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.

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 closedloop 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.drug delivery | focused ultrasound | acoustic cavitation | treatment control | blood-brain barrier

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 for 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 has a pronounced therapeutic effect in this rat glioma model.

Summary Being able to noninvasively modulate brain activity, where and when an experimenter desires, with an immediate path towards 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.

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.

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 70 days from the first session and brains were examined in histology. We found that clinically-relevant concentrations of doxorubicin (4.8 ± 0.5 µg/g) were delivered to the brain with the sonication parameters (0.69 MHz; 0.55–0.81 MPa; 10 ms bursts; 1 Hz PRF; 60s duration), microbubble concentration (Definity, 10 µl/kg), and the administered Lipo-DOX dose (5.67 mg/kg) used. The resulting concentration of Lipo-DOX was reduced by 32% when it was injected 10 minutes 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: 0.5–2 mm) at the focal targets with evidence of minor prior damage, either a small scar (n=4) and 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.

Author Contributions QZ and RDA designed the experiments. QZ performed the chemistry of nanoparticle production and physicochemical characterization, carried out the pharmacokinetics study and in vivo biodistribution of the nanoparticles, and performed the in vitro/in vivo ultrasonic drug uncaging experiments, with guidance from MA and assistance from JBW and AK. Cryo-TEM imaging was performed by MAB, RHC, and NHN. QZ and BCY performed the ultrasound imaging and ultrasonic nicardipine uncaging experiments and their analysis. TI and KFW designed and performed the ultra-high-speed optical imaging and passive cavitation detection of the nanoemulsions. QZ, BCY, and RDA prepared the figures and wrote the manuscript, with input and edits from all the authors.

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 690 kHz 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 5.67 mg kg−1. The resulting DOX concentration was measured via fluorometry at 1 or 24 hours 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 24 hours after sonication. The DOX concentrations were significantly larger (P<0.01) in the control tumors harvested 1 hour after FUS than in those harvested at 24 hours, 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 24 hours 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 24 hours. 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.