Noninvasive ultrasonic induction of cerebrospinal fluid flow enhances intrathecal drug delivery.

Intrathecal drug delivery is routinely used in the treatment and prophylaxis of varied central nervous system conditions, as doing so allows drugs to directly bypass the blood-brain barrier. However, the utility of this route of administration is limited by poor brain and spinal cord parenchymal drug uptake from the cerebrospinal fluid. We demonstrate that a simple noninvasive transcranial ultrasound protocol can significantly increase influx of cerebrospinal fluid into the perivascular spaces of the brain, to enhance the uptake of intrathecally administered drugs. Specifically, we administered small (~1 kDa) and large (~155 kDa) molecule agents into the cisterna magna of rats and then applied low, diagnostic-intensity focused ultrasound in a scanning protocol throughout the brain. Using real-time magnetic resonance imaging and ex vivo histologic analyses, we observed significantly increased uptake of small molecule agents into the brain parenchyma, and of both small and large molecule agents into the perivascular space from the cerebrospinal fluid. Notably, there was no evidence of brain parenchymal damage following this intervention. The low intensity and noninvasive approach of transcranial ultrasound in this protocol underscores the ready path to clinical translation of this technique. In this manner, this protocol can be used to directly bypass the blood-brain barrier for whole-brain delivery of a variety of agents. Additionally, this technique can potentially be used as a means to probe the causal role of the glymphatic system in the variety of disease and physiologic processes to which it has been correlated.

Emerging Therapeutic Strategies for Brain Tumors.

Nearly thirty thousand incidences of primary and 300 thousand incidences of metastatic brain cancer are diagnosed in the USA each year. It has a high mortality rate and is often unresponsive to the standard of care, which includes surgical resection, radiation, and chemotherapy. These treatment strategies are also hindered by their invasiveness and toxic effects on healthy cells and tissues. Furthermore, the blood-brain/tumor barrier severely limits delivery of anti-cancer therapeutics administered intravenously to brain tumors, resulting in poor tumor response to the treatment. There is a critical need to develop new approaches to brain cancer therapy that can overcome these limitations. Focused ultrasound has emerged as a modality that addresses many of these limitations and has the potential to alter the treatment paradigm for brain cancer. Ultrasound transmitted through the skull can be focused on tumors and used for targeted ablation or opening the vascular barriers for drug delivery. This review provides insight on the current status of these unique ultrasound techniques, different strategies of using this technique for brain cancer, experience in preclinical models, and potential for clinical translation. We also debate the safety perspective of these techniques and discuss potential avenues for future work in noninvasive planning, monitoring, and evaluation of the ultrasonic neurointervention.

Closed-loop trans-skull ultrasound hyperthermia leads to improved drug delivery from thermosensitive drugs and promotes changes in vascular transport dynamics in brain tumors.

Effective drug delivery in brain tumors remains a major challenge in oncology. Although local hyperthermia and stimuli-responsive delivery systems, such as thermosensitive liposomes, represent promising strategies to locally enhance drug delivery in solid tumors and improve outcomes, their application in intracranial malignancies remains unexplored. We hypothesized that the combined abilities of closed-loop trans-skull Magnetic Resonance Imaging guided Focused Ultrasound (MRgFUS) hyperthermia with those of thermosensitive drugs can alleviate challenges in drug delivery and improve survival in gliomas. Methods: To conduct our investigations, we first designed a closed loop MR-guided Focused Ultrasound (MRgFUS) system for localized trans-skull hyperthermia (ΔT < 0.5 °C) in rodents and established safety thresholds in healthy mice. To assess the abilities of the developed system and proposed therapeutic strategy for FUS-triggered chemotherapy release we employed thermosensitive liposomal Dox (TSL-Dox) and tested it in two different glioma tumor models (F98 in rats and GL261 in mice). To quantify Dox delivery and changes in the transvascular transport dynamics in the tumor microenvironment we combined fluorescent microscopy, dynamic contrast enhanced MRI (DCE-MRI), and physiologically based pharmacokinetic (PBPK) modeling. Lastly, to assess the therapeutic efficacy of the system and of the proposed therapeutic strategy we performed a survival study in the GL261 glioma bearing mice. Results: The developed closed-loop trans-skull MRgFUS-hyperthermia system that operated at 1.7 MHz, a frequency that maximized the brain (FUS-focus) to skull temperature ratio in mice, was able to attain and maintain the desired focal temperature within a narrow range. Histological evidence (H&E and Nissl) suggests that focal temperature at 41.5 ± 0.5 °C for 10 min is below the threshold for tissue damage. Quantitative analysis of doxorubicin delivery from TSLs with MRgFUS-hyperthermia demonstrated 3.5-fold improvement in cellular uptake in GL261 glioma mouse tumors (p < 0.001) and 5-fold increase in delivery in F98 glioma rat tumors (p < 0.05), as compared to controls (TSL-Dox-only). Moreover, PBPK modeling of drug transport that was calibrated using the experimental data indicated that thermal stress could lead to significant improvement in the transvascular transport (2.3-fold increase in the vessel diffusion coefficient; P < 0.001), in addition to promoting targeted Dox release. Prospective experimental investigations with DCE-MRI during FUS-hyperthermia, supported these findings and provided evidence that moderate thermal stress (≈41 °C for up to 10 min) can promote acute changes in the vascular transport dynamics in the brain tumor microenvironment (Ktrans value for control vs. FUS was 0.0097 and 0.0148 min-1, respectively; p = 0.026). Crucially, survival analysis demonstrated significant improvement in the survival in the TSL-Dox-FUS group as compared to TSL-Dox-only group (p < 0.05), providing supporting evidence on the therapeutic potential of the proposed strategy. Conclusions: Our investigations demonstrated that spatially controlled thermal stress can be attained and sustained in the mouse brain, using a trans-skull closed-loop MRgFUS system, and used to promote the effective delivery of chemotherapy in gliomas from thermosensitive drugs. This system also allowed us to conduct mechanistic investigations that resulted in the refinement of our understanding on the role of thermal stress in augmenting mass and drug transport in brain tumors. Overall, our study established a new paradigm for effective drug delivery in brain tumors based on closed-loop ultrasound-mediated thermal stress and thermosensitive drugs.

Molecular imaging of a fluorescent antibody against epidermal growth factor receptor detects high-grade glioma.

The prognosis for high-grade glioma (HGG) remains dismal and the extent of resection correlates with overall survival and progression free disease. Epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGG. We assessed the feasibility of detecting HGG using near-infrared fluorescent antibody targeting EGFR. Mice bearing orthotopic HGG xenografts with modest EGFR expression were imaged in vivo after systemic panitumumab-IRDye800 injection to assess its tumor-specific uptake macroscopically over 14 days, and microscopically ex vivo. EGFR immunohistochemical staining of 59 tumor specimens from 35 HGG patients was scored by pathologists and expression levels were compared to that of mouse xenografts. Intratumoral distribution of panitumumab-IRDye800 correlated with near-infrared fluorescence and EGFR expression. Fluorescence distinguished tumor cells with 90% specificity and 82.5% sensitivity. Target-to-background ratios peaked at 14 h post panitumumab-IRDye800 infusion, reaching 19.5 in vivo and 7.6 ex vivo, respectively. Equivalent or higher EGFR protein expression compared to the mouse xenografts was present in 77.1% HGG patients. Age, combined with IDH-wildtype cerebral tumor, was predictive of greater EGFR protein expression in human tumors. Tumor specific uptake of panitumumab-IRDye800 provided remarkable contrast and a flexible imaging window for fluorescence-guided identification of HGGs despite modest EGFR expression.

Focused Ultrasound for Noninvasive, Focal Pharmacologic Neurointervention.

A long-standing goal of translational neuroscience is the ability to noninvasively deliver therapeutic agents to specific brain regions with high spatiotemporal resolution. Focused ultrasound (FUS) is an emerging technology that can noninvasively deliver energy up the order of 1 kW/cm2 with millimeter and millisecond resolution to any point in the human brain with Food and Drug Administration-approved hardware. Although FUS is clinically utilized primarily for focal ablation in conditions such as essential tremor, recent breakthroughs have enabled the use of FUS for drug delivery at lower intensities (i.e., tens of watts per square centimeter) without ablation of the tissue. In this review, we present strategies for image-guided FUS-mediated pharmacologic neurointerventions. First, we discuss blood-brain barrier opening to deliver therapeutic agents of a variety of sizes to the central nervous system. We then describe the use of ultrasound-sensitive nanoparticles to noninvasively deliver small molecules to millimeter-sized structures including superficial cortical regions and deep gray matter regions within the brain without the need for blood-brain barrier opening. We also consider the safety and potential complications of these techniques, with attention to temporal acuity. Finally, we close with a discussion of different methods for mapping the ultrasound field within the brain and describe future avenues of research in ultrasound-targeted drug therapies.

MRI Monitoring and Quantification of Ultrasound-Mediated Delivery of Liposomes Dually Labeled with Gadolinium and Fluorophore through the Blood-Brain Barrier.

Magnetic resonance image-guided focused ultrasound has emerged as a viable non-invasive technique for the treatment of central nervous system-related diseases/disorders. Application of mechanical and thermal effects associated with focused transcranial ultrasound has been studied extensively in pre-clinical models, which has paved the way for clinical trials. However, in vivo treatment evaluation techniques on drug delivery application via blood-brain barrier opening has not been fully explored. Current treatment evaluation techniques via magnetic resonance imaging are hindered by systemic toxicity resulting from free gadolinium delivery. Here we propose a novel treatment evaluation strategy to overcome limitations by (i) synthesizing liposomes that are dually labeled with gadolinium, a magnetic resonance imaging (MRI) contrast agent, and rhodamine, a fluorophore; (ii) applying a focused ultrasound (FUS)-mediated BBB opening technique to deliver the liposomes across vascular barriers, achieving local gadolinium enhancement while reducing systemic and unwanted regional toxic effects associated with free gadolinium; and (iii) utilizing the MRI modality to confirm the delivery as it is already included in the FUS treatment in clinic. Liposomes were secondarily labeled with a fluorescent marker to confirm results obtained by MRI quantification postmortem. Two different sizes, 77.5 nm (group A) and 140 nm (group B), of gadolinium- and fluorescence-labeled liposomes were fabricated using thin-film hydration followed by extrusion methods and determined their stability up to 6 h under physiologic conditions. Gadolinium signal was detected on contrast-enhanced T1-weighted MRI 5 h after the delivery of liposomes via the BBB opening approach with an ultrasound pulse of 0.42 MPa (estimate in water) combined with microbubbles. MRI contrast was enhanced significantly in sonicated regions compared with non-sonicated regions of the brain. This was due to the accumulation of labeled liposomes, which was confirmed by detection of rhodamine fluorescence in histologic sections. The relative increase in MRI signal intensity was greater for smaller liposomes (mean diameter = 77.5 nm) than larger liposomes (mean diameter = 140 nm), which suggested a greater accumulation of the smaller liposomes in the brain after ultrasound-mediated opening of the BBB. Our findings suggest that the dual-labeled nanocarrier platform can be established, the FUS-mediated BBB opening approach can be used to deliver it through vascular barriers and MRI can be used to evaluate the extent of nanocarrier delivery.

Noninvasive Ultrasonic Drug Uncaging Maps Whole-Brain Functional Networks.

Being able to noninvasively modulate brain activity, where and when an experimenter desires, with an immediate path toward human translation is a long-standing goal for neuroscience. To enable robust perturbation of brain activity while leveraging the ability of focused ultrasound to deliver energy to any point of the brain noninvasively, we have developed biocompatible and clinically translatable nanoparticles that allow ultrasound-induced uncaging of neuromodulatory drugs. Utilizing the anesthetic propofol, together with electrophysiological and imaging assays, we show that the neuromodulatory effect of ultrasonic drug uncaging is limited spatially and temporally by the size of the ultrasound focus, the sonication timing, and the pharmacokinetics of the uncaged drug. Moreover, we see secondary effects in brain regions anatomically distinct from and functionally connected to the sonicated region, indicating that ultrasonic drug uncaging could noninvasively map the changes in functional network connectivity associated with pharmacologic action at a particular brain target.

Closed-loop control of targeted ultrasound drug delivery across the blood-brain/tumor barriers in a rat glioma model.

Cavitation-facilitated microbubble-mediated focused ultrasound therapy is a promising method of drug delivery across the blood-brain barrier (BBB) for treating many neurological disorders. Unlike ultrasound thermal therapies, during which magnetic resonance thermometry can serve as a reliable treatment control modality, real-time control of modulated BBB disruption with undetectable vascular damage remains a challenge. Here a closed-loop cavitation controlling paradigm that sustains stable cavitation while suppressing inertial cavitation behavior was designed and validated using a dual-transducer system operating at the clinically relevant ultrasound frequency of 274.3 kHz. Tests in the normal brain and in the F98 glioma model in vivo demonstrated that this controller enables reliable and damage-free delivery of a predetermined amount of the chemotherapeutic drug (liposomal doxorubicin) into the brain. The maximum concentration level of delivered doxorubicin exceeded levels previously shown (using uncontrolled sonication) to induce tumor regression and improve survival in rat glioma. These results confirmed the ability of the controller to modulate the drug delivery dosage within a therapeutically effective range, while improving safety control. It can be readily implemented clinically and potentially applied to other cavitation-enhanced ultrasound therapies.

Effects on P-Glycoprotein Expression after Blood-Brain Barrier Disruption Using Focused Ultrasound and Microbubbles.

Many blood-borne substances attempting to pass through the luminal membrane of brain endothelial cells are acted upon by a variety of metabolizing enzymes or are actively expelled back into the capillary lumen by embedded efflux transporters, such as Permeability-glycoprotein (Pgp). Overexpression of this protein has also been linked to multidrug resistance in cancer cells. Previous studies have shown that focused ultrasound (FUS), when combined with a microbubble agent, has ability to temporarily disrupt blood-brain barrier (BBBD). In this work, we investigated whether modulation of Pgp expression is part of the FUS-induced effects. We found that ultrasound can temporarily suppress Pgp expression. When BBBD was produced at 0.55 MPa, Pgp was suppressed up to 48 hours and restored by 72 hours. At 0.81 MPa, suppression can last 72 hours or longer. These findings support the idea that microbubble-enhanced FUS disrupts the functional components of the BBB through suppression of drug efflux.

Evaluation of permeability, doxorubicin delivery, and drug retention in a rat brain tumor model after ultrasound-induced blood-tumor barrier disruption.

Drug delivery in brain tumors is challenging because of the presence of blood-brain barrier (BBB) and the blood-tumor barrier (BTB). Focused ultrasound (FUS) combined with microbubbles can enhance the permeability of the BTB in brain tumors, as well as disrupting the BBB in the surrounding tissue. In this study, dynamic contrast-enhanced Magnetic Resonance Imaging (DCE-MRI) was used to characterize FUS-induced permeability changes in a rat glioma model and in the normal brain and to investigate the relationship between these changes and the resulting concentration of the chemotherapy agent doxorubicin (DOX). 9L gliosarcoma cells were implanted in both hemispheres in male rats. At day 10-12 after implantation, FUS-induced BTB disruption using 690kHz ultrasound and Definity microbubbles was performed in one of the tumors and in a normal brain region in each animal. After FUS, DOX was administered at a dose of 5.67mg/kg. The resulting DOX concentration was measured via fluorometry at 1 or 24h after FUS. The transfer coefficient Ktrans describing extravasation of the MRI contrast agent Gd-DTPA was significantly increased in both the sonicated tumors and in the normal brain tissue (P<0.001) between the two DCE-MRI acquisitions obtained before and after FUS, while no significant difference was found in the controls (non-sonicated tumor/normal brain tissue). DOX concentrations were also significantly larger than controls in both the sonicated tumors and in the normal tissue volumes at 1 and 24h after sonication. The DOX concentrations were significantly larger (P<0.01) in the control tumors harvested 1h after FUS than in those harvested at 24h, when the tumor concentrations were not significantly different than in the non-sonicated normal brain. In contrast, there was no significant difference in the DOX concentrations between the tumors harvested at 1 and 24h after FUS or in the concentrations measured in the brain at these time points. The transfer coefficient Ktrans for Gd-DTPA and the drug concentrations showed a good linear correlation (R2=0.56). Overall, these data suggest that FUS and microbubbles can not only increase DOX delivery across the BBB and BTB, but that it is retained in the tissue at significantly enhanced levels for at least 24h. Such enhanced retention may increase the potency of this chemotherapy agent and allow for reduced systemic doses. Furthermore, MRI-based estimates of Gd-DTPA transport across these barriers might be useful to estimate local DOX concentrations in the tumor and in the surrounding normal tissue.

Multiple sessions of liposomal doxorubicin delivery via focused ultrasound mediated blood-brain barrier disruption: a safety study.

Transcranial MRI-guided focused ultrasound is a rapidly advancing method for delivering therapeutic and imaging agents to the brain. It has the ability to facilitate the passage of therapeutics from the vasculature to the brain parenchyma, which is normally protected by the blood-brain barrier (BBB). The method's main advantages are that it is both targeted and noninvasive, and that it can be easily repeated. Studies have shown that liposomal doxorubicin (Lipo-DOX), a chemotherapy agent with promise for tumors in the central nervous system, can be delivered into the brain across BBB. However, prior studies have suggested that doxorubicin can be significantly neurotoxic, even at small concentrations. Here, we studied whether multiple sessions of Lipo-DOX administered after FUS-induced BBB disruption (FUS-BBBD) induces severe adverse events in the normal brain tissues. First, we used fluorometry to measure the doxorubicin concentrations in the brain after FUS-BBBD to ensure that a clinically relevant doxorubicin concentration was achieved in the brain. Next, we performed three weekly sessions with FUS-BBBD±Lipo-DOX administration. Five to twelve targets were sonicated each week, following a schedule described previously in a survival study in glioma-bearing rats (Aryal et al., 2013). Five rats received three weekly sessions where i.v. injected Lipo-DOX was combined with FUS-BBBD; an additional four rats received FUS-BBBD only. Animals were euthanized 70days from the first session and brains were examined in histology. We found that clinically-relevant concentrations of doxorubicin (4.8±0.5µg/g) were delivered to the brain with the sonication parameters (0.69MHz; 0.55-0.81MPa; 10ms bursts; 1Hz PRF; 60s duration), microbubble concentration (Definity, 10µl/kg), and the administered Lipo-DOX dose (5.67mg/kg) used. The resulting concentration of Lipo-DOX was reduced by 32% when it was injected 10min after the last sonication compared to cases where the agent was delivered before sonication. In histology, the severe neurotoxicity observed in some previous studies with doxorubicin by other investigators was not observed here. However, four of the five rats who received FUS-BBBD and Lipo-DOX had regions (dimensions: 0.5-2mm) at the focal targets with evidence of minor prior damage, either a small scar (n=4) or a small cyst (n=1). The focal targets were unaffected in rats who received FUS-BBBD alone. The result indicates that while delivery of Lipo-DOX to the rat brain might result in minor damage, the severe neurotoxicity seen in earlier works does not appear to occur with delivery via FUS-BBB disruption. The damage may be related to capillary damage produced by inertial cavitation, which might have resulted in excessive doxorubicin concentrations in some areas.

Enhancement in blood-tumor barrier permeability and delivery of liposomal doxorubicin using focused ultrasound and microbubbles: evaluation during tumor progression in a rat glioma model.

Effective drug delivery to brain tumors is often challenging because of the heterogeneous permeability of the 'blood tumor barrier' (BTB) along with other factors such as increased interstitial pressure and drug efflux pumps. Focused ultrasound (FUS) combined with microbubbles can enhance the permeability of the BTB in brain tumors, as well as the blood-brain barrier in the surrounding tissue. In this study, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to characterize the FUS-induced permeability changes of the BTB in a rat glioma model at different times after implantation. 9L gliosarcoma cells were implanted in both hemispheres in male rats. At day 9, 14, or 17 days after implantation, FUS-induced BTB disruption using 690 kHz ultrasound and definity microbubbles was performed in one tumor in each animal. Before FUS, liposomal doxorubicin was administered at a dose of 5.67 mg kg(-1). This chemotherapy agent was previously shown to improve survival in animal glioma models. The transfer coefficient Ktrans describing extravasation of the MRI contrast agent Gd-DTPA was measured via DCE-MRI before and after sonication. We found that tumor doxorubicin concentrations increased monotonically (823 ± 600, 1817 ± 732 and 2432 ± 448 ng g(-1)) in the control tumors at 9, 14 and 17 d. With FUS-induced BTB disruption, the doxorubicin concentrations were enhanced significantly (P < 0.05, P < 0.01, and P < 0.0001 at days 9, 14, and 17, respectively) and were greater than the control tumors by a factor of two or more (2222 ± 784, 3687 ± 796 and 5658 ± 821 ng g(-1)) regardless of the stage of tumor growth. The transfer coefficient Ktrans was significantly (P < 0.05) enhanced compared to control tumors only at day 9 but not at day 14 or 17. These results suggest that FUS-induced enhancements in tumor drug delivery are relatively consistent over time, at least in this tumor model. These results are encouraging for the use of large drug carriers, as they suggest that even large/late-stage tumors can benefit from FUS-induced drug enhancement. Corresponding enhancements in Ktrans were found to be variable in large/late-stage tumors and not significantly different than controls, perhaps reflecting the size mismatch between the liposomal drug (~100 nm) and Gd-DTPA (molecular weight: 938 Da; hydrodynamic diameter: ≃2 nm). It may be necessary to use a larger MRI contrast agent to effectively evaluate the sonication-induced enhanced permeabilization in large/late-stage tumors when a large drug carrier such as a liposome is used.

Ultrasound-mediated blood-brain barrier disruption for targeted drug delivery in the central nervous system.

The physiology of the vasculature in the central nervous system (CNS), which includes the blood-brain barrier (BBB) and other factors, complicates the delivery of most drugs to the brain. Different methods have been used to bypass the BBB, but they have limitations such as being invasive, non-targeted or requiring the formulation of new drugs. Focused ultrasound (FUS), when combined with circulating microbubbles, is a noninvasive method to locally and transiently disrupt the BBB at discrete targets. This review provides insight on the current status of this unique drug delivery technique, experience in preclinical models, and potential for clinical translation. If translated to humans, this method would offer a flexible means to target therapeutics to desired points or volumes in the brain, and enable the whole arsenal of drugs in the CNS that are currently prevented by the BBB.

Multiple treatments with liposomal doxorubicin and ultrasound-induced disruption of blood-tumor and blood-brain barriers improve outcomes in a rat glioma model.

The blood-brain-barrier (BBB) prevents the transport of most anticancer agents to the central nervous system and restricts delivery to infiltrating brain tumors. The heterogeneous vascular permeability in tumor vessels, along with several other factors, creates additional barriers for drug treatment of brain tumors. Focused ultrasound (FUS), when combined with circulating microbubbles, is an emerging noninvasive method to temporarily permeabilize the BBB and the "blood-tumor barrier". Here, we tested the impact of three weekly sessions of FUS and liposomal doxorubicin (DOX) in 9L rat glioma tumors. Animals that received FUS+DOX (N=8) had a median survival time that was increased significantly (P<0.001) compared to animals who received DOX only (N=6), FUS only (N=8), or no treatment (N=7). Median survival for animals that received FUS+DOX was increased by 100% relative to untreated controls, whereas animals who received DOX alone had only a 16% improvement. Animals who received only FUS showed no improvement. No tumor cells were found in histology in 4/8 animals in the FUS+DOX group, and in two animals, only a few tumor cells were detected. Adverse events in the treatment group included skin toxicity, impaired activity, damage to surrounding brain tissue, and tissue loss at the tumor site. In one animal, intratumoral hemorrhage was observed. These events are largely consistent with known side effects of doxorubicin and with an extensive tumor burden. Overall this work demonstrates that multiple sessions using this FUS technique to enhance the delivery of liposomal doxorubicin have a pronounced therapeutic effect in this rat glioma model.