Molecules of Various Pharmacologically-Relevant Sizes Can Cross the Ultrasound-Induced Blood-Brain Barrier Opening in vivo

Abstract

Focused ultrasound (FUS) is hereby shown to noninvasively and selectively deliver compounds at pharmacologically relevant molecular weights through the opened blood-brain barrier (BBB). A complete examination on the size of the FUS-induced BBB opening, the spatial distribution of the delivered agents and its dependence on the agent's molecular weight were imaged and quantified using fluorescence microscopy. BBB opening in mice (n=13) was achieved in vivo after systemic administration of microbubbles and subsequent application of pulsed FUS (frequency: 1.525 MHz, peak-rarefactional pressure in situ: 569 kPa) to the left murine hippocampus through the intact skin and skull. BBB-impermeant, fluorescent-tagged dextrans at three distinct molecular weights spanning over several orders of magnitude were systemically administered and acted as model therapeutic compounds. First, dextrans of 3 and 70 kDa were delivered trans-BBB while 2000 kDa dextran was not. Second, compared to 70 kDa dextran, a higher concentration of 3 kDa dextran was delivered through the opened BBB. Third, the 3 and 70 kDa dextrans were both diffusely distributed throughout the targeted brain region. However, high concentrations of 70 kDa dextran appeared more punctated throughout the targeted region. In conclusion, FUS combined with microbubbles opened the BBB sufficiently to allow passage of compounds of at least 70 kDa, but not greater than 2000 kDa, into the brain parenchyma. This noninvasive and localized BBB opening technique could thus provide a unique means for the delivery of compounds of several magnitudes of kDa that include agents with shown therapeutic promise in vitro, but whose in vivo translation has been hampered by their associated BBB impermeability.

Noninvasive and localized neuronal delivery using short ultrasonic pulses and microbubbles

“…Previous work by our group and others typically optimized a few ultrasonic parameters, namely the PRP, frequency, PRF, and PL. However, increasing the delivered dose by modifying one of these parameters was also associated with either poorer distribution characteristics (17) or the onset of cellular damage (11). This paper explored a wider range of parameters that, to our knowledge, has not been used for drug delivery.…”

“…FUSinduced BBB disruption has been previously demonstrated to successfully deliver large molecular agents such as 70-kDa dextrans (17), Herceptin (24), and Doxorubicin (25). Nevertheless, concerns remain with the dose and distribution of the agents delivered (14,17) and the safety associated with enhancing these characteristics (11). For instance, increased dose can be achieved with higher PRPs and PLs, but higher PRPs have been associated with cellular damage (11) and longer PLs have been associated with increased molecular accumulation at or near larger vessels, thus indicating off-target and inhomogeneous distribution (16,17).…”

“…Nevertheless, concerns remain with the dose and distribution of the agents delivered (14,17) and the safety associated with enhancing these characteristics (11). For instance, increased dose can be achieved with higher PRPs and PLs, but higher PRPs have been associated with cellular damage (11) and longer PLs have been associated with increased molecular accumulation at or near larger vessels, thus indicating off-target and inhomogeneous distribution (16,17). The short PL sequences used in this paper produced a more homogeneous distribution of molecular delivery throughout the targeted volume when compared to longer 20-ms PLs, especially for the 3-kDa dextran (Figs.…”

“…1 and 2). However, drug penetration and distribution are a greater concern for larger molecular weight agents because attempts to deliver 70-kDa dextran using a 20-ms PL resulted in punctate regions of high concentrations in the larger vessels (i.e., longitudinal and transverse hippocampal vessels and posterior cerebral artery) in addition to low levels of diffuse fluorescence (17). Although short PLs have not eliminated the presence of these punctate regions, evidence suggests that their locations have penetrated deeper into the vascular branches and into smaller vessels such as arterioles.…”

“…Most of the aforementioned studies have used long PLs of 10 or 20 ms. However, recent work has shown that these PLs distribute the molecules heterogeneously throughout the target volume, with a greater accumulation near larger vessels (17). Reduction of the PL decreased not only the likelihood of BBB disruption, but also the delivered dose.…”