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.

Liposome delivery to the brain with rapid short-pulses of focused ultrasound and microbubbles.

Liposomes are clinically used drug carriers designed to improve the delivery of drugs to specific tissues while minimising systemic distribution. However, liposomes are unable to cross the blood-brain barrier (BBB) and enter the brain, mostly due to their large size (ca. 100 nm). A noninvasive and localised method of delivering liposomes across the BBB is to intravenously inject microbubbles and apply long pulses of ultrasound (pulse length: >1 ms) to a targeted brain region. Recently, we have shown that applying rapid short pulses (RaSP) (pulse length: 5 µs) can deliver drugs with an improved efficacy and safety profile. However, this was tested with a relatively smaller 3-kDa molecule (dextran). In this study, we examine whether RaSP can deliver liposomes to the murine brain in vivo. Fluorescent DiD-PEGylated liposomes were synthesized and injected intravenously alongside microbubbles. The left hippocampus of mice was then sonicated with either a RaSP sequence (5 µs at 1.25 kHz in groups of 10 ms at 0.5 Hz) or a long pulse sequence (10 ms at 0.5 Hz), with each pulse having a 1-MHz centre frequency (0.35 and 0.53 MPa). The delivery and distribution of the fluorescently-labelled liposomes were assessed by fluorescence imaging of the brain sections. The safety profile of the sonicated brains was assessed by histological staining. RaSP was shown to locally deliver liposomes across the BBB at 0.53 MPa with a more diffused and safer profile compared to the long pulse ultrasound sequence. Cellular uptake of liposomes was observed in neurons and microglia, while no uptake within astrocytes was observed in both RaSP and long pulse-treated brains. This study shows that RaSP allows a targeted and safe delivery of liposomal drugs into the murine brain with potential to deliver drugs into neuronal and glial targets.

Modulation of amyloid-ß aggregation by metal complexes with a dual binding mode and their delivery across the blood-brain barrier using focused ultrasound.

One of the key hallmarks of Alzheimer's disease is the aggregation of the amyloid-ß peptide to form fibrils. Consequently, there has been great interest in studying molecules that can disrupt amyloid-ß aggregation. While a handful of molecules have been shown to inhibit amyloid-ß aggregation in vitro, there remains a lack of in vivo data reported due to their inability to cross the blood-brain barrier. Here, we investigate a series of new metal complexes for their ability to inhibit amyloid-ß aggregation in vitro. We demonstrate that octahedral cobalt complexes with polyaromatic ligands have high inhibitory activity thanks to their dual binding mode involving π-π stacking and metal coordination to amyloid-ß (confirmed via a range of spectroscopic and biophysical techniques). In addition to their high activity, these complexes are not cytotoxic to human neuroblastoma cells. Finally, we report for the first time that these metal complexes can be safely delivered across the blood-brain barrier to specific locations in the brains of mice using focused ultrasound.

Combination Strategies to Improve Targeted Radionuclide Therapy.

In recent years, targeted radionuclide therapy (TRT) has emerged as a promising strategy for cancer treatment. In contrast to conventional radiotherapy, TRT delivers ionizing radiation to tumors in a targeted manner, reducing the dose that healthy tissues are exposed to. Existing TRT strategies include the use of 177Lu-DOTATATE, 131I-metaiodobenzylguanidine, Bexxar, and Zevalin, clinically approved agents for the treatment of neuroendocrine tumors, neuroblastoma, and non-Hodgkin lymphoma, respectively. Although promising results have been obtained with these agents, clinical evidence acquired to date suggests that only a small percentage of patients achieves complete response. Consequently, there have been attempts to improve TRT outcomes through combinations with other therapeutic agents; such strategies include administering concurrent TRT and chemotherapy, and the use of TRT with known or putative radiosensitizers such as poly(adenosine diphosphate ribose) polymerase and mammalian-target-of-rapamycin inhibitors. In addition to potentially achieving greater therapeutic effects than the respective monotherapies, these strategies may lead to lower dosages or numbers of cycles required and, in turn, reduce unwanted toxicities. As of now, several clinical trials have been conducted to assess the benefits of TRT-based combination therapies, sometimes despite limited preclinical evidence being available in the public domain to support their use. Although some clinical trials have yielded promising results, others have shown no clear survival benefit from particular combination treatments. Here, we present a comprehensive review of combination strategies with TRT reported in the literature to date and evaluate their therapeutic potential.

Molecular recognition of bisphosphonate-based drugs by di-zinc receptors in aqueous solution and on gold nanoparticles.

Metal-based anion receptors have several important applications in sensing, separation and transport of negatively charged species. Amongst these receptors, di-zinc(ii) complexes are of particular interest for the recognition of oxoanions, in particular phosphate derivatives. Herein we report the synthesis of a di-zinc(ii) receptor and show that it has high affinity and selectivity for bisphosphonates such as alendronate and etidronate - which are used to treat a number of skeletal disorders as well as showing interesting anticancer properties. The binding mode of the di-zinc(ii) receptor with alendronate and etidronate has been unambiguously established by single crystal X-ray crystallography. In addition, by modifying the backbone of the receptor, we show that the drug-loaded receptor can be attached onto gold nanoparticles as potential drug-delivery vehicles.

Neuron labeling with rhodamine-conjugated Gd-based MRI contrast agents delivered to the brain via focused ultrasound.

Gadolinium-based magnetic resonance imaging contrast agents can provide information regarding neuronal function, provided that these agents can cross the neuronal cell membrane. Such contrast agents are normally restricted to extracellular domains, however, by attaching cationic fluorescent dyes, they can be made cell-permeable and allow for both optical and magnetic resonance detection. To reach neurons, these agents also need to cross the blood-brain barrier. Focused ultrasound combined with microbubbles has been shown to enhance the permeability of this barrier, allowing molecules into the brain non-invasively, locally and transiently. The goal of this study was to investigate whether combining fluorescent rhodamine with a gadolinium complex would form a dual-modal contrast agent that could label neurons in vivo when delivered to the mouse brain with focused ultrasound and microbubbles. Methods: Gadolinium complexes were combined with a fluorescent, cationic rhodamine unit to form probes with fluorescence and relaxivity properties suitable for in vivo applications. The left hemisphere of female C57bl/6 mice (8-10 weeks old; 19.07 ± 1.56 g; n = 16) was treated with ultrasound (centre frequency: 1 MHz, peak-negative pressure: 0.35 MPa, pulse length: 10 ms, repetition frequency: 0.5 Hz) while intravenously injecting SonoVue microbubbles and either the 1 kDa Gd(rhodamine-pip-DO3A) complex or a conventionally-used lysine-fixable Texas Red® 3 kDa dextran. The opposite right hemisphere was used as a non-treated control region. Brains were then extracted and either sectioned and imaged via fluorescence or confocal microscopy or imaged using a 9.4 T magnetic resonance imaging scanner. Brain slices were stained for neurons (NeuN), microglia (Iba1) and astrocytes (GFAP) to investigate the cellular localization of the probes. Results: Rhodamine fluorescence was detected in the left hemisphere of all ultrasound treated mice, while none was detected in the right control hemisphere. Cellular uptake of Gd(rhodamine-pip-DO3A) was observed in all the treated regions with a uniform distribution (coefficient of variation = 0.4 ± 0.05). Uptake was confirmed within neurons, whereas the probe did not co-localize with microglia and astrocytes. Compared to the dextran molecule, Gd(rhodamine-pip-DO3A) distributed more homogeneously and was less concentrated around blood vessels. Furthermore, the dextran molecule was found to accumulate unselectively in microglia as well as neurons, whereas our probe was only taken up by neurons. Gd(rhodamine-pip-DO3A) was detected via magnetic resonance imaging ex vivo in similar regions to where fluorescence was detected. Conclusion: We have introduced a method to image neurons with a dual-modal imaging agent delivered non-invasively and locally to the brain using focused ultrasound and microbubbles. When delivered to the mouse brain, the agent distributed homogeneously and was only uptaken by neurons; in contrast, conventionally used dextran distributed heterogeneously and was uptaken by microglia as well as neurons. This result indicates that our probe labels neurons without microglial involvement and in addition the probe was found to be detectable via both ex vivo MRI and fluorescence. Labeling neurons with such dual-modal agents could facilitate the study of neuronal morphology and physiology using the advantages of both imaging modalities.

Rapid Short-pulse Ultrasound Delivers Drugs Uniformly across the Murine Blood-Brain Barrier with Negligible Disruption.

Background Previous work has demonstrated that drugs can be delivered across the blood-brain barrier by exposing circulating microbubbles to a sequence of long ultrasound pulses. Although this sequence has successfully delivered drugs to the brain, concerns remain regarding potentially harmful effects from disrupting the brain vasculature. Purpose To determine whether a low-energy, rapid, short-pulse ultrasound sequence can efficiently and safely deliver drugs to the murine brain. Materials and Methods Twenty-eight female wild-type mice underwent focused ultrasound treatment after injections of microbubbles and a labeled model drug, while three control mice were not treated (May-November 2017). The left hippocampus of 14 mice was exposed to low-energy short pulses (1 MHz; five cycles; peak negative pressure, 0.35 MPa) of ultrasound emitted at a rapid rate (1.25 kHz) in bursts (0.5 Hz), and another 14 mice were exposed to standard long pulses (10 msec, 0.5 Hz) containing 150 times more acoustic energy. Mice were humanely killed at 0 (n = 5), 10 (n = 3), or 20 minutes (n = 3) after ultrasound treatment. Hematoxylin-eosin (H-E) staining was performed on three mice. The delivered drug dose and distribution were quantified with the normalized optical density and coefficient of variation. Safety was assessed by H-E staining, the amount of albumin released, and the duration of permeability change in the blood-brain barrier. Statistical analysis was performed by using the Student t test. Results The rapid short-pulse sequence delivered drugs uniformly throughout the parenchyma. The acoustic energy emitted from the microbubbles also predicted the delivered dose (r = 0.97). Disruption in the blood-brain barrier lasted less than 10 minutes and 3.4-fold less albumin was released into the brain than with long pulses. No vascular or tissue damage from rapid short-pulse exposure was observable using H-E staining. Conclusion The rapid short-pulse ultrasound sequence is a minimally disruptive and efficient drug delivery method that could improve the treatment, diagnosis, and study of neurologic diseases. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Klibanov and McDannold in this issue.

Targeted Delivery of DNA-Au Nanoparticles across the Blood-Brain Barrier Using Focused Ultrasound.

Nanoparticles have been widely studied as versatile platforms for in vivo imaging and therapy. However, their use to image and/or treat the brain is limited, as they are often unable to cross the blood-brain barrier (BBB). To overcome this problem, herein we report the use of focused ultrasound in vivo to successfully deliver DNA-coated gold nanoparticles to specific locations in the brains of mice.