Focused ultrasound-enabled brain tumor liquid biopsy

Abstract 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

The Physical Mechanism of Blood-Brain Barrier Opening Using Focused Ultrasound and Microbubbles

The key to effective treatment of neurological diseases resides in the safe opening of the blood-brain barrier (BBB), a specialized structure that impedes the delivery of therapeutic agents to the parenchyma. Despite the fact that several approaches have been successful in overcoming the BBB impermeability, none of them can induce localized BBB opening noninvasively except for focused ultrasound (FUS) in conjunction with microbubbles. The physical mechanism behind the opening, however, has not been identified. Insight into the mechanism can be critical for delineating the safety profile for in both small and large animals alike. Therefore the purpose of this dissertation is to first determine the physical mechanism of FUS-induced BBB opening in mice and then translate this approach to non-human primates. To accomplish this goal, an in vivo transcranial cavitation detection system was developed and tested, built in phantoms and in vivo, to monitor the behavior of the microbubbles in the FUS bean, and to determine the type of cavitation, i.e., the activation of bubbles in an acoustic field, during BBB opening. We showed that the inertial cavitation (IC), a collapse of a bubble, which can vary from a fragmentation of the bubble to shock wave and liquid jets depending on the pressure, thereby damaging the endothelial cells of the brain capillaries, was not required to induce BBB opening in mice. With this system, the role of microbubble properties, including the diameter and shell components, in the BBB opening were determined. When the BBB opens with stable cavitation (SC), i.e., relatively moderate amplitude changes in the bubble size, the bubble diameter is similar to the capillary diameter (i.e., at 4-5, 6-8 µm) while with inertial cavitation it is not (i.e., at 1-2 µm). The bubble may thus have to be in closer proximity to the capillary wall to induce BBB opening without IC. The BBB opening properties, such as volume and permeability, however, were not affected by the shell component of the microbubbles in mice. The connection between the physical and physiological mechanism was then investigated to identify the lowest peak rarefactional pressure BBB opening threshold at 1.5 MHz (0.18 MPa). A sufficiently long pulse (pulse length = 0.5 ms) was required for the SC to induce BBB opening at the lowest pressure. However, the tight junctions, the main formation of the BBB, were found not to be disrupted after sonication at both low (0.18 MPa) and high (0.45 MPa) pressures. Therefore, the transcellular pathway may be the main route of the FUS-induced BBB opening. Finally, the cavitation-guided BBB opening system was used to induce reversible BBB opening in non-human primates. This is a major step towards clinical feasibility. In conclusion, a transcranial cavitation detection system was developed, in order to characterize the physical mechanism, the role of the microbubbles, and the corresponding physiological response of the FUS-induced BBB opening

Quantitative analysis of the focused ultrasound-induced blood-brain barrier opening with applications in neurodegenerative disorders

The blood-brain barrier poses a formidable impediment to the treatment of adult-onset neurodegenerative disorders, by prevention of most drugs from gaining access to the brain parenchyma. Focused ultrasound (FUS), in conjunction with systemically administered microbubbles, has been shown to open the blood-brain barrier (BBB) locally, reversibly and non invasively both in rodents and in non-human-primates. Initially, we demonstrate a monotonic increase of the BBB opening volume with close to normal incidence angle, detectable by diffusion tensor imaging; the employed contrast-free magnetic resonance protocol that revealed the anisotropic nature of the diffusion gradient. Implementation of this optimized BBB opening technique in Parkinsonian mice, coupled with the administration of trophic growth factors, induced restorative effects in the dopaminergic neurons, the main cellular target of the pathological process in Parkinson’s disease. The immune response initiated by the FUS-induced BBB disruption has been proven pivotal in reducing proteinaceous aggregates from the brain through the activation of a gliosis cascade. Therefore, we investigated this immunomodulatory effect in Alzheimer’s disease. The neuropathological hallmarks of Alzheimer’s disease include aggregation of amyloid beta into plaques and accumulation of tau protein into neurofibrillary tangles. Tau pathology correlates well with impaired neuronal activity and dementia and was found to be attenuated after the application of ultrasound that correlated with increased microglia activity. Given the beneficial effect of this methodology on the Alzheimer’s pathologies when studied separately, we explored the application of FUS in brains subjected concurrently to amyloidosis and tau phosphorylation. Our findings indicate the reduction of tau protein and decrease in the amyloid load from brains treated with ultrasound, accompanied by spatial memory improvement. Overall, in this dissertation, we established an optimized targeting and detection protocol, pre-clinical implementation of which confirmed its ameliorative effects as a drug-delivery adjuvant or an immune response stimulant. These preclinical findings support the immense potential of such a methodology that significantly contributes to the treatment of different neurodegenerative disorders curbing their progression

Ultrasounds induce blood-brain barrier opening across a sonolucent polyolefin plate in an in vitro isolated brain preparation

The blood-brain barrier (BBB) represents a major obstacle to the delivery of drugs to the central nervous system. The combined use of low-intensity pulsed ultrasound waves and intravascular microbubbles (MB) represents a promising solution to this issue, allowing reversible disruption of the barrier. In this study, we evaluate the feasibility of BBB opening through a biocompatible, polyolefin-based plate in an in vitro whole brain model. Twelve in vitro guinea pig brains were employed; brains were insonated using a planar transducer with or without interposing the polyolefin plate during arterial infusion of MB. Circulating MBs were visualized with an ultrasonographic device with a linear probe. BBB permeabilization was assessed by quantifying at confocal microscopy the extravasation of FITC-albumin perfused after each treatment. US-treated brains displayed BBB permeabilization exclusively in the volume under the US beam; no significant differences were observed between brains insonated with or without the polyolefin plate. Control brains not perfused with MB did not show signs of FITC-albumin extravasation. Our preclinical study suggests that polyolefin cranial plate could be implanted as a skull replacement to maintain craniotomic windows and perform post-surgical repeated BBB opening with ultrasound guidance to deliver therapeutic agents to the central nervous system

Quantification of transient increase of the blood–brain barrier permeability to macromolecules by optimized focused ultrasound combined with microbubbles

Radioimmunotherapy using a radiolabeled monoclonal antibody that targets tumor cells has been shown to be efficient for the treatment of many malignant cancers, with reduced side effects. However, the blood–brain barrier (BBB) inhibits the transport of intravenous antibodies to tumors in the brain. Recent studies have demonstrated that focused ultrasound (FUS) combined with microbubbles (MBs) is a promising method to transiently disrupt the BBB for the drug delivery to the central nervous system. To find the optimal FUS and MBs that can induce reversible increase in the BBB permeability, we employed minimally invasive multiphoton microscopy to quantify the BBB permeability to dextran-155 kDa with similar molecular weight to an antibody by applying different doses of FUS in the presence of MBs with an optimal size and concentration. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a micro-grinder. About 5 minutes after applying the FUS on the thinned skull in the presence of MBs for 1 minute, TRITC (tetramethylrhodamine isothiocyanate)-dextran-155 kDa in 1% bovine serum albumin in mammalian Ringer’s solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump. Simultaneously, the temporal images were collected from the brain parenchyma ~100–200 μm below the pia mater. Permeability was determined from the rate of tissue solute accumulation around individual microvessels. After several trials, we found the optimal dose of FUS. At the optimal dose, permeability increased by ~14-fold after 5 minutes post-FUS, and permeability returned to the control level after 25 minutes. FUS without MBs or MBs injected without FUS did not change the permeability. Our method provides an accurate in vivo assessment for the transient BBB permeability change under the treatment of FUS. The optimal FUS dose found for the reversible BBB permeability increase without BBB disruption is reliable and can be applied to future clinical trials

Rapid short-pulses of focused ultrasound and microbubbles deliver a range of agent sizes to the brain

Focused ultrasound and microbubbles can non-invasively and locally deliver therapeutics and imaging agents across the blood–brain barrier. Uniform treatment and minimal adverse bioeffects are critical to achieve reliable doses and enable safe routine use of this technique. Towards these aims, we have previously designed a rapid short-pulse ultrasound sequence and used it to deliver a 3 kDa model agent to mouse brains. We observed a homogeneous distribution in delivery and blood–brain barrier closing within 10 min. However, many therapeutics and imaging agents are larger than 3 kDa, such as antibody fragments and antisense oligonucleotides. Here, we evaluate the feasibility of using rapid short-pulses to deliver higher-molecular-weight model agents. 3, 10 and 70 kDa dextrans were successfully delivered to mouse brains, with decreasing doses and more heterogeneous distributions with increasing agent size. Minimal extravasation of endogenous albumin (66.5 kDa) was observed, while immunoglobulin (~ 150 kDa) and PEGylated liposomes (97.9 nm) were not detected. This study indicates that rapid short-pulses are versatile and, at an acoustic pressure of 0.35 MPa, can deliver therapeutics and imaging agents of sizes up to a hydrodynamic diameter between 8 nm (70 kDa dextran) and 11 nm (immunoglobulin). Increasing the acoustic pressure can extend the use of rapid short-pulses to deliver agents beyond this threshold, with little compromise on safety. This study demonstrates the potential for deliveries of higher-molecular-weight therapeutics and imaging agents using rapid short-pulses

Recent advances on ultrasound contrast agents for blood-brain barrier opening with focused ultrasound

The blood-brain barrier is the primary obstacle to efficient intracerebral drug delivery. Focused ultrasound, in conjunction with microbubbles, is a targeted and non-invasive way to disrupt the blood-brain barrier. Many commercially available ultrasound contrast agents and agents specifically designed for therapeutic purposes have been investigated in ultrasound-mediated blood-brain barrier opening studies. The new generation of sono-sensitive agents, such as liquid-core droplets, can also potentially disrupt the blood-brain barrier after their ultrasound-induced vaporization. In this review, we describe the different compositions of agents used for ultrasound-mediated blood-brain barrier opening in recent studies, and we discuss the challenges of the past five years related to the optimal formulation of agents

Engineering Lipid-stabilized Microbubbles for Magnetic Resonance Imaging guided Focused Ultrasound Surgery

Lipid-stabilized microbubbles are gas-filled microspheres encapsulated with a phospholipid monolayer shell. Because of the high echogenicity provided by its highly compressible gas core, these microbubbles have been adapted as ultrasound contrast agents for a variety of applications such as contrast-enhanced ultrasonography (CEUS), targeted drug delivery and metabolic gas transport. Recently, these lipid-stabilized microbubbles have demonstrated increased potential as theranostic (therapy + diagnostics) agents for non-invasive surgery with focused ultrasound (FUS). For instance, their implementation has reduced the acoustic intensity threshold needed to open the blood-brain-barrier (BBB) with FUS, which potentially allows for the localized delivery of drugs to treat neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases. However, the effectiveness of microbubbles for this application is dependent on successful microbubble engineering. One necessary improvement is the development and utilization of monodisperse microbubbles of varying size classes. Another design improvement is the development of a microbubble construct whose fragmentation state during or after FUS surgery can be tracked by magnetic resonance imaging (MRI). Thus, in this thesis, we describe a method to generate and select lipid-coated gas-filled microbubbles of specific size fractions based on their migration in a centrifugal field. We also detail the design and characterization of size-selected lipid-coated microbubbles with shells containing the magnetic resonance (MR) contrast media Gadolinium (Gd(III)), for utility in both MR and ultrasound imaging. Initial characterization of the lipid headgroup labeled Gd(III)-microbubbles by MRI revealed that the Gd(III) relaxivity increased after microbubble fragmentation into non-gas-containing lipid vesicles. This behavior was explained to stem from an increase in interaction between water protons and the Gd(III)-bound lipid fragments due to an increase in lipid headgroup area after microbubble fragmentation. To explore this hypothesis, an alternative construct consisting of Gd(III) preferentially bound to the protective poly(ethylene glycol) (PEG) brush of the lipid shell architecture was also designed and compared to the lipid headgroup-labeled Gd(III)-microbubbles. Nuclear magnetic resonance (NMR) analysis revealed that, in contrast to the headgroup labeled Gd(III)-microbubbles, the relaxivity of the PEG-labeled Gd(III)-microbubbles decreased after microbubble fragmentation. NMR analysis also revealed an independent concentration-dependent enhancement of the transverse MR signal by virtue of the microbubble gas core. The results of this study illustrated the roles that Gd(III) placement on the lipid shell and the presence of the gas core may play on the MR signal when monitoring Gd(III)-microbubble cavitation during non-invasive surgery with FUS

In vivo evaluation of the efficacy and safety of rapid short-pulse sequences for ultrasound-medicated delivery of agents to the brain

The blood-brain barrier is essential to the maintenance of homeostasis in the brain, but it also prevents 98% of small molecule drugs and imaging agents from entering the brain. Focused ultrasound in combination with microbubbles is a method that can increase the permeability of the blood-brain barrier in a non-invasive, localised and transient manner, allowing drugs and imaging agents into the brain. In conventional ultrasound methods, a sequence of long pulses is applied to the brain, which can cause undesired effects, such as uneven drug distributions and a barrier altered for several hours, exposing the brain to unwanted bloodborne substances. In this thesis, we have investigated whether the efficacy and safety of drug delivery can be improved in vivo by emitting ultrasound in a Rapid Short-Pulse (RaSP) sequence. We first investigated the differences in performance and safety between emitting a RaSP sequence and a long pulse sequence to deliver a dextran model drug. We found that a more uniform drug distribution was achieved using RaSP, with a delivered dose comparable to that of long pulses. The barrier permeability was altered for less than 10 minutes, minimising the amount of endogenous proteins entering the brain, while no tissue damage was observed. We then investigated whether RaSP could deliver large 100 nm liposomes into the brain. We showed that RaSP can achieve this with an improved safety profile, although higher pressures were needed compared to long pulses. Finally, we evaluated whether a dual-modal MRI-optical probe could be delivered into the brain, using long pulses, to image neurons. We confirmed uptake within neurons and detected both fluorescence and MRI signals ex vivo. This work demonstrates that ultrasound sequences can be designed to improve the efficacy and safety of drug delivery for the diagnosis and treatment of brain diseases.Open Acces