In Vitro Assessment of Radiopharmaceutical Uptake in Brain Tumor Cells Using Focused Ultrasound Stimulation.

Physical blood-brain barrier disruption induced by focused ultrasound does not overcome the transporter-mediated efflux of erlotinib

Recently, blood-brain barrier disruption (BBBD) has been performed by focused ultrasound (FUS) combining with microbubbles (MBs). The outcome of BBBD enhances local drug or gene delivery for improving the treatment efficiency of brain diseases. However, over-excitation of FUS may cause brain damage such as shutdown blood flow, intracerebral hemorrhage and brain edema. Therefore, it is essential to develop a an imaging system to assess dynamic perfusion changes during FUS-induced BBBD process. Here, we used the high-frequency destruction/reperfusion contrast-enhanced imaging technique to observe the cerebral perfusion under the cases of with/without hemorrhage in BBBD procedure. The BBB was disrupted by a 2.25 MHz FUS combining with MBs at 0.5-0.7 MPa (pulse repetition frequency: 1 Hz, pulse length: 1 ms, sonication time: 60 s). The results showed that the velocity of blood flow decreased after BBBD induced by FUS sonication. Particularly, the plateau of time-intensity curve was higher than prior to MBs destruction at 20 s after sonication and the blood flow would be obstructed due to the blood coagulates at 60s after sonication. The pattern of hemorrhagic damage caused by FUS can be monitored by the TIC. In addition, the location of blood flow velocity decrease was consistent with the areas of BBBD and the variation of blood flow depends on the applied acoustic pressure. In conclusion, the blood flow velocity changes have potential as an in vivo tool for quantifying the extent of the FUS-induced BBBD and detecting intracerebral hemorrhage occurrence.

Focused ultrasound blood-brain barrier disruption in high-grade gliomas: Scoping review of clinical studies

Focused ultrasound (FUS) with microbubbles (MBs) has been approved to achieve local blood-brain barrier disruption (BBBD), increasing the delivery of therapeutic agents into brain. Previous reports have demonstrated the use of medical imaging system (ultrasound, CT, MRI, SPECT, or PET) to observe the redistribution of the delivered substance. However, the redistribution of elements such as zinc or copper has not yet been discovered, nor their effects and roles to CNS after FUS sonications. Laser ablation/inductively coupled plasma mass spectrometry (LA-ICP-MS) is a sensitive analytical technique and is capable of quantitatively providing solid inorganic matrices distribution in tissues. In this study, LA-ICPMS was used to explore the feasibility of detecting the delivered iron (i.e. SPIO-conjugated MBs, SPIO-MBs) and its redistribution after magnetic targeting (MT), as well as the elements redistribution. The 1-MHz FUS (acoustic energy = 0.4-1.0 MPa, duty cycle = 10%, sonication time = 1 min) was exposed to left brain of rats following SPIO-MBs injection to induce BBBD (SPIO payload was 200 ± 19.6 μg by ICP-AES). A 0.5 T permanent magnet was attached to the rat's scalp for performing MT. The spatial elemental maps were performed with the LA-ICP-MS system excited by 213 nm Nd-YAG laser (energy = 1.6 mJ, frequency = 10 Hz, scanning speed = 90 μm/s). The distribution and concentration of elements including iron, copper, and zinc were analyzed. Results showed that LA-ICPMS performed high sensitivity in the detection of the elements distribution in the brain. The iron mapping revealed that SPIOs were highly concentrated at FUS exposed and MT performed sites. No obvious zinc and copper deposition within sonication regions were found after FUS sonication. This study confirmed the potential of LA-ICP-MS for providing highly-sensitive detection method for solid inorganic matrices in brain tissues, and tracing the redistribution of elements. Moreover, concurrent use of SPIO-MBs with MT further enhanced the SPIO delivery into the BBBD area. Future work includes using SPIO-MBs as a surrogate tracer to estimate drug delivery efficiency during BBBD.

Background: Focused ultrasound (FUS) blood brain barrier disruption (BBBD) permits the noninvasive, targeted, and repeatable delivery of drugs to the brain. FUS BBBD also elicits secondary responses capable of augmenting immunotherapies, clearing amyloid-β and hyperphosphorylated tau, and driving neurogenesis. Leveraging these secondary effects will benefit from an understanding of how they correlate to the magnitude of FUS BBBD and are differentially affected by the mechanical and biochemical stimuli imparted during FUS BBBD.Methods: We aggregated 75 murine transcriptomes in a multiple regression framework to identify genes expressed in proportion to biochemical (i.e. contrast MR image enhancement (CE)) or mechanical (i.e. harmonic acoustic emissions from MB-activation (MBA)) stimuli associated with FUS BBBD. Models were constructed to control for potential confounders, such as sex, anesthesia, and sequencing batch.Results: MBA and CE differentially predicted expression of 1,124 genes 6 h or 24 h later. While there existed overlap in the transcripts correlated with MBA vs CE, MBA was principally predictive of expression of genes associated with endothelial reactivity while CE chiefly predicted sterile inflammation gene sets. Over-representation analysis identified transcripts not previously linked to BBBD, including actin filament organization, which is likely important for BBB recovery. Transcripts and pathways associated with neurogenesis, microglial activation, and amyloid-β clearance were significantly correlated to BBBD metrics.Conclusions: The secondary effects of BBBD may have the potential to be tuned by modulating FUS parameters during BBBD, and MBA and CE may serve as independent predictors of transcriptional reactions in the brain.

Focused ultrasound (FUS) in combination with microbubbles has been studied extensively as a blood-brain barrier (BBB) disruption tool for the delivery of drugs in the blood circulation to the brain parenchyma. Recently, we introduced the hypothesis that FUS-mediated BBB disruption could be viewed as a tool for enhancing “two-way trafficking” between brain and blood. While circulating molecules can be allowed to enter the brain using FUS-mediated BBB disruption, brain tumor biomarkers can also be released into the blood circulation for liquid biopsies. Based on this hypothesis, we proposed to develop FUS-enabled brain tumor liquid biopsy technique (FUS-LBx), which uses FUS in combination with microbubbles to enhance the release of biomarkers from brain tumors into the blood circulation for liquid biopsies. We performed a proof-of-concept study and demonstrated the feasibility of FUS-LBx for the local release of mRNA from glioblastoma tumors in mice into the bloodstream for liquid biopsies. We also optimize the FUS-LBx technique by investigating the effects of FUS acoustic pressure on the tumor biomarker release level and potentially associated hemorrhage burden. We found that FUS-LBx technique can be optimized to be a safe and effective image-guided biomarker release technique. We developed an MR-compatible FUS system for FUS-LBx application in a porcine model and demonstrated the feasibility and safety of FUS-LBx in the large animal model. In summary, FUS-LBx is a promising tool for brain tumor diagnosis.

Glial cell line-derived neurotrophic factor (GDNF) is a potent agent for treating Parkinson's disease (PD). Microbubbles (MBs) have been exploited as a carrier for GDNF delivery with focused ultrasound (FUS) destruction. However, GDNF supplementation only relieves PD symptoms rather than cure it. Thus, design of an endogenous GDNF generative mechanism is an urgent concern for novel PD treatment. Here, we synthesized a cationic MBs (cMBs) with high GDNF plasmid (GDNFp) binding to achieve blood-brain barrier disruption (BBBD) with FUS, and currently enhance the GDNFp transfection for PD therapy. PD lesions were established by implanting 6-hydroxydopamine into left brain of rats. A 1-MHz FUS (0.7 MPa, duty factor = 5 %, PRF = 1 Hz, sonication time = 90 s) was sonicated at striatum and substantia nigra after GcMBs injection. Sites of gene expression were confirmed by firefly luciferase gene-loaded cMBs (LcMBs) with bioluminescence imaging, individually. The motor behavior and dopamine (DA) levels of the treated rats were traced to estimate treatment outcome. Result showed that the positively charge of cMBs (+39.48 ± 9.28 mV) made them significant for DNA binding. With FUS, GcMBs provided sufficient BBBD area for enhanced gene transfection in PD lesion. Bioluminescence data showed that the released Luc-gene expressed started from 24 h after treatment. The GDNF level of PD rats restored following the combing treatment (66.6 % of recovery). The treatment reduced rotations by 92.2 ± 5.7%, and improved of DA levels by 164.9 ± 1.23%. This study confirmed that GcMBs with FUS provides a novel strategy for local gene therapy in PD rats.

To investigate the correlation between the contrast-enhanced magnetic resonance imaging (MRI) signal and the duration of blood-brain barrier (BBB) disruption induced by focused ultrasound (FUS). FUS was applied to 45 rat brains in the presence of microbubbles, and these rats were scanned on a 3T MRI system at several timepoints. The rat brains were then studied using contrast-enhanced spin echo T1-weighted images. At the same time, BBB disruption was evaluated based on Evans blue (EB) extravasation. The relationship between the normalized signal intensity change of the MRI and EB extravasation was analyzed by least-squares linear regression and the calculation of correlation coefficients. When MRI enhancement was quantitatively evaluated by EB extravasation, a strong correlation between the normalized signal intensity change of the MRI and EB extravasation was identified during BBB disruption after sonication. However, the correlation coefficient decreased as BBB closure occurred after sonication ended. The contrast-enhanced MRI signal can potentially be used to evaluate the amount of chemotherapeutic agents entering the targeted tissue, but the accuracy of the assessment will be affected by the time interval since sonication.

Transcranial Photoacoustic Detection of Blood-Brain Barrier Disruption Following Focused Ultrasound-Mediated Nanoparticle Delivery.

Focused ultrasound (FUS) with microbubbles has been discovered to locally increase the permeability of blood-brain barrier (BBB). However, bioeffects such as erythrocyte extravasations and brain tissue damage may accompany. Therefore, development of a real-time imaging method to visualize occurrence of brain injury during opening of BBB by FUS is chief to improve the safety of this technology. The purpose of this study is to investigate the feasibility of using the high-frequency ultrasound imaging technique including contrast-enhanced and B-mode two images to monitor the transient responses of BBB disruption (BBBD) induced by FUS. We observed that BBBD with extensive hemorrhage and ischemia in 1-MHz FUS with 1.1 MPa. The bright spots from the blood clots scattering appeared in B-mode images and correspond to the map without perfusion in the contrast-enhanced images. In addition, a relative safe power to BBBD without such bioeffects was 0.3 MPa. The results indicated that location of hyperechoic spots correlates with hemorrhage areas and the ischemia region consistent with the BBBD areas under the cases of high FUS power. That reflected high-frequency ultrasound can provide the information of microhemorrhage and transient ischemia information during BBB open by FUS.

Focused ultrasound (FUS) with microbubbles (MBs) has been confirmed to achieve the blood-brain barrier disruption (BBBD). Magnetic resonance imaging (MRI) can monitor the contrast agent leakage such as superparamagnetic iron oxide (SPIO) after BBBD, which relies on indirect drug-contrast agent relationship and fails to perform direct drug quantification/distribution monitoring. This study proposed a novel doxorubicin-loaded and SPIO-conjugated MB (DOX-SPIO-MB) that can open BBB and concurrently perform drug delivery. Moreover, we also illustrate the feasibility of using external magnetic targeting (MT) to enhance the drug delivery in rat GBM model. Rats implanted with C6 glioma cells were used. At 11 days postimplantation, FUS (frequency = 0.5 MHz, energy = 4 W, duty cycle = 5%, sonication = 6 min) was performed following DOX-SPIO-MB injection. A 0.5 T magnet was attached to the rat's scalp for acting MT. The BBBD were confirmed by Evans blue stained imaging. Results showed that DOX-SPIO-MB indeed have the superparamagnetic property and good acoustic stability. Combing with FUS sonication, DOX-SPIO-MBs can concurrently perform DOX delivery and BBBD. This study confirmed that the DOX-SPIO-MB with FUS exposure can concurrently serve as an excellent theranostic tools to increase blood-brain tumor permeability and enhance brain tumor drug delivery.

Boronophenylalanine has been applied in clinical boron neutron capture therapy for the treatment of high-grade gliomas. The purpose of this study was to evaluate the pharmacokinetics of 4-borono-2-(18)F-fluoro-L-phenylalanine-fructose ((18)F-FBPA-Fr) in F98 glioma-bearing Fischer 344 rats by means of intravenous injection of (18)F-FBPA-Fr both with and without blood-brain barrier disruption (BBB-D) induced by focused ultrasound (FUS). Dynamic PET imaging of (18)F-FBPA-Fr was performed on the ninth day after tumor implantation. Blood samples were collected to obtain an arterial input function for tracer kinetic modeling. Ten animals were scanned for approximately 3 h to estimate the uptake of (18)F radioactivity with respect to time for the pharmacokinetic analysis. Rate constants were calculated by use of a 3-compartment model. The accumulation of (18)F-FBPA-Fr in brain tumors and the tumor-to-contralateral brain ratio were significantly elevated after intravenous injection of (18)F-FBPA-Fr with BBB-D. (18)F-FBPA-Fr administration after sonication showed that the tumor-to-contralateral brain ratio for the sonicated tumors (3.5) was approximately 1.75-fold higher than that for the control tumors (2.0). Furthermore, the K1/k2 pharmacokinetic ratio after intravenous injection of (18)F-FBPA-Fr with BBB-D was significantly higher than that after intravenous injection without BBB-D. This study demonstrated that radioactivity in tumors and the tumor-to-normal brain ratio after intravenous injection of (18)F-FBPA-Fr with sonication were significantly higher than those in tumors without sonication. The K1/k2 ratio may be useful for indicating the degree of BBB-D induced by FUS. Further studies are needed to determine whether FUS may be useful for enhancing the delivery of boronophenylalanine in patients with high-grade gliomas.

PurposeAmyloid-β (Aβ) peptides, the main component of amyloid plaques found in the Alzheimer's disease (AD) brain, are implicated in its pathogenesis, and are considered a key target in AD therapeutics. We herein propose a reliable strategy for non-invasively delivering a specific anti-Aβ antibody in a mouse model of AD by microbubbles-enhanced Focused Ultrasound (FUS)-mediated Blood–brain barrier disruption (BBBD), using a simple single stage MR-compatible positioning device.MethodsThe initial experimental work involved wild-type mice and was devoted to selecting the sonication protocol for efficient and safe BBBD. Pulsed FUS was applied using a single-element FUS transducer of 1 MHz (80 mm radius of curvature and 50 mm diameter). The success and extent of BBBD were assessed by Evans Blue extravasation and brain damage by hematoxylin and eosin staining. 5XFAD mice were divided into different subgroups; control (n = 1), FUS + MBs alone (n = 5), antibody alone (n = 5), and FUS + antibody combined (n = 10). The changes in antibody deposition among groups were determined by immunohistochemistry.ResultsIt was confirmed that the antibody could not normally enter the brain parenchyma. A single treatment with MBs-enhanced pulsed FUS using the optimized protocol (1 MHz, 0.5 MPa in-situ pressure, 10 ms bursts, 1% duty factor, 100 s duration) transiently disrupted the BBB allowing for non-invasive antibody delivery to amyloid plaques within the sonicated brain regions. This was consistently reproduced in ten mice.ConclusionThese preliminary findings should be confirmed by longer-term studies examining the antibody effects on plaque clearance and cognitive benefit to hold promise for developing disease-modifying anti-Aβ therapeutics for clinical use.

Focused ultrasound (FUS) mediated blood–brain barrier disruption (BBBD) targets the delivery of systemically‐administered therapeutics to the central nervous system. Preclinical investigations of BBBD have been performed on different anesthetic backgrounds; however, the influence of the choice of anesthetic on the molecular response to BBBD is unknown, despite its potential to critically affect interpretation of experimental therapeutic outcomes. Here, using bulk RNA sequencing, we comprehensively examined the transcriptomic response of both normal brain tissue and brain tissue exposed to FUS‐induced BBBD in mice anesthetized with either isoflurane with medical air (Iso) or ketamine/dexmedetomidine (KD). In normal murine brain tissue, Iso alone elicited minimal differential gene expression (DGE) and repressed pathways associated with neuronal signaling. KD alone, however, led to massive DGE and enrichment of pathways associated with protein synthesis. In brain tissue exposed to BBBD (1 MHz, 0.5 Hz pulse repetition frequency, 0.4 MPa peak‐negative pressure), we systematically evaluated the relative effects of anesthesia, microbubbles, and FUS on the transcriptome. Of particular interest, we observed that gene sets associated with sterile inflammatory responses and cell–cell junctional activity were induced by BBBD, regardless of the choice of anesthesia. Meanwhile, gene sets associated with metabolism, platelet activity, tissue repair, and signaling pathways, were differentially affected by BBBD, with a strong dependence on the anesthetic. We conclude that the underlying transcriptomic response to FUS‐mediated BBBD may be powerfully influenced by anesthesia. These findings raise considerations for the translation of FUS‐BBBD delivery approaches that impact, in particular, metabolism, tissue repair, and intracellular signaling.

Methods to improve drug delivery efficiency through blood-brain barrier disruption (BBBD) based on microbubbles and focused ultrasound (FUS) are continuously being studied. However, most studies are being conducted in preclinical trial environments using small animals. The use of the human skull shows differences between the clinical and preclinical trials. BBBD results from preclinical trials are difficult to represent in clinical trials because various distortions of ultrasound by the human skull are excluded in the former. Therefore, in our study, a clinical validation platform based on a preclinical trial environment, using a human skull fragment and a rat model, was developed to induce BBBD under conditions similar to clinical trials. For this, a human skull fragment was inserted between the rat head and a 250 kHz FUS transducer, and optimal ultrasound parameters for the free field (without human skull fragment) and human skull (with human skull fragment) were derived by 300 mVpp and 700 mVpp, respectively. BBBD was analyzed according to each case using magnetic resonance images, Evans blue dye, cavitation, and histology. Although it was confirmed using magnetic resonance images and Evans blue dye that a BBB opening was induced in each case, multiple BBB openings were observed in the brain tissues. This phenomenon was analyzed by numerical simulation, and it was confirmed to be due to standing waves owing to the small skull size of the rat model. The stable cavitation doses (SCDh and SCDu) in the human skull decreased by 13.6- and 5.3-fold, respectively, compared to those in the free field. Additionally, the inertial cavitation dose in the human skull decreased by 1.05-fold compared to that of the free field. For the histological analysis, although some extravasated red blood cells were observed in each case, it was evaluated as recoverable based on our previous study results. Therefore, our proposed platform can help deduct optimal ultrasound parameters and BBBD results for clinical trials in the preclinical trials with small animals because it considers variables relevant to the human skull.