Real-Time Intravital Imaging of Acoustic Cluster Therapy-Induced Vascular Effects in the Murine Brain.

OBJECTIVE: The blood-brain barrier (BBB) is an obstacle for cerebral drug delivery. Controlled permeabilization of the barrier by external stimuli can facilitate the delivery of drugs to the brain. Acoustic Cluster Therapy (ACT®) is a promising strategy for transiently and locally increasing the permeability of the BBB to macromolecules and nanoparticles. However, the mechanism underlying the induced permeability change and subsequent enhanced accumulation of co-injected molecules requires further elucidation. METHODS: In this study, the behavior of ACT® bubbles in microcapillaries in the murine brain was observed using real-time intravital multiphoton microscopy. For this purpose, cranial windows aligned with a ring transducer centered around an objective were mounted to the skull of mice. Dextrans labeled with 2 MDa fluorescein isothiocyanate (FITC) were injected to delineate the blood vessels and to visualize extravasation. DISCUSSION: Activated ACT® bubbles were observed to alter the blood flow, inducing transient and local increases in the fluorescence intensity of 2 MDa FITC-dextran and subsequent extravasation in the form of vascular outpouchings. The observations indicate that ACT® induced a transient vascular leakage without causing substantial damage to the vessels in the brain. CONCLUSION: The study gave novel insights into the mechanism underlying ACT®-induced enhanced BBB permeability which will be important considering treatment optimization for a safe and efficient clinical translation of ACT®.

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain. Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

Acoustic Cluster Therapy (ACT®) enhances accumulation of polymeric micelles in the murine brain.

The restrictive nature of the blood-brain barrier (BBB) prevents efficient treatment of many brain diseases. Focused ultrasound in combination with microbubbles has shown to safely and transiently increase BBB permeability. Here, the potential of Acoustic Cluster Therapy (ACT®), a microbubble platform specifically engineered for theranostic purposes, to increase the permeability of the BBB and improve accumulation of IRDye® 800CW-PEG and core-crosslinked polymeric micelles (CCPM) in the murine brain, was studied. Contrast enhanced magnetic resonance imaging (MRI) showed increased BBB permeability in all animals after ACT®. Near infrared fluorescence (NIRF) images of excised brains 1 h post ACT® revealed an increased accumulation of the IRDye® 800CW-PEG (5.2-fold) and CCPM (3.7-fold) in ACT®-treated brains compared to control brains, which was retained up to 24 h post ACT®. Confocal laser scanning microscopy (CLSM) showed improved extravasation and penetration of CCPM into the brain parenchyma after ACT®. Histological examination of brain sections showed no treatment related tissue damage. This study demonstrated that ACT® increases the permeability of the BBB and enhances accumulation of macromolecules and clinically relevant nanoparticles to the brain, taking a principal step in enabling improved treatment of various brain diseases.

Focused Ultrasound and Microbubble Treatment Increases Delivery of Transferrin Receptor-Targeting Liposomes to the Brain.

The blood-brain barrier (BBB) is a major obstacle to treating several brain disorders. Focused ultrasound (FUS) in combination with intravascular microbubbles increases BBB permeability by opening tight junctions, creating endothelial cell openings, improving endocytosis and increasing transcytosis. Here we investigated whether combining FUS and microbubbles with transferrin receptor-targeting liposomes would result in enhanced delivery to the brain of post-natal rats compared with liposomes lacking the BBB-targeting moiety. For all animals, increased BBB permeability was observed after FUS treatment. A 40% increase in accumulation of transferrin receptor-targeting liposomes was observed in the FUS-treated hemisphere, whereas the isotype immunoglobulin G liposomes showed no increased accumulation. Confocal laser scanning microscopy of brain sections revealed that both types of liposomes were mainly observed in endothelial cells in the FUS-treated hemisphere. The results demonstrate that FUS and microbubble treatment combined with BBB-targeting liposomes could be a promising approach to enhance drug delivery to the brain.

Ultrasound-mediated delivery enhances therapeutic efficacy of MMP sensitive liposomes.

To improve therapeutic efficacy of nanocarrier drug delivery systems, it is essential to improve their uptake and penetration in tumour tissue, enhance cellular uptake and ensure efficient drug release at the tumour site. Here we introduce a tumour targeting drug delivery system based on the ultrasound-mediated delivery of enzyme sensitive liposomes. These enzyme sensitive liposomes are coated with cleavable poly(ethylene glycol) (PEG) which will be cleaved by two members of the enzyme matrix metalloproteinase family (MMP-2 and MMP-9). Cleavage of the PEG coat can increase cellular uptake and will destabilize the liposomal membrane which can result in accelerated drug release. The main aim of the work was to study the effect of focused ultrasound and microbubbles on the delivery and therapeutic efficacy of the MMP sensitive liposome. The performance of the MMP sensitive liposome was compared to a non-MMP sensitive version and Doxil-like liposomes. In vitro, the cellular uptake and cytotoxicity of the liposomes were studied, while in vivo the effect of ultrasound and microbubbles on the tumour accumulation, biodistribution, microdistribution, and therapeutic efficacy were investigated. For all tested liposomes, ultrasound and microbubble treatment resulted in an improved tumour accumulation, increased extravasation, and increased penetration of the liposomes from blood vessels into the extracellular matrix. Surprisingly, penetration depth was independent of the ultrasound intensity used. Ultrasound-mediated delivery of free doxorubicin and the Doxil-like and MMP sensitive liposome resulted in a significant reduction in tumour volume 28 days post the first treatment and increased median survival. The MMP sensitive liposome showed better therapeutic efficacy than the non-MMP sensitive version indicating that cleaving the PEG-layer is important. However, the Doxil-like liposome outcompeted the MMP and non-MMP sensitive liposome, both with and without the use of ultrasound and microbubbles.