in 1.29 to 2.06 folds higher efficiency than other strategies as evaluated by Evans blue extravasation. The average coefficient of variation of the Evans blue delivery was 0.66 for interleaved sonication with bolus injection, compared to 0.68-0.88 for all other strategies. Similar trend was also observed in the quantified total cavitation dose and coefficient of variance of the cavitation dose. This strategy was then applied to deliver fluorescently labeled aPD-L1 which was quantified using fluorescence imaging. A strong segmented linear correlation (RFindings from this study suggest that efficient and homogeneous large-volume FUS-BBBO can be achieved by interleaved sonication combined with bolus injection of microbubbles, and the efficiency and homogeneity can be monitored by PCI.

Torpor is an energy-conserving state in which animals dramatically decrease their metabolic rate and body temperature to survive harsh environmental conditions. Here, we report the noninvasive, precise and safe induction of a torpor-like hypothermic and hypometabolic state in rodents by remote transcranial ultrasound stimulation at the hypothalamus preoptic area (POA). We achieve a long-lasting (>24 h) torpor-like state in mice via closed-loop feedback control of ultrasound stimulation with automated detection of body temperature. Ultrasound-induced hypothermia and hypometabolism (UIH) is triggered by activation of POA neurons, involves the dorsomedial hypothalamus as a downstream brain region and subsequent inhibition of thermogenic brown adipose tissue. Single-nucleus RNA-sequencing of POA neurons reveals TRPM2 as an ultrasound-sensitive ion channel, the knockdown of which suppresses UIH. We also demonstrate that UIH is feasible in a non-torpid animal, the rat. Our findings establish UIH as a promising technology for the noninvasive and safe induction of a torpor-like state.

The glymphatic system is a perivascular fluid transport system for waste clearance. Glymphatic transport is believed to be driven by the perivascular pumping effect created by the pulsation of the arterial wall caused by the cardiac cycle. Ultrasound sonication of circulating microbubbles (MBs) in the cerebral vasculature induces volumetric expansion and contraction of MBs that push and pull on the vessel wall to generate a MB pumping effect. The objective of this study was to evaluate whether glymphatic transport can be mechanically manipulated by focused ultrasound (FUS) sonication of MBs. The glymphatic pathway in intact mouse brains was studied using intranasal administration of fluorescently labeled albumin as fluid tracers, followed by FUS sonication at a deep brain target (thalamus) in the presence of intravenously injected MBs. Intracisternal magna injection, the conventional technique used in studying glymphatic transport, was employed to provide a comparative reference. Three-dimensional confocal microscopy imaging of optically cleared brain tissue revealed that FUS sonication enhanced the transport of fluorescently labeled albumin tracer in the perivascular space (PVS) along microvessels, primarily the arterioles. We also obtained evidence of FUS-enhanced penetration of the albumin tracer from the PVS into the interstitial space. This study revealed that ultrasound combined with circulating MBs could mechanically enhance glymphatic transport in the brain.

Cavitation is the fundamental physical mechanism of various focused ultrasound (FUS)-mediated therapies in the brain. Accurately knowing the three-dimensional (3-D) location of cavitation in real-time can improve the targeting accuracy and avoid off-target tissue damage. Existing techniques for 3-D passive transcranial cavitation detection require the use of expensive and complicated hemispherical phased arrays with 128 or 256 elements. The objective of this study was to investigate the feasibility of using four sensors for transcranial 3-D localization of cavitation. Differential microbubble cavitation detection combined with the time difference of arrival algorithm was developed for the localization using the four sensors. Numerical simulation using k-Wave toolbox was performed to validate the proposed method for transcranial cavitation source localization. The sensors with a center frequency of 2.25 MHz and a 6 dB bandwidth of 1.39 MHz were used to locate cavitation generated by FUS (500 kHz) sonication of microbubbles that were injected into a tube positioned inside an ex vivo human skullcap. Cavitation emissions from the microbubbles were detected transcranially using the four sensors. Both simulation and experimental studies found that the proposed method achieved accurate 3-D cavitation localization. When the cavitation source was located within 30 mm from the geometric center of the sensor network, the accuracy of the localization method with the skull was measured to be 1.9±1.0 mm, which was not significantly different from that without the skull (1.7 ± 0.5 mm). The accuracy decreased as the cavitation source was away from the geometric center of the sensor network. It also decreased as the pulse length increased. Its accuracy was not significantly affected by the sensor position relative to the skull. In summary, four sensors combined with the proposed localization algorithm offer a simple approach for 3-D transcranial cavitation localization.

Focused ultrasound combined with circulating microbubbles (FUS+MB) can transiently enhance blood-brain barrier (BBB) permeability at targeted brain locations. Its great promise in improving drug delivery to the brain is reflected by a rapidly growing number of clinical trials using FUS+MB to treat various brain diseases. As the clinical applications of FUS+MB continue to expand, it is critical to have a better understanding of the molecular and cellular effects induced by FUS+MB to enhance the efficacy of current treatment and enable the discovery of new therapeutic strategies. Existing studies primarily focus on FUS+MB-induced effects on brain endothelial cells, the major cellular component of BBB. However, bioeffects induced by FUS+MB expand beyond the BBB to cells surrounding blood vessels, including astrocytes, microglia, and neurons. Together these cell types comprise the neurovascular unit (NVU). In this review, we examine cell-type-specific bioeffects of FUS+MB on different NVU components, including enhanced permeability in endothelial cells, activation of astrocytes and microglia, as well as increased intraneuron protein metabolism and neuronal activity. Finally, we discuss knowledge gaps that must be addressed to further advance clinical applications of FUS+MB.

The glymphatic system is a perivascular fluid transport system for waste clearance. Glymphatic transportation is believed to be driven by the perivascular pumping effect generated by arterial wall pulsation induced by the cardiac cycle. Ultrasound sonication of circulating microbubbles in the cerebral vasculature induces volumetric expansion and contraction of microbubbles that push and pull on the vessel wall to generate a microbubble pumping effect. The objective of this study was to evaluate whether glymphatic transportation can be mechanically manipulated by focused ultrasound (FUS) sonication of microbubbles. The glymphatic pathway in intact mouse brains was studied using intranasal administration of fluorescently labeled albumin as a fluid tracer followed by FUS sonication at a deep brain target (thalamus) in the presence of intravenously injected microbubbles. Three-dimensional confocal microscopy imaging of optically cleared brain tissue revealed that FUS sonication enhanced the transport of fluorescently labeled albumin tracer in the perivascular space along microvessels, primarily the arterioles. We also obtained evidence of FUS-enhanced penetration of the albumin tracer from the perivascular space into the interstitial space. This study revealed that ultrasound combined with circulating microbubbles could noninvasively enhance glymphatic transportation in the brain.Significance StatementThe glymphatic system is a waste clearance system in the brain analogous to the lymphatic system in peripheral organs. Glymphatic system impairment might contribute to brain disease pathologies, including those in neurodegenerative diseases, traumatic brain injury, and stroke. This study revealed that ultrasound could mechanically enhance glymphatic transportation. This result opens opportunities for using ultrasound to probe the role of the glymphatic system in brain function and brain diseases. Findings from this study suggest that ultrasound can be utilized as a noninvasive/nonpharmacological approach to mitigate brain diseases caused by impaired glymphatic function.

BACKGROUND: Critical advances in the investigation of brain functions and treatment of brain disorders are hindered by our inability to selectively target neurons in a noninvasive manner in the deep brain. OBJECTIVE: This study aimed to develop sonothermogenetics for noninvasive, deep-penetrating, and cell-type-specific neuromodulation by combining a thermosensitive ion channel TRPV1 with focused ultrasound (FUS)-induced brief, non-noxious thermal effect. METHODS: The sensitivity of TRPV1 to FUS sonication was evaluated in vitro. It was followed by in vivo assessment of the success rate of sonothermogenetics in the activation of genetically defined neurons in the mouse brain by two-photon microscopic calcium imaging. Behavioral response evoked by sonothermogenetic stimulation at a deep brain target was recorded in freely moving mice. Immunohistochemistry staining of ex vivo brain slices was performed to evaluate the safety of FUS sonication. RESULTS: TRPV1 was found to be an ultrasound-sensitive ion channel. FUS sonication at the mouse brain in vivo selectively activated neurons that were genetically modified to express TRPV1. Temporally precise activation of TRPV1-expressing neurons was achieved with its success rate linearly correlated with the peak temperature within the FUS-targeted brain region as measured by in vivo magnetic resonance thermometry. FUS stimulation of TRPV1-expressing neurons at the striatum repeatedly evoked locomotor behavior in freely moving mice. FUS sonication was confirmed to be safe based on inspection of neuronal integrity, inflammation, and apoptosis markers. CONCLUSIONS: This noninvasive and cell-type-specific neuromodulation approach with the capability to target the deep brain has the promise to advance the study of the intact nervous system and uncover new ways to treat neurological disorders.

Background Leptomeningeal disease and hydrocephalus are present in up to 30% of patients with diffuse intrinsic pontine glioma (DIPG), however there are no animal models of cerebrospinal fluid (CSF) dissemination. As the tumor–CSF–ependymal microenvironment may play an important role in tumor pathogenesis, we identified characteristics of the Nestin-tumor virus A (Nestin-Tva) genetically engineered mouse model that make it ideal to study the interaction of tumor cells with the CSF and its associated pathways with implications for the development of treatment approaches to address CSF dissemination in DIPG. Methods A Nestin-Tva model of DIPG utilizing the 3 most common DIPG genetic alterations (H3.3K27M, PDGF-B, and p53) was used for this study. All mice underwent MR imaging and a subset underwent histopathologic analysis with H&E and immunostaining. Results Tumor dissemination within the CSF pathways (ventricles, leptomeninges) from the subependyma was present in 76% (25/33) of mice, with invasion of the choroid plexus, disruption of the ciliated ependyma and regional subependymal fluid accumulation. Ventricular enlargement consistent with hydrocephalus was present in 94% (31/33). Ventricle volume correlated with region-specific transependymal CSF flow (periventricular T2 signal), localized anterior to the lateral ventricles. Conclusions This is the first study to report CSF pathway tumor dissemination associated with subependymal tumor in an animal model of DIPG and is representative of CSF dissemination seen clinically. Understanding the CSF–tumor–ependymal microenvironment has significant implications for treatment of DIPG through targeting mechanisms of tumor spread within the CSF pathways.

Intranasal delivery provides non-invasive delivery of drugs from the nose to the brain bypassing the blood-brain barrier (BBB). We previously reported the focused ultrasound (FUS)-mediated intranasal delivery (FUSIN) of various agents in mice using FUS. However, no study has investigated the feasibility of FUSIN in large animals. The objective of this study was to investigate the feasibility of MR-guided FUS-mediated intranasal delivery in a large animal model. Pigs were used as the large animal model due to their clinical relevance to humans. Fluorescence-labeled albumin was mixed with an MRI contrast agent and administrated intranasally to the pig using a catheter. Followed by intravenous injection of microbubbles, FUS sonication was performed at two brain regions, the cortex and brainstem. T1-weighted MR images showed enhancement of MRI contrast agent at the olfactory region of the pig nose. Fluorescence imaging displayed enhanced fluorescence intensity at the FUS-targeted brain regions. These findings suggest that MR-guided FUS can achieve noninvasive and localized delivery of intranasally administered agents to the brain in large animals.

The glymphatic system, a perivascular network in the brain, regulates the exchange of cerebrospinal fluid and interstitial fluid in the perivascular space. Focused ultrasound combined with microbubbles (FUSMB) has been recently shown feasible to manipulate the glymphatic transportation by enhancing the intranasal delivery of agents in the perivascular space. The objective of this study was to reveal the dynamics of FUSMB-enhanced agent transport in the glymphatic system. A ring-shaped FUS transducer was confocally aligned with an objective of a two-photon microscope (2PM). Fluorescently labeled albumin was administered to the mouse nose, where it transported along olfactory nerve and trigeminal nerve from nose to brain and then spread in perivascular space in the brain. FUS sonication was performed after intravenous injection of microbubbles. In vivo 2PM recorded the dynamics of agent transportation before, during, and after FUS sonication. Time-lapse recording showed that FUSMB enhanced the agent accumulation at the perivascular space. FUS significantly increased the accumulation of the agent in the perivascular space by 1.2-folds immediately after the sonication (p = 0.0325), and 1.6-folds at 5 min after the sonication (p

Focused ultrasound-mediated intranasal (FUSIN) delivery is a recently proposed technique that bypasses the blood-brain barrier to achieve noninvasive and localized brain drug delivery. The goal of this study was to characterize FUSIN drug delivery outcomes in mice with regard to its dependency on several critical experimental factors, including the time interval between IN administration and FUS sonication (T(lag1)), the FUS pressure, and the time for sacrificing the mice post-FUS (T(lag2)). Wild-type mice were treated by FUSIN delivery of near-infrared fluorescent dye-labeled bovine serum albumin (800CW-BSA, used as a model agent). 800CW-BSA was intranasally administered to the mice in vivo, followed by intravenous injection of microbubbles and FUS sonication at the brainstem. Fluorescence imaging of ex vivo mouse brain slices was used to quantify the delivery outcomes of 800CW-BSA. Major organs, along with the nasal tissue and trigeminal nerve, were harvested to assess the biodistribution of 800CW-BSA. The delivery outcome of 800CW-BSA was the highest at the brainstem when T(lag1) was 0.5 h, which was on average 24.5-fold, 5.4-fold, and 21.6-fold higher than those of the IN only, T(lag1) = 1.5 h, and T(lag1) = 4.0 h, respectively. The FUSIN delivery outcome at the lowest pressure level, 0.43 MPa, was on average 1.8-fold and 3.7-fold higher than those at 0.56 MPa and 0.70 MPa, respectively. The mean concentration of 800CW-BSA in the brainstem after FUSIN delivery decreased from 0.5 h to 4.0 h post-FUS. The accumulation of 800CW-BSA was low in the heart, lung, spleen, kidneys, and liver, but high in the stomach and intestines. This study revealed the unique characteristics of FUSIN as a noninvasive, efficient, and localized brain drug delivery technique.

Diffuse intrinsic pontine glioma (DIPG) is an invasive pediatric brainstem malignancy exclusively in children without effective treatment due to the often-intact blood-brain tumor barrier (BBTB), an impediment to the delivery of therapeutics. Herein, we used focused ultrasound (FUS) to transiently open BBTB and delivered radiolabeled nanoclusters ((64)Cu-CuNCs) to tumors for positron emission tomography (PET) imaging and quantification in a mouse DIPG model. First, we optimized FUS acoustic pressure to open the blood-brain barrier (BBB) for effective delivery of (64)Cu-CuNCs to pons in wildtype mice. Then the optimized FUS pressure was used to deliver radiolabeled agents in DIPG mouse. Magnetic resonance imaging (MRI)-guided FUS-induced BBTB opening was demonstrated using a low molecular weight, short-lived (68)Ga-DOTA-ECL1i radiotracer and PET/CT before and after treatment. We then compared the delivery efficiency of (64)Cu-CuNCs to DIPG tumor with and without FUS treatment and demonstrated the FUS-enhanced delivery and time-dependent diffusion of (64)Cu-CuNCs within the tumor.

The blood-brain barrier (BBB) is the major obstacle for brain drug delivery and limits the treatment options for central nervous system diseases. To circumvent the BBB, we introduce focused ultrasound-mediated intranasal brain drug delivery (FUSIN). FUSIN utilizes the nasal route for direct nose-to-brain drug administration, bypassing the BBB and minimizing systemic exposure to the major organs, such as heart, lung, liver, and kidney [1]. It also uses transcranial ultrasound energy focused at a targeted brain region to induce microbubble cavitation, enhancing the transport of intranasally administered agents at the FUS-targeted brain location. FUSIN is unique because it can achieve noninvasive and localized brain drug delivery with minimized systemic toxicity to other major organs. The goal of this paper is to provide a detailed protocol for FUSIN delivery to the mouse brain.•FUSIN delivery utilizes the nose-to-brain pathway for brain drug delivery.•FUSIN utilizes FUS combined with microbubble to significantly enhance the delivery efficiency of intranasally administered drugs to the FUS targeted brain regions.•FUSIN achieves efficient brain delivery with minimized systemic exposure in the major organs., Graphical abstract Image, graphical abstract

Diffuse intrinsic pontine glioma (DIPG) arising in the brainstem is the deadliest pediatric brain cancer with nearly 100% fatality and a median survival of

Significance: The blood–brain barrier (BBB) is a major obstacle to detecting and treating brain tumors. Overcoming this challenge will facilitate the early and accurate detection of brain lesions and guide surgical resection of tumors. Aim: We generated an orthotopic brain tumor model that simulates the pathophysiology of gliomas at early stages; determine the BBB integrity and breakdown over the time course of tumor progression using generic and cancer-targeted near-infrared (NIR) fluorescent molecular probes. Approach: We developed an intracranial tumor xenograft model that rapidly reestablished BBB integrity and monitored tumor progression by bioluminescence imaging. Sham control mice were injected with phosphate-buffered saline only. Fluorescence molecular tomography (FMT) was used to quantify the uptake of tumor-targeted and passive NIR fluorescent imaging agents in orthotopic glioma (U87-GL-GFP PDE7B H217Q cells) tumor model. Cancer-induced and transient (with focused ultrasound, FUS) disruption of BBB integrity was monitored with NIR fluorescent dyes. Results: Stereotactic injection of 50,000 cells into mouse brain allowed rapid reestablishment of BBB integrity within a week, as determined by the inability of both tumor-targeted and generic NIR imaging agents to extravasate into the brain. Tumor-induced BBB disruption was observed 7 weeks after tumor implantation. FUS achieved a similar effect at any time point after reestablishing BBB integrity. While tumor uptake and retention of the passive NIR dye, indocyanine green, was negligible, both actively tumor-targeting agents exhibited selective accumulation in the tumor region. The tumor-targeting molecular probe that clears rapidly from nontumor brain tissue exhibits higher contrast than the analogous vascular-targeting agent and helps delineate tumors from sham control. Conclusions: We highlight the utility of FMT imaging for longitudinal assessment of brain tumors and the interplay between the stages of BBB disruption and molecular probe retention in tumors, with potential application to other neurological diseases.

Sonogenetics, which uses ultrasound to noninvasively control cells genetically modified with ultrasound-sensitive ion channels, can be a powerful tool for investigating intact brain circuits. Here we show that TRPV1 is an ultrasound-sensitive ion channel that can modify the activity of TRPV1-expressing cells in vitro when exposing to focused ultrasound. We also show that focused ultrasound exposure at the mouse brain in vivo can selectively activate neurons that are genetically modified to express TRPV1. We demonstrate that precise manipulation of neural activity via TRPV1-based sonogenetics can be achieved by spatiotemporal control of ultrasound-induced heating. The focused ultrasound exposure is safe based on our inspection of neuronal integrity, apoptosis, and inflammation markers. This sonogenetic tool enables noninvasive, cell-type specific, spatiotemporally controlled modulation of mammalian brain activity.

Focused ultrasound combined with microbubble-mediated intranasal delivery (FUSIN) is a new brain drug delivery technique. FUSIN utilizes the nasal route for direct nose-to-brain drug administration, thereby bypassing the blood-brain barrier (BBB) and minimizing systemic exposure. It also uses FUS-induced microbubble cavitation to enhance transport of intranasally (IN) administered agents to the FUS-targeted brain location. Previous studies have provided proof-of-concept data showing the feasibility of FUSIN to deliver dextran and the brain-derived neurotrophic factor to the caudate putamen of mouse brains. The objective of this study was to evaluate the biodistribution of IN administered gold nanoclusters (AuNCs) and assess the feasibility and short-term safety of FUSIN for the delivery of AuNCs to the brainstem. Three experiments were performed. First, the whole-body biodistribution of IN administered (64)Cu-alloyed AuNCs ((64)Cu-AuNCs) was assessed using in vivo positron emission tomography/computed tomography (PET/CT) and verified with ex vivo gamma counting. Control mice were intravenously (IV) injected with the (64)Cu-AuNCs. Second, (64)Cu-AuNCs and Texas red-labeled AuNCs (TR-AuNCs) were used separately to evaluate FUSIN delivery outcome in the brain. (64)Cu-AuNCs or TR-AuNCs were administered to mice through the nasal route, followed by FUS sonication at the brainstem in the presence of systemically injected microbubbles. The spatial distribution of (64)Cu-AuNCs and TR-AuNCs were examined by autoradiography and fluorescence microscopy of ex vivo brain slices, respectively. Third, histological analysis was performed to evaluate any potential histological damage to the nose and brain after FUSIN treatment. The experimental results revealed that IN administration induced significantly lower (64)Cu-AuNCs accumulation in the blood, lungs, liver, spleen, kidney, and heart compared with IV injection. FUSIN enhanced the delivery of (64)Cu-AuNCs and TR-AuNCs at the FUS-targeted brain region compared with IN delivery alone. No histological-level tissue damage was detected in the nose, trigeminal nerve, and brain. These results suggest that FUSIN is a promising technique for noninvasive, spatially targeted, and safe delivery of nanoparticles to the brain with minimal systemic exposure.

The goal of this study was to establish the feasibility of integrating focused ultrasound (FUS)-mediated delivery of (64)Cu-integrated gold nanoclusters ((64)Cu-AuNCs) to the pons for in vivo quantification of the nanocluster brain uptake using positron emission tomography (PET) imaging. FUS was targeted at the pons for the blood-brain barrier (BBB) disruption in the presence of systemically injected microbubbles, followed by the intravenous injection of (64)Cu-AuNCs. The spatiotemporal distribution of the (64)Cu-AuNCs in the brain was quantified using in vivo microPET/CT imaging at different time points post injection. Following PET imaging, the accumulation of radioactivity in the pons was further confirmed using autoradiography and gamma counting, and the gold concentration was quantified using inductively coupled plasma-mass spectrometry (ICP-MS). We found that the noninvasive and localized BBB opening by the FUS can successfully deliver the (64)Cu-AuNCs to the pons. We also demonstrated that in vivo real-time microPET/CT imaging was a reliable method for monitoring and quantifying the brain uptake of (64)Cu-AuNCs delivered by the FUS. This drug delivery platform that integrates FUS, radiolabeled nanoclusters, and PET imaging provides a new strategy for noninvasive and localized nanoparticle delivery to the pons with concurrent in vivo quantitative imaging to evaluate delivery efficiency. The long-term goal is to apply this drug delivery platform to the treatment of pontine gliomas.

Although blood-based liquid biopsies have emerged as a promising non-invasive method to detect biomarkers in various cancers, limited progress has been made for brain tumors. One major obstacle is the blood-brain barrier (BBB), which hinders efficient passage of tumor biomarkers into the peripheral circulation. The objective of this study was to determine whether FUS in combination with microbubbles can enhance the release of biomarkers from the brain tumor to the blood circulation. Two glioblastoma tumor models (U87 and GL261), developed by intracranial injection of respective enhanced green fluorescent protein (eGFP)-transduced glioblastoma cells, were treated by FUS in the presence of systemically injected microbubbles. Effect of FUS on plasma eGFP mRNA levels was determined using quantitative polymerase chain reaction. eGFP mRNA were only detectable in the FUS-treated U87 mice and undetectable in the untreated U87 mice (maximum cycle number set to 40). This finding was replicated in GL261 mice across three different acoustic pressures. The circulating levels of eGFP mRNA were 1,500–4,800 fold higher in the FUS-treated GL261 mice than that of the untreated mice for the three acoustic pressures. This study demonstrated the feasibility of FUS-enabled brain tumor liquid biopsies in two different murine glioma models across different acoustic pressures.

Immune checkpoint inhibitors have great potential for the treatment of gliomas, however, their therapeutic efficacy has been partially limited by their inability to efficiently cross the blood–brain barrier (BBB). The objective of this study was to evaluate the capability of focused-ultrasound-mediated intranasal brain drug delivery (FUSIN) in achieving the locally enhanced delivery of anti-programmed cell death-ligand 1 antibody (aPD-L1) to the brain. Both non-tumor mice and mice transcranially implanted with GL261 glioma cells at the brainstem were used in this study. aPD-L1 was labeled with a near-infrared fluorescence dye (IRDye 800CW) and administered to mice through the nasal route to the brain, followed by focused ultrasound sonication in the presence of systemically injected microbubbles. FUSIN enhanced the accumulation of aPD-L1 at the FUS-targeted brainstem by an average of 4.03- and 3.74-fold compared with intranasal (IN) administration alone in the non-tumor mice and glioma mice, respectively. Immunohistochemistry staining found that aPD-L1 was mainly located within the perivascular spaces after IN delivery, while FUSIN further enhanced the penetration depth and delivery efficiency of aPD-L1 to the brain parenchyma. The delivered aPD-L1 was found to be colocalized with the tumor cells after FUSIN delivery to the brainstem glioma. These findings suggest that FUSIN is a promising technique to enhance the delivery of immune checkpoint inhibitors to gliomas.

Background Leptomeningeal disease and hydrocephalus are present in up to 30% of patients with diffuse intrinsic pontine glioma (DIPG), however there are no animal models of cerebrospinal fluid (CSF) dissemination. As the tumor-CSF-ependymal microenvironment may play an important role in tumor pathogenesis, we identified characteristics of the Nestin-tumor virus A (Nestin-Tva) genetically engineered mouse model (GEMM) that make it ideal to study the interaction of tumor cells with the CSF and its associated pathways with implications for the development of treatment approaches to address CSF dissemination in DIPG. Methods A Nestin-TVa model of DIPG utilizing the three most common DIPG genetic alterations (H3.3K27M, PDGF-B, p53) was used for this study. All animals underwent MR imaging and a subset underwent histopathologic analysis with H&E and beta-IV tubulin. Results Tumor dissemination within the CSF pathways (ventricles, leptomeninges) was present in 76% (25/33) of animals, with invasion of the choroid plexus, disruption of the ciliated ependyma and regional subependymal fluid accumulation. Ventricular enlargement consistent with hydrocephalus was present in 94% (31/33). Ventricle volume correlated with region specific transependymal CSF flow (periventricular T2 signal), localized anterior to the lateral ventricles. Subependymal tumor cells were also present subjacent to the 4th ventricle in a post-mortem human specimen. Conclusions This is the first study to report CSF pathway tumor dissemination an animal model of DIPG and is representative of CSF dissemination seen clinically. Understanding the CSF-tumor-ependymal microenvironment has significant implications for treatment of DIPG through targeting mechanisms of tumor spread within the CSF pathways.

Understanding dissolution kinetics of a colloidal particle in an aqueous solution is of great importance in many pharmaceutical and biochemical applications. We present theoretical analysis of low Reynolds number flow dynamics and mass transfer of a dissolving spherical particle in a unidirectional oscillatory flow field. The coupling of fluid flow and passive motion of the particle are solved analytically, and the transient mass transfer associated with the oscillation of the particle is computed numerically. The flow patterns, basic characteristics of mass transport, and the simplified long-time dissolution process under various saturation concentrations and relative flow conditions are discussed in details.

The static magnetic field of an MRI scanner was found to dampen the microbubble cavitation activity and decrease the focused ultrasound-combined with microbubble-mediated blood-brain barrier opening.

Theoretical and experimental investigations are presented for the precision measurement of evaporation kinetics of binary mixtures using a quartz crystal resonator. A thin layer of light alcohol mixture including a volatile (methanol) and a much less volatile (1-butanol) components is deployed on top of a crystal resonator for the evaporation experiment. A one-dimensional theoretical model is developed to describe the underlying mass transfer and interfacial transport phenomena. Along with the theoretical analysis, the transient evaporation kinetics, moving interface, and the stratification of viscosity of the liquid mixture during evaporation can be simultaneously measured by the impedance response of the shear and longitudinal waves emitted from the resonator. The result on the binary mixture presents a simplified model system for further investigations of complicated evaporation kinetics involving complex fluids or multi-component fuel systems.

Cavitation is widely existing in focused ultrasound (FUS)-mediated therapies in the brain, such as FUS in combination with microbubble-induced blood-brain barrier disruption, nonthermal ablation, as well as transcranial histotripsy therapy. Accurately knowing the 3-D location of cavitation in real time can improve the treatment targeting accuracy and avoid off-target tissue damage. However, the skull induces strong phase and amplitude aberration to the cavitation signals and presents a significant challenge to the transcranial cavitation localization. Existing techniques for 3-D cavitation localization use hemispherical multi-element arrays combined with passive beamforming and adaptive skull-specific correction algorithm. However, these techniques require expensive equipment and treatment planning. Their time-consuming computations limit applications in real-time cavitation monitoring, which is critically needed to ensure the safety and efficacy of the FUS treatment. The object of this study was to investigate the feasibility of using a four-sensor network to transcranially locate the cavitation source in 3-D by time difference of arrival algorithm. The positional error of transcranial cavitation localization with the human skull along x, y, and z axes were 1.7 ± 1.2 mm, 1.6 ± 1.7 mm, and 4.1 ± 1.5 mm, respectively. For comparison, the positional error of without the human skull were 1.2 ± 1.8 mm, 0.9 ± 1.6 mm, and 3.1 ± 2.3 mm, respectively.

Focused ultrasound combined with microbubble for blood-brain barrier disruption (FUS-BBBD) is a promising technique for noninvasive and localized brain drug delivery. This study demonstrates that passive cavitation imaging (PCI) is capable of predicting the location and concentration of nanoclusters delivered by FUS-BBBD. During FUS-BBBD treatment of mice, the acoustic emissions from FUS-activated microbubbles were passively detected by an ultrasound imaging system and processed offline using a frequency-domain PCI algorithm. After the FUS treatment, radiolabeled gold nanoclusters, 64Cu-AuNCs, were intravenously injected into the mice and imaged by positron emission tomography/computed tomography (PET/CT). The centers of the stable cavitation dose (SCD) maps obtained by PCI and the corresponding centers of the 64Cu-AuNCs concentration maps obtained by PET coincided within 0.3 ± 0.4 mm and 1.6 ± 1.1 mm in the transverse and axial directions of the FUS beam, respectively. The SCD maps were found to be linearly correlated with the 64Cu-AuNCs concentration maps on a pixel-by-pixel level. These findings suggest that SCD maps can spatially “paint” the delivered nanocluster concentration, a technique that we named as cavitation dose painting. This PCI-based cavitation dose painting technique in combination with FUS-BBBD opens new horizons in spatially targeted and modulated brain drug delivery.

Focused ultrasound-enabled intranasal brain drug delivery (FUSIN)utilizes the nose-to-brain pathway for drug administration and FUS in combination with microbubbles for enhancing the accumulation of the administered drugs at the FUS-targeted brain location. FUSIN is different from the established FUS-induced BBB disruption (FUS-BBBD)technique. The objective of this study was to compare the delivery efficiency, systemic exposure, and delivery pathways of FUSIN and FUS-BBBD. Taxes red-labeled gold nanoclusters (TR-AuNCs)were used as the model agent and FUS was targeted at the brainstem. Mice were divided into four groups: FUSIN, FUS-BBBD, IN only, and IV only. The delivery efficiency of TR-AuNCs to the mouse brain and exposure to other major organs, as well as the blood, nasal tissue, and trigeminal nerve, were quantified using inductively coupled plasma-mass spectrometry (ICP-MS)analysis of Au concentration. It was found that FUSIN achieved one order of magnitude higher delivery efficiency and one order of magnitude lower systemic exposure in major organs except stomach and intestines than FUS-BBBD. It also verified that FUSIN delivered the AuNCs through the nose-to-brain pathway, while FUS-BBBD utilized the blood-to-brain pathway. These findings suggested that FUSIN is a promising technique for noninvasive and localized brain drug delivery with minimized systemic exposure.

Focused ultrasound (FUS)-induced cavitation-mediated brain therapies have become emerging therapeutic modalities for neurologic diseases. Cavitation monitoring is essential to ensure the safety of all cavitation-mediated therapeutic techniques as inertial cavitation can be associated with tissue damage. The objective of this study was to reveal the correlation between the inertial cavitation dose, quantified by passive cavitation imaging (PCI), and brain tissue histologic-level damage induced by FUS in combination with microbubbles. An ultrasound image-guided FUS system consisting of a single-element FUS transducer (1.5 MHz) and a co-axially aligned 128-element linear ultrasound imaging array was used to perform FUS treatment of mice. Mice were sonicated by FUS with different peak negative pressures (0.5 MPa, 1.1 MPa, 4.0 MPa and 6.5 MPa) in the presence of systemically injected microbubbles. The acoustic emissions from the FUS-activated microbubbles were passively detected by the imaging array. The pre-beamformed channel data were acquired and processed offline using the frequency-domain delay, sum and integration algorithm to generate inertial cavitation maps. All the mice were sacrificed after the FUS treatment, and their brains were harvested and processed for hematoxylin and eosin staining. The obtained inertial cavitation maps revealed the dynamic changes of microbubble behaviors during FUS treatment at different pressure levels. It was found that the inertial cavitation dose quantified based on PCI had a linear correlation with the scale of histologic-level tissue damage. Findings from this study suggested that PCI can be used to predict histologic-level tissue damage associated with the FUS-induced cavitation.

Intranasal route provides therapeutics direct access to the brain through the nose-to-brain pathway, bypassing the BBB and minimizing systemic exposure. However, nasal delivery is limited by its low delivery efficiency and non-localized delivery. Focused ultrasound-enhanced intranasal (FUSIN) delivery is a new technique for noninvasive and localized delivery of therapeutic agents to the brain. Previous studies have shown its feasibility in the delivery of dextrans and brain-derived neurotrophic factor to the brain. The goal of this study was to demonstrate the application of FUSIN for the delivery of temozolomide (TMZ), a first-line drug for treating glioma tumors, for the treatment of glioblastoma in an orthotopic mouse model. U87 glioblastoma cells were trancranially implanted to nude mice. The tumor mice were divided into four groups: (1) FUSIN + TMZ (n = 7); (2) IN TMZ (n = 6); (3) Oral TMZ (n = 6); and (4) IN vehicle (n = 6), and were treated once each week for 4 weeks. Mice were weighed once a week. A body weight loss reaching 20% was selected as the endpoint criteria for the study. Mice in FUSIN group showed statistically significant higher survival rate when compared with mice in all other groups. This pilot study suggested that FUSIN can potentially be an effective technique for the treatment of brain diseases.Intranasal route provides therapeutics direct access to the brain through the nose-to-brain pathway, bypassing the BBB and minimizing systemic exposure. However, nasal delivery is limited by its low delivery efficiency and non-localized delivery. Focused ultrasound-enhanced intranasal (FUSIN) delivery is a new technique for noninvasive and localized delivery of therapeutic agents to the brain. Previous studies have shown its feasibility in the delivery of dextrans and brain-derived neurotrophic factor to the brain. The goal of this study was to demonstrate the application of FUSIN for the delivery of temozolomide (TMZ), a first-line drug for treating glioma tumors, for the treatment of glioblastoma in an orthotopic mouse model. U87 glioblastoma cells were trancranially implanted to nude mice. The tumor mice were divided into four groups: (1) FUSIN + TMZ (n = 7); (2) IN TMZ (n = 6); (3) Oral TMZ (n = 6); and (4) IN vehicle (n = 6), and were treated once each week for 4 weeks. Mice were weighed once a week....

Nanomedicine is the next-generation technology for cancer therapy. Liposomes are one of such nanomedicine that have well-established clinical applications. However, their large sizes make it challenging to deliver them to the brain due to the existence of the blood-brain barrier (BBB). Previous studies have demonstrated the potential of integrating focused ultrasound (FUS)-induced BBB opening with liposomes for brain drug delivery. However, no study has characterized the spatial distributions of the liposomes and released drugs in the brain parenchyma at a microscopic level after the BBB was disrupted by the FUS. In this study, we manufactured liposomes with a fluorescent dye embedded in the lipid layer and chemotherapeutic drug doxorubicin encapsulated in the core. These liposomes were intravenously injected into the mouse followed by FUS sonication of a targeted brain location in the presence of microbubbles. The mouse brains were coronally sectioned and imaged using fluorescence microscopy. Preliminary results confirmed the successful delivery of doxorubicin to the brain. However, the liposome distribution as indicated by the dye embedded in the lipid shell was not spatially correlated with the distribution of the doxorubicin. Further studies are needed to better understand the mechanism of FUS-enabled liposome delivery.Nanomedicine is the next-generation technology for cancer therapy. Liposomes are one of such nanomedicine that have well-established clinical applications. However, their large sizes make it challenging to deliver them to the brain due to the existence of the blood-brain barrier (BBB). Previous studies have demonstrated the potential of integrating focused ultrasound (FUS)-induced BBB opening with liposomes for brain drug delivery. However, no study has characterized the spatial distributions of the liposomes and released drugs in the brain parenchyma at a microscopic level after the BBB was disrupted by the FUS. In this study, we manufactured liposomes with a fluorescent dye embedded in the lipid layer and chemotherapeutic drug doxorubicin encapsulated in the core. These liposomes were intravenously injected into the mouse followed by FUS sonication of a targeted brain location in the presence of microbubbles. The mouse brains were coronally sectioned and imaged using fluorescence microscopy. Preliminary...