Oluyemi Olumolade

Activation of signaling pathways following localized delivery of systemically administered neurotrophic factors across the blood–brain barrier using focused ultrasound and microbubbles

The brain-derived neurotrophic factor (BDNF) has been shown to have broad neuroprotective effects in addition to its therapeutic role in neurodegenerative disease. In this study, the efficacy of delivering exogenous BDNF to the left hippocampus is demonstrated in wild-type mice (n = 7) through the noninvasively disrupted blood-brain barrier (BBB) using focused ultrasound (FUS). The BDNF bioactivity was found to be preserved following delivery as assessed quantitatively by immunohistochemical detection of the pTrkB receptor and activated pAkt, pMAPK, and pCREB in the hippocampal neurons. It was therefore shown for the first time that systemically administered neurotrophic factors can cross the noninvasively disrupted BBB and trigger neuronal downstream signaling effects in a highly localized region in the brain. This is the first time that the administered molecule is tracked through the BBB and localized in the neuron triggering molecular effects. Additional preliminary findings are shown in wild-type mice with two additional neurotrophic factors such as the glia-derived neurotrophic factor (n = 12) and neurturin (n = 2). This further demonstrates the impact of FUS for the early treatment of CNS diseases at the cellular and molecular level and strengthens its premise for FUS-assisted drug delivery and efficacy.

Targeted drug delivery with focused ultrasound-induced blood-brain barrier opening using acoustically-activated nanodroplets.

Focused ultrasound (FUS) in the presence of systemically administered microbubbles has been shown to locally, transiently and reversibly increase the permeability of the blood-brain barrier (BBB), thus allowing targeted delivery of therapeutic agents in the brain for the treatment of central nervous system diseases. Currently, microbubbles are the only agents that have been used to facilitate the FUS-induced BBB opening. However, they are constrained within the intravascular space due to their micron-size diameters, limiting the delivery effect at or near the microvessels. In the present study, acoustically-activated nanodroplets were used as a new class of contrast agents to mediate FUS-induced BBB opening in order to study the feasibility of utilizing these nanoscale phase-shift particles for targeted drug delivery in the brain. Significant dextran delivery was achieved in the mouse hippocampus using nanodroplets at clinically relevant pressures. Conventional microbubbles with the same lipid shell composition and perfluorobutane core as the nanodroplets were also used to compare the efficiency of FUS-induced dextran delivery. It was found that nanodroplets had a higher BBB opening pressure threshold but a lower stable cavitation threshold than microbubbles, indicating that contrast agent-dependent acoustic emission monitoring should be carried out. More homogeneous dextran delivery within the targeted hippocampus was achieved using nanodroplets without inducing inertial cavitation or compromising safety. Our results offered a new means of developing the FU-Sinduced BBB opening technology for potential extravascular targeted drug delivery in the brain, extending the potential drug delivery region beyond the cerebral vasculature.

Noninvasive, neuron-specific gene therapy can be facilitated by focused ultrasound and recombinant adeno-associated virus.

Recombinant adeno-associated virus (rAAV) has shown great promise as a potential cure for neurodegenerative diseases. The existence of the blood–brain barrier (BBB), however, hinders efficient delivery of the viral vectors. Direct infusion through craniotomy is the most commonly used approach to achieve rAAV delivery, which carries increased risks of infection and other complications. Here, we report a focused ultrasound (FUS)-facilitated noninvasive rAAV delivery paradigm that is capable of producing targeted and neuron-specific transductions. Oscillating ultrasound contrast agents (microbubbles), driven by FUS waves, temporarily ‘unlock’ the BBB, allowing the systemically administrated rAAVs to enter the brain parenchyma, while maintaining their bioactivity and selectivity. Taking the advantage of the neuron-specific promoter synapsin, rAAV gene expression was triggered almost exclusively (95%) in neurons of the targeted caudate–putamen region. Both behavioral assessment and histological examination revealed no significant long-term adverse effects (in the brain and several other critical organs) for this combined treatment paradigm. Results from this study demonstrated the feasibility and safety for the noninvasive, targeted rAAV delivery, which might have open a new avenue in gene therapy in both preclinical and clinical settings.

Effects of the microbubble shell physicochemical properties on ultrasound-mediated drug delivery to the brain.

Lipid-shelled microbubbles have been used in ultrasound-mediated drug delivery. The physicochemical properties of the microbubble shell could affect the delivery efficiency since they determine the microbubble mechanical properties, circulation persistence, and dissolution behavior during cavitation. Therefore, the aim of this study was to investigate the shell effects on drug delivery efficiency in the brain via blood-brain barrier (BBB) opening in vivo using monodisperse microbubbles with different phospholipid shell components. The physicochemical properties of the monolayer were varied by using phospholipids with different hydrophobic chain lengths (C16, C18, and C24). The dependence on the molecular size and acoustic energy (both pressure and pulse length) were investigated. Our results showed that a relatively small increase in the microbubble shell rigidity resulted in a significant increase in the delivery of 40-kDa dextran, especially at higher pressures. Smaller (3 kDa) dextran did not show significant difference in the delivery amount, suggesting the observed shell effect was molecular size-dependent. In studying the impact of acoustic energy on the shell effects, it was found that they occurred most significantly at pressures causing microbubble fragmentation (450 kPa and 600 kPa); by increasing the pulse length to deliver the 40-kDa dextran, the difference between C16 and C18 was eliminated while C24 achieved the highest delivery efficiency. These findings indicated that the acoustic parameters could be adjusted to modulate the shell effects. The acoustic cavitation emission revealed the physical mechanisms associated with different shells. Overall, lipid-shelled microbubbles with long hydrophobic chain length could achieve high delivery efficiency for larger molecules especially with high acoustic energy. Our study offered, for the first time, evidence directly linking the microbubble monolayer shell with their efficacy for drug delivery in vivo.

Focused ultrasound-facilitated brain drug delivery using optimized nanodroplets: Vaporization efficiency dictates large molecular delivery

Focused ultrasound with nanodroplets could facilitate localized drug delivery after vaporization with potentially improved in vivo stability, drug payload, and minimal interference outside of the focal zone compared with microbubbles. While the feasibility of blood-brain barrier (BBB) opening using nanodroplets has been previously reported, characterization of the associated delivery has not been achieved. It was hypothesized that the outcome of drug delivery was associated with the droplet sensitivity to acoustic energy, and can be modulated with the boiling point of the liquid core. Therefore, in this study, highly-efficient octafluoropropane (OFP) and less-efficient decafluorobutane (DFB) nanodroplets were used in vivo for delivering molecules with a size relevant to proteins (40-kDa dextran) to the murine brain. It was found that successful delivery was achieved with OFP droplets at 300 kPa or higher safely using 1/4 dosage compared to DFB droplets at 900 kPa where inertial cavitation caused damage. As a result, the OFP droplets due to the higher vaporization efficiency served as better acoustic agents to deliver large molecules safely and efficiently to the brain compared with the DFB droplets.

Enhancement of direct brain infusion with focused ultrasound and microbubbles

Direct infusion to the brain is a frequently used technique in pre-clinical neuroscience research as well as several clinical applications. The relatively high intracranial pressure is one of the limiting factors that hinder efficient diffusion from the cannula tip. In this study, we utilized focused ultrasound (FUS) and microbubbles to condition the brain prior to performing the direct infusion. The acoustic parameters used for sonications were: 0.45 MPa peak rarefactional pressure, 6.7 ms pulse length, 5 Hz pulse repetition frequency, and a duration of 60 s. A 9.4 T magnetic resonance imaging (MRI) system was used to monitor the diffusion of an albumin-tagged MR contrast agent up to two hours. In addition, the diffusion of a commonly used gene therapy vector - adeno-associated virus (AAV) was evaluated via fluorescence imaging. Our results revealed that Pre-treatment with FUS and microbubbles significantly enhanced the total volume (P<;0.001) and volume increase (P<;0.05) of MR contrast agent in vivo.

Focused ultrasound-facilitated brain drug delivery using optimized nanodroplets

Focused ultrasound with nanodroplets could facilitate localized drug delivery after vaporization with potentially improved in vivo stability, drug payload, and minimal interference outside of the focal zone compared with microbubbles. While the feasibility of blood-brain barrier (BBB) opening using nanodroplets has been previously reported, characterization of the associated delivery has not been achieved. It was hypothesized that the outcome of drug delivery was associated with the droplet sensitivity to acoustic energy, and can be modulated with the boiling point of the liquid core. Therefore, in this study, highly-efficient octafluoropropane (OFP) and less-efficient decafluorobutane (DFB) nanodroplets were used in vivo for delivering molecules with a size relevant to proteins (40-kDa dextran) to the murine brain. It was found that successful delivery was achieved with OFP droplets at 300 kPa or higher safely using 1/4 dosage compared to DFB droplets at 900 kPa where inertial cavitation caused damage. As a result, the OFP droplets due to the higher vaporization efficiency served as better acoustic agents to deliver large molecules safely and efficiently to the brain compared with the DFB droplets.

Focused ultrasound-facilitated molecular delivery to the brain using drug-loaded nanodroplets

Acoustically-activated nanodroplets facilitate localized drug delivery after vaporization with improved in vivo stability, drug payload, and minimal interference outside of the ultrasound focal zone compared with microbubbles. They are new acoustic mediators to induce blood-brain barrier (BBB) opening for drug delivery to the brain, with promising potential of extravasation to enhance targeted delivery in the extravascular space due to the nano sizes.)

Effects of microbubble shell physicochemical properties on ultrasound-mediated drug delivery to the brain

Lipid-shelled microbubbles have been used in ultrasound-mediated drug delivery. The physicochemical properties of the microbubble shell could affect the delivery efficiency since they determine the microbubble mechanical properties, circulation persistence, and dissolution behavior during cavitation. Therefore, the aim of this study was to investigate the shell effects on drug delivery efficiency in the brain via blood-brain barrier (BBB) opening in vivo using monodisperse microbubbles with different phospholipid shell components. The physicochemical properties of the monolayer were varied by using phospholipids with different hydrophobic chain lengths (C16, C18, and C24). The dependence on the molecular size and acoustic energy (both pressure and pulse length) were investigated. Our results showed that a relatively small increase in the microbubble shell rigidity resulted in a significant increase in the delivery of 40-kDa dextran, especially at higher pressures. Smaller (3 kDa) dextran did not show significant difference in the delivery amount, suggesting the observed shell effect was molecular size-dependent. In studying the impact of acoustic energy on the shell effects, it was found that they occurred most significantly at pressures causing microbubble fragmentation (450 kPa and 600 kPa); by increasing the pulse length to deliver the 40-kDa dextran, the difference between C16 and C18 was eliminated while C24 achieved the highest delivery efficiency. These findings indicated that the acoustic parameters could be adjusted to modulate the shell effects. The acoustic cavitation emission revealed the physical mechanisms associated with different shells. Overall, lipid-shelled microbubbles with long hydrophobic chain length could achieve high delivery efficiency for larger molecules especially with high acoustic energy. Our study offered, for the first time, evidence directly linking the microbubble monolayer shell with their efficacy for drug delivery in vivo.

Neurotrophic delivery in Alzheimer’s-model mice

Alzheimer’s disease (AD), which has emerged as one of the most common brain disorders, begins in the hippocampal formation and gradually spreads to the remaining brain at its most advanced stages, and is characterized partly by deposition of amyloid plaques in the brain tissue but also in the blood vessels themselves. Our studies have dealt with both the delivery of neurotrophic factors through the FUS-induced blood-brain barrier (BBB) opening to the hippocampus in both the presence and absence of disease in AD mouse models. The Brain-Derived Neurotrophic Factor (BDNF) is widely and abundantly expressed in the CNS and is available to some peripheral nervous system neurons that uptake the neurotrophin produced by peripheral tissues. BDNF can modulate neuronal synaptic strength and has been implicated in hippocampal mechanisms of learning and memory. As BDNF has been proven to serve as a neuroprotective agent, delivery of exogenous BDNF is a good candidate for therapeutic treatment of several CNS disorders.